Advances in Protein Chemistry, Volume 39

ADVANCES IN PROTEIN CHEMISTRY Volume 39

This Page Intentionally Left Blank

ADVANCES IN PROTEIN CHEMISTRY EDITED BY

C. B. ANFINSEN

JOHN T. EDSALL

Department of Biology The Johns Hopkins University B8ltimore, Maryland

Department of Biochemistry 8nd Molecular B i o l ~ y Harvard University C8mbridge, MaSS8ChuSetlS

FREDERIC M. RICHARDS

DAVID S. EISENBERG

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California

VOLUME 39

ACADEMIC PRESS, INC. Harcourt Brace Jovanovlch, Publlrhorr

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9 8 7 6 5 4 3 2 1

CONTENTS PREFACE

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Basement Membrane Proteins: Molecular Structure and Function GEORGER. MARTIN,RUPERTTIMPL, AND KLAUSKUHN

Introduction . . . . Morphology and Ultrastructure. Components . . . . Self-Assembly and Interaction between Components . . V. Biological Aspects . . . References . . . . .

I. 11. 111. IV.

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51 53 68 101 118

Design of Peptides and Proteins WILLIAMF. DEGRADO

I. 11. 111. JV.

Introduction . . . . Design of Small Peptides . Design of Medium-Sized Peptides . . . Protein Design . References . . . . .

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Weakly Polar Interactions in Proteins S. K. BURLEYAND G. A. PETSKO

I. 11, 111. IV. V. VI. VII.

Introduction . . . . . . Electrostatic Interactions in Proteins . Weakly Polar Interactions in Proteins . Interactions: A Summary . . . Hydrophobic Interactions in Proteins . Discussion . . . . . . . Conclusion . . . . . . . References . . . . . . . V

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125 126 152 176 177 181 183 186

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CONTENTS

Stability of Protein Structure and Hydrophobic Interaction PETERL. PRIVALOV AND STANLEY J. GILL

I. Introduction . . . . . . . . 11. Calorimetric Studies of Protein Denaturation . 111. Studies of Dissolution of Nonpolar Substances into Water . . . . . , . . . IV. Hydration of Nonpolar Molecules . . . V. Comparison of Results on Protein Denaturation and Hydrocarbon Dissolution in Water . . VI. Mechanism of Stabilization of Compact Protein Structures . . . . . . . . . References . . . . . . . . .

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Abstract of a Review on Chemistry of Peanut Proteins R. BHUSHAN, G. P.

Abstract References AUTHOR INDEX SUBJECT INDEX

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REDDY, AND

K. R. N. REDDY

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PREFACE

We are happy to announce that David S. Eisenberg has agreed to join us as an editor of Advances in Protein Chemisty. He has been concerned with protein chemistry since he worked with one of us on a laboratory thesis during his undergraduate work at Harvard. As a Rhodes scholar at Oxford, working with C. A. Coulson, he broadened his knowledge of fundamental physical chemistry. Returning to Princeton, he wrote, with Walter Kauzmann, an influential book on water and its properties. Since then, in his years at Cal Tech and especially at the University of California at Los Angeles, he has become a leader in many aspects of protein chemistry. As an X-ray crystallographer he has elucidated the structures of two complicated and important enzymes: glutamine synthetase and ribulose-bisphosphatecarboxylase. He has been deeply involved in the study of hydrophobic interactions, and his formulation of the concept of hydrophobic moment and its application to peptides and proteins of diverse functions have been illuminating. He has been involved in the design of new peptides and proteins with specific conformations and functions, a topic of one of the articles in this volume. He has served as the first president of the recently formed Protein Society. Although he had no part in the planning of this volume, he has already, in discussions with us, done extensive planning for possible authors and subjects for future volumes. The first article in this volume, by George R. Martin, Rupert Timpl, and Klaus Kuhn, deals with the biologically important subject of basement membrane proteins. These membranes, which underlie most epithelia and are also involved in the structure of nerve, muscle, and fat cells, are thin sheets with complex structures. They involve such unusual proteins as type IV collagen, the large glycoprotein laminin, and the heparan sulfate proteoglycans, calcium-binding proteins, and others. In addition to dealing with these individual constituents, Martin et al. consider the structure and organization of the basement membrane system as a whole. William F. DeGrado treats the problems of designing new peptides and proteins to extend and deepen our understanding of the molecules found in nature and to determine the structural and conformational basis of biologically significant activities. Sometimes the synthetic peptides surpass, in some specific activity, the natural molecules to which they are related. The importance of such studies for the future of physiology, pharmacology, and medicine is obvious. Important applications vii

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Vlll

PREFACE

to the structure and activity of natural proteins have already been made, and much greater ones are certain to follow in the future. S. K. Burley and G. A. Petsko cover the field of noncovalent interactions of proteins, with particular emphasis on weakly polar interactions. Their presentation of the whole field of electrostatic interactions should be of value to many workers in protein chemistry, but their special concern is with the weaker, but very important, interactions involving aromatic side chains, their orientation relative to one another, to oxygen and sulfur atoms, to amino groups, and to aromatic ligands that may bind to the protein. These interactions, only recently recognized for their influence on protein structure, play an important part in the formation of aromatic clusters in the interior of globular proteins and in other features of structure. The authors provide numerous illustrations of the principles involved, from recently determined structures, of both small molecules and proteins. In the last full review article, Peter L. Privalov and Stanley J. Gill propose a drastic revision of our concepts relating to the role of hydrophobic interactions in protein structure and stability. Recent studies of reversible heat denaturation of proteins and of the interaction of small nonpolar molecules with water, over a wide range of temperature, appear to compel a reconsideration of the role of entropic factors in protein structure and stability. Privalov and Gill conclude that the maintenance of the compact folded state in native proteins is primarily due to hydrogen bonding and to van der Waals interactions between the nonpolar side chains in the protein interior. They infer that water solvation of nonpolar groups actually destabilizes the compact folded state. This destabilizing action increases with decreasing temperature, leading to the phenomenon of cold denaturation. This article is likely to arouse controversy, but the conclusions drawn are based on much recent experimental evidence, which potential critics must assimilate before attempting to draw different conclusions. The final brief report on peanut proteins, a subject treated by J. C. Arthur in Volume 8 of Advances in Protein Chemistry, is an abstract of a more extensive review by R. Bhushan, G. P. Reddy, and K. R. N. Reddy, which appeared in a new Indian journal. Since this journal is apparently not covered at present by any abstracting service, the authors have prepared, at our request, an abstract of the longer review so that workers interested in the subject may become aware of a study that they might otherwise miss. In the future, we may consider including other such abstracts to call attention to a relevant article that might otherwise be “lost.” C. B. ANFINSEN

T. EDSALL FREDERIC M. RICHARDS

JOHN

BASEMENT MEMBRANE PROTEINS: MOLECULAR STRUCTURE AND FUNCTION By GEORGE R.

MARTIN;.’

RUPERT TIMPL,t and KLAUS KUHNt

*Laboratory of Developmental Blology and Anomallea, National Institute of Dental Rerearch, Natlonal Inrtltuter of Health, Betherda, Maryland 20892 tMax-Planck-lnrtltut fur Blochemlo, D-8033 Martlnrrled be1 Miinchen, Federal Republlc of Germany

.............................. ..... ..... A. Type IV Collagen. . . . . . . . . . . . . . . . . . . . . . . . . . . B.Laminin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. NidogenlEntactin. . . . . . . . . . . . . . . . . . . . . . . . . . . D. Heparan Sulfate Proteoglycans. . . . . . . . . . . . . . . . . . . . . E. Calcium-Binding Proteins . . . . . . . . . . . . . . . . . . . . . . . F. Other Basement Membrane Components . . . . . . . . . . . . . . . . IV. Self-Assembly and Interaction between Components. . . . . . . . . . . . . A.Laminin.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Multiple Interactions . . . . . . . . . . . . . . . . . . . . . . . . . V. Biological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structural Functions . . . . . . . . . . . . . . . . . . . . . . . . . B. Cellular Receptors for Collagen IV and Laminin . . . . . . . . . . . . . C. Turnover and Degradation. . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction.

11. Morphology and Ultrastructure . . . . . . . . . . . . . . . . . 111. Components. . . . . . . . . . . . . . . . . . . . . . . . . .

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3 6 7 21 26 28 32 33 36 38 38 40 40 41 42 43

I. INTRODUCTION Basement membranes, first described over 100 years ago, are the principal extracellular matrices for most epithelial cells and for peripheral nerve, muscle, and fat cells. Their presence at the basal surface of epithelial tissues gave rise to the term basement membrane. These structures have been the subject of many histological studies, in part because they are very prominent after staining with the periodic acid-Schiff reagent. Such studies suggest that basement membranes are rather homogeneous sheets, perhaps contiguous with the cell surface itself. However, this is incorrect, since basement membranes are very thin extracellular matrices (50-250 nm) whose structural diversity cannot be resolved by light microscopy (Martinez-Hernandez and Amenta, 1983). Chemical studies on most basement membranes are also difficult, since the basement memPortions of this article were prepared at the Max-Planck-Institut while G . R. Martin was the recipient of an Alexander von Humboldt award. I ADVANCES IN PROTEIN CHEMISTRY. Vol. 39

Copyright 0 I988 hy Academic Press. Inc. All rights of reproduction in any form reserved.

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GEORGE R. MARTIN ET AL.

brane is not readily separated from adjacent connective tissue or from cellular elements. Careful dissection or physical separations involving sieving, sonication, and flotation are often utilized to obtain small amounts of purified material from normal tissues (Krakower and Greenspon, 1978). Analyses of material isolated in this way demonstrated the presence of amino acids such as hydroxyproline and hydroxylysine, which are relatively specific markers for collagen (Kefalides, 1973). However, ultrastructural studies did not show the presence of typical collagen fibers. Recent studies have shown that all basement membranes contain a unique collagen arranged in an unusual network structure as well as certain glycoproteins and proteoglycans that occur only in basement membranes (Laurie et al., 1983). The cooccurrence of these proteins in basement membranes is due both to their synthesis by the cells that lie along the basement membrane and to strong and specific interactions that maintain the components together in situ. Also, the cells on basement membranes have specific receptors for these components (Yamada, 1983; von der Mark and Kiihl, 1985; Kleinman et al., 1985; Martin and Timpl, 1987). Basement membrane is the first extracellular matrix produced during development, and it is required for the initial events in morphogenesis (Leivo, 1983). Various components of basement membranes have been found to influence cellular adhesion, migration, growth, and differentiation. Basement membranes also provide a physical support for a wide variety of cells, create barriers that maintain tissue structure, and form a molecular filter that prevents the passage of proteins (Farquhar, 1981) in kidney glomeruli, in capillaries, and in other sites. The structure and function of basement membranes are altered in certain diseases. Grossly thickened basement membranes are observed in diabetic individuals, and these thickened basement membranes are believed to induce the degeneration of ocular, vascular, and renal tissues in long-term diabetes (Brownlee and Cerami, 1981). Further, the ability of tumor cells to penetrate basement membranes signals the transformation of such cells from benign to malignant status (Liotta et al., 1986). Such observations have made this a major research front-more than 5000 publications on basement membranes were retrieved by a computer search of the literature from 1979 to 1986. In recent years, many excellent reviews and books have been published on basement membranes (Kefalides, 1973, 1978; Kefalides et al., 1979; Heathcote and Grant, 1981; Kuhn et al., 1982; Timpl and Martin, 1982; Martinez-Hernandez and Amenta, 1983; Porter and Whelan, 1984; Shibata, 1985; Timpl and Dziadek, 1986). Our review will not attempt to be inclusive but will deal with a description of basement membrane compo-

BASEMENT MEMBRANE PROTEINS

3

nents, their structures, and the interactions that relate to the formation and maintenance of this matrix. 11.

MORPHOLOGY AND ULTRASTRUCTURE

Basement membranes show characteristic arrangements in tissues. They lie at the base of various epithelial tissues, such as the epidermis and glands, and of endothelial tissues, thereby separating the cells of these tissues from the adjacent stromal connective tissue. This arrangement creates a distinct polarity, with nutrient exchange occurring at the basal surface of the cells and secretion of cellular products occurring at their apical surface. Other cells, including Schwann, cardiac, striated and smooth muscle, and fat cells, are completely encircled by a basement membrane. The basement membrane in such sites can show extensive convolutions, as at neuromuscular junctions where multiple interdigitations occur in the muscle. In the glomerulus, lung, and some other sites, the basement membranes adjacent to endothelial and epithelial cells are fused. A scanning electron micrograph of a section of human skin is shown in Fig. 1A. In this case, the papillary dermis with attached basement membrane has separated from the epidermis. The basement membrane is seen as a smooth continuous sheet. A transmission electron micrograph of a section cut through a part of the eridermis and dermis is shown in Fig. 1B and C. In general, three distinct zones2 have been delineated in such basement membranes: an electron-lucent zone next to the epithelial cells (the lamina lucida), an electron-dense zone (the lamina densa), and the reticular layer (the lamina fibroreticularis),which is an incompletely delineated zone continuous with and also containing connective tissue fibers. In a few basement membranes such as Reichert's membrane (a fetal basement membrane) and lens capsule, there may be little or no lamina lucida. In tissues where epithelial and endothelial cells are directly apposed, as in glomerular and alveolar basement membrane, the two fuse to form a single, thickened basement membrane. There is little to note in the way of structures in the lamina lucida with the exception of strands of material that extend from the lamina densa to the cell surface and appear to be extensions of the lamina densa (Laurie et al., 1984). These and other data (Goldberg and Escaig-Haye, 1986) indicate that the lamina lucida could be a fixation artifact. 2 Diverse terms have been used to describe the zones in basement membranes, including basal lamina and lamina rara. The terminology used here follows the recommendation of the International Anatomical Nomenclature Committee (Laurie and Leblond, 1985).

4

GEORGE R. MARTIN ET AL.

FIG.I . (A) Scanning electron micrograph of human skin. The epidermis has pulled away from part of the basement membrane. (B and C)Transmission electron micrograph through the epidermal-dermal junction of human skin. Keratinocytes (KF) are the cells in the human epidermis. LD, The lamina densa of the basement membrane; LL, the lamina lucida. Typical anchoring fibrils (AF) formed from type VII collagen are shown at higher power in c. Courtesy of Dr. K. Holbrook, University of Washington.

The lamina densa is a well-delineated, highly stained zone whose presence is the sine qua non of a basement membrane. Many investigators use the term basement membrane to refer to this sheet-like structure, particularly where the presence of unique basement membrane components, such as collagen IV and laminin, can be established with specific antibodies. At higher magnification, the lamina densa can be seen to be a threedimensional network of 3- to 8-nm cords (Fig. 2). In addition, in such preparations one can usually see tubule-like structures (basotubules) as well as small parallel rods known as double pegs (Inoue et al., 1983; Laurie el al., 1984). The reticular layer of basement membrane in a tissue such as the skin contains a variety of matrix structures. Strands of basement membrane may project down to type I collagen fibers. Anchoring fibers, banded fibers composed of type VII collagen, extend into the basement mem-

BASEMENT MEMBRANE PROTEINS

FIG.IB and C.

5

6

GEORGE R. MARTIN ET AL.

FIG.2. A high-power view of the basement membrane zone. The lamina densa (D)is composed of poorly delineated cord structures that vary from 3 to 8 nm in diameter. Strands from the lamina densa (arrow) are seen to cross the lamine lucida (L) to the surface of the epidermal cells (Ep). Careful examination of the lamina densa has shown the presence of 8-nm hollow tubes (see circled structure in the lower right quadrant): the basotubules. These are formed of pentagonal units stacked one over the other and are believed to contain amyloid P. The insets at the lower left of the figure show double peg structures (arrows) that occur throughout basement membranes. Courtesy of Dr. S.Inoue and Dr. C. P. Leblond, McGill University.

brane and help to fix it in place (Burgeson et al., 1985; Sakai et al., 1986). Fibers of various other collagens may also be in close association with the lamina densa. The absence of certain proteins from these zones or the deposition of antibodies against components of these regions occur in certain disorders and induce blistering due to a loss of firm connections (Pate1 et al., 1983; Goldsmith and Briggaman, 1983; Fine et al., 1984). 111. COMPONENTS

Those components common to all basement membrane and believed to be integral to it rather than adventitiously associated with it are listed in

7

BASEMENT MEMBRANE PROTEINS

TABLE I Intrinsic Basement Membrane Components Component

Chain structure

Major function

Collagen IV Larninin Heparan sulfate proteoglycans

Cul(IV), a2(IV) BI, 8 2 , A Low density (M,, 500-650K); 3-4 heparan sulfate chains 130-250K); High density (M,, 4 heparan sulfate chains 15OK) Single chain (M,, Single chain (M,, 67K) Single chain (M,, 35K)

Structural support Cell attachment Filtration

Nidogedentactin Laminin receptor BM-40/osteonectin/SPARC

Filtration Larninin binding Laminin binding Calcium binding

Table I. They include collagen IV, laminin, heparan sulfate proteoglycan, and nidogen (entactin). Electron micrographs of collagen IV, laminin, and nidogen are shown in Fig. 3. Other components are known to be present in certain basement membranes, and they are presumed to be unique contributions from the resident cells. These include acetylcholinesterase in neuromuscular junctions and the bullous pemphigoid antigen in the epidermal basement membrane. Other constituents, such as a small calcium-binding glycoprotein (osteonectin/BM-40/SPARC), are apparently not restricted to basement membranes but are also found in both mineralizing and nonmineralizing extracellular matrices. Still other components may arise in distant sites, be transported through the blood, and lodge in the basement membrane due to specific binding or to mechanical trapping. These latter substances may include fibronectin, Clq bound to antigenantibody complexes, and albumin. A . Type IV Collagen Analyses of highly purified preparations of basement membrane showed the presence of amino acids, particularly hydroxyproline and hydroxylysine, that are characteristic of collagens (Kefalides, 1973), and the collagen content of various basement membranes has been estimated as 50% or higher on the basis of amino acid analyses. Attempts were made to extract collagen from basement membrane, but it proved difficult to isolate and to characterize due to a low solubility in ordinary solvents and the very limited amounts of basement membrane available. A major advance in this work was the solubilization of a collagenous protein from anterior lens capsule by digestion with pepsin, a procedure that produced a protein with d i k e chains somewhat larger than the chains of interstitial collagens (Kefalides, 1973; Dehm and Kefalides, 1978). Somewhat earlier it had been discovered that there were genetically distinct collagens, often

8

GEORGE R. MARTIN ET AL.

FIG.3. Electron micrographs of type IV collagen (a), laminin (b), and nidogen (c) molecules after rotary shadowing. Bar in a-c, 50 nm.

with a preferential distribution among the tissues (see Table I1 for an updated list). Since the collagen from basement membrane had a distinctive cyanogen bromide peptide pattern, Kefalides (1972a, 1973)concluded that basement membrane contained a distinctive collagen, which he designated type IV collagen, since three other collagens had been described and enumerated. He also proposed that the molecule was triple helical, since it resisted protease digestion and was composed of three identical chains. This model assumed that pepsin had cleaved some nonhelical

BASEMENT MEMBRANE PROTEINS

9

TABLEI1 Types of Collagenous Proteins Type

Helix (nm)

Tissue form

Characteristics, special function, and distribution

I I1 I11

300 300 300

Large, 67-nm banded fibers Abundant; structural; skin, bone, tendon, etc. Small, 67-nm banded fibers Abundant; structural; cartilage Small, 67-nm banded fibers Abundant; reticulin-like; blood vessels, internal organs

IV V VI

390 300 I05

Nonfibrillar network Small fibers Microfibrils, zebra bodies, 105-nm banded tactoids

Basement membrane specific; structural Pericellular; in most interstitial tissues Links constituents of interstitial tissues

VII VIII

450

-

Short, banded straps Unknown

Anchoring fibers for basement membranes Some endothelial cells

IX

200

Unknown

X

I50 300

Unknown Unknown

Throughout cartilage; contains chondroitin sulfate Hypertrophic and mineralizing cartilage Cartilage

XI

regions of the protein and left the main triple helix intact. However, these results, i.e., the isolation of a collagen with three identical chains, were not readily reproduced and were controversial, since multiple collagenous components could be isolated from glomerular basement membrane using essentially identical conditions (Daniels and Chu, 1975). In fact, it was found later that the structure of the basement membrane collagen was quite different from that of the fiber-forming collagens (Table 11). In retrospect, much of the problem in isolating basement membrane collagen was due to the methods applied. The mechanical sieving and sonication used in isolating basement membranes, as well as the pepsin used to solubilize basement membrane collagen, caused extensive degradation of the protein and created artifactual heterogeneity (Heathcote and Grant, 1981; Timpl and Martin, 1982). Model systems, including tumors and cultured cells, proved to be more suitable sources of intact basement membrane proteins. A transplantable mouse tumor, the EHS tumor,3was found to produce a matrix composed solely of basement membrane components that were readily extracted and served as an abundant source of these proteins (Orkin et al., 1977). By light microscopy, the matrix of the 3 This tumor was discovered by Professor Engelbreth-Holm in Denmark and extensively studied by Dr. Richard Swarm at the National Cancer Institute in Bethesda, Maryland. The EHS prefix is derived from the initials of these two investigators.

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GEORGE R. MARTIN ET AL.

FIG.4. Transmission electron micrograph through a portion of the EHS tumor. A cell is seen at the bottom of the figure. At a distance from the cell (d), the basement membrane is seen to take on a laminated appearance (arrows), which is not apparent in the matrix proximal (p) to the cell. Courtesy of Dr. S. lnoue and Dr. C. P. Leblond, McGin University.

EHS tumor appears quite homogeneous, although areas with a repetitive lamellar structure are readily discerned by electron microscopy (Fig. 4). Comparable rat tumors have been identified (Martinez-Hernandezet al., 1982; Engvall et al., 1983). Other tumors, derived from parietal yolk sac (PYS cells), had been found even earlier to produce basement membrane, but these tumors grow only in less common strains of mice, are toxic for the host, and produce less tissue when transplanted (Pierce et al., 1982). However, the parietal yolk sac cells grow well in culture and continue to produce collagen IV, laminin, and other basement membrane components and are frequently used for biosynthetic studies on basement membrane (Oohira et al., 1982; Oberbaumer et al., 1982; Leivo et al., 1982). Certain teratocarcinoma cell lines, such as the F9 line of mouse tumor cells, are also widely used, since retinoic acid plus cyclic AMP induces their differentiation to parietal yolk sac cells with a concomitant transcription of the genes for basement membrane proteins (Strickland et al., 1980; Howe and Solter, 1980; Prehm et al., 1982; Carlin et al., 1983; Durkin et al., 1986). The fact that the molecules produced in such model systems are compara-

11

BASEMENT MEMBRANE PROTEINS

ble to those occurring in normal tissues is usually inferred from the reaction of antibodies prepared to the tumor proteins with tissue basement membranes and not by more direct methods. 1 . Molecular Structure The collagen IV molecule contains two distinct chains, aI(IV) and a2(IV), which form heterotrimers with the composition [aI(IV)]2a2(IV). The conclusion that most collagen IV molecules contain two distinct types of chains and are heteropolymers is based on the ability of monoclonal antibodies to react with native molecules containing both chains (Odermatt et al., 1984; Mayne et al., 1984), on the finding that the two chains comigrate when the native protein is chromatographed on ionexchange columns (Mayne and Zettergren, 1980; Trueb et al., 1982), and by the identification of a trimeric, cyanogen-bromide peptide from a region approximately 70 nm from the 7 S domain in which fragments of two al(1V) and one a2(IV) chain are cross-linked by disulfide bonds (Dieringer et al., 1985). However, some molecules may also exist as homopolymers (Gehron-Robey and Martin, 1981; Timpl et al., 1979a; Haralson et al., 1985). Collagen IV molecules have been visualized by electron microscopy after spreading on cleaved mica and rotary shadowing (Oberbaumer et al., 1982; Bachinger et al., 1982). A typical molecule appears as a long strand terminating in a distinct globule (Fig. 3a). Several major domains (Fig. 5 ) have been defined in the molecule. These include NC1, a terminal signal pept ide

I S I30nml 1 :

1H 1360nml

NCl

I 1

I

FIG. 5 . Schematic representation of the collagen IV molecule, which consists of two al(IV) chains and one a2(IV) chain. The non-triple-helical interruptions of the triple helix are indicated by black bars. The cysteine residues (C) and lysine or hydroxylysine (K) residues putatively involved in intra- or intermolecular bonds are shown. CHO designates a N-glycosidically bound oligosaccharide chain. The subscript numerals indicate the number of residues in a distinct region, summarized for all three a-chains. P designates a main pepsin cleavage site. In interruption 13, the ar2(IV) chain forms a 21-residue-long loop, stabilized by an interchain disulfide bridge. NCI, Noncollagenous domain I ; TH, triple-helical domain; 7 S, carboxyl-terminal domain.

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GEORGE R. MARTIN ET AL.

globule at the carboxyl end. The main triple-helical domain extends from the NC1 globule to about 30 nm from the N-terminal portion of the molecule. The amino-terminal domain that participates in intramolecular cross-linking is termed the 7 S domain (Kuhn et al., 1981; Timpl et al., 1981; Mayne et al., 1982).Additionally, a second noncollagenous domain (NC2) has been inferred to occur some 90 nm from the amino terminus of the molecule (Timpl et al., 1981), although it may actually represent a region with unusual resistance to'collagenase. Current information indicates that three chains extend in register the entire length of the molecule and form the helical domains (390 nm versus 300 nm in the interstitial collagens). A similar alignment of chains is observed in interstitial procollagens (types I-111), but these have noncollagenous domains that occur at both ends of the molecule and are removed enzymatically prior to deposition of these collagens into fibers. No such processing is observed with collagen IV molecules (reviewed in Timpl and Dziadek, 1986). In addition, collagen IV molecules are considerably more flexible than collagen I, 11, and I11 (Hofmann et al., 1984). Some sequence studies, discussed below, indicate that the greater flexibility of the molecules and their susceptibility to proteases is due to the presence of many nonhelical sequences within the major helical domain (Fig. 5). The helices of interstitial collagens have repetitive units that are about 234 residues long and that regulate the lateral alignment of molecules and allow the molecules to assemble into ordered fibrils. Such repetitive units do not occur in collagen IV, a situation that may account in part for their inability to form such fibrils, although current models for the structure of collagen IV molecules in the matrix suggest that some lateral associations occur (Yurchenco and Furthmayr, 1984). The complete amino acid sequence of the human al(1V) and (w2(IV) chains has been obtained (Babel and Glanville, 1984; Pihlajaniemi et al., 1985; Brinker et al., 1985; Glanville et al., 1985; Braze1 et al., 1987; Siebold et al., 1987; K. Kuhn, unpublished). Including a signal peptide, the al(1V) chain comprises 1669 amino acids and the a2(IV) chain has about 1712 amino acids. The carboxyl ends of both chains are formed by the globular NC1 domains, which occupy 229 amino acid residues that are homologous to one another but not, for example, to the carboxyl-terminal propeptides of collagens 1-111. Each individual NC1 domain consists of two repeating units (Schwarz-Magdolen et al., 1986). Each repeat is stabilized by three disulfide bridges, which force the peptide chain into two large and one small loop. Figure 6 indicates how the two repeats of a NC1 domain can be schematically arranged in a rather symmetrical four-leaf clover-like structure. These structures have been conserved during evolution since 60% sequence identity is observed between the NCl domain of Drosophila, mouse, and human (Blumberg et al., 1987). Interestingly, the

BASEMENT MEMBRANE PROTEINS

13

FIG.6 . Loop structures in the NCI domain of collagen IV chains. The beginning portion of the triple-helical domain is shown as a jagged line labeled N. The NCI domain contains two repeating structures, each comprising a large loop, followed by a small loop, and then another large loop. The location of the intrachain disulfide bonds has been deduced (Sieboldt et a / . , unpublished).

subdomains of NCI do not coincide with the exon structure of human aI(1V) chain gene (Soininen et al., 1986b), a finding indicating that this homology is not the result of simple gene duplications. The major helical domain is about 360 nm long. The al(1V) chain contains 21 and the a2(IV) chain 23 nontripeptide interruptions (Fig. 5 ) . These include deletions of a single glycine residue from the Gly-X-Y triplet as well as stretches of as many as 13 residues lacking the Gly-X-Y repeat. Not all of the nontripeptide interruptions in the two chains are aligned, thus creating 26 separate interruptions in the triple helix of the molecule (Schwarz et al., 1986; K. Kiihn, unpublished). These non-triplehelical segments can be divided into flexible sites, bends, and neutral substitutions (Table 111). Such sequences could serve in the attachment of other proteins to the collagen IV network and also serve as sites of cleavage by various proteases. The amino-terminal region of the collagen IV molecule was isolated from proteolytic digests of basement membrane-rich tissue in the form of a cross-linked tetrameric complex, the so-called 7 S collagen. The aI(1V)

14

GEORGE R. MARTIN ET AL.

and a2(IV)chains in this 7 S domain both consist of a short (30 nm) helical region flanked on each side by nonhelical regions (Fig. 7A). The short nonhelical N-terminal segment on each chain contains cysteines and hy-

TABLEI11 Some Nontripeptide Sections of al(1V) and a2(IV)

Flexible sites 1 al a2 a1 2 a2 3 al a2 al 4 a2 Bends I al

G G G G G G G G

K i b S M D H V D M G S M K G Q K P 6 - P L I L P G M K D I K G E K W A E L f i G I U f D T V D L - P G S P T 6 8 H P - V E G P i T S P P S N G G S P P b E P Y D V I K G E P I P Q K I A V Q P G T L

G Q P ! G S ) K

a2

G F I K G V K 2 al G E V Y G F h G a2 G E A N T L P G Neutral substitutions 1 al G L P b G F t a2 G V D U G D P al G L i 8 I P G V 2 a2 G P N A L P G I 3 al G F D 8 A P G Q a2 G V S A V P G F

L A P V

+

K K K R

The nontriplet regions of the al(IV) and the a2(IV) chains are underline!. P denotes 4-hydroxyproline; K denotes fully hydroxylated and glycosylated lysine residues; (-) denotes deletions which had to be introduced to ensure optimal matching of the tripeptide structure of both chains. The non-triple-helical sequences are underlined. Three different types of nontriple-helical interruptions have been observed: ( I ) longer nonhelical sequences, expected to create flexible sites sensitive to proteases; (2) an extra amino acid in one of the two chains expected to create a bend; (3) neutral substitutions expected to disturb only slightly the structure of the triple helix.

15

BASEMENT MEMBRANE PROTEINS

droxylysines that are used to generate intra- and intermolecular crosslinks. Adjacent to this nonhelical sequence is a helical segment of 114 residues. Both of these segments form the overlapping, antiparallel strucA

Helical X-linking site

Cop site

CHO

+

+

+

+

a1

GPrGPTGPTGPr

a2

GRPGPPGPPGPP

a2 K F D V P C G GR D C S G G COC Y P E k G G R NonhelicolX-linking site

Nonhelicol bend

B I

7s I

Overlap Zone

FIG.7. (A) A detailed model of the amino-terminal aI(IV) and a2(IV) chains containing the 7 S domain and an adjacent segment of the main triple helix. TheJerminal sequence of both chains is nonhelical and contains lysines (K), hydroxylysines (K),and cysteines (C) that participate in intra- and intermolecular cross-linking. A helical cross-linking site is located about 30 nm from the amino terminus (N) of the molecule and contains cysteines and a hydroxylysine in the al(IV) chain involved in cross-linking plus a complex carbohydrate attachment site (CHO).The “cap” site within the p i n triple helix identifies a series of four triplets containing proline (P) and hydroxyproline (P), a composition that would be expected to form a very stable helical structure. [Data from Glanville et al. (1985); Siebold et al. (1987)]. (9) The antiparallel arrangement of type IV molecules with alignment of crosslinking sites.

16

GEORGE R. MARTIN ET AL.

tures and contain cross-linking sites where cysteine and hydroxylysines residues form intramolecular crosslinks (Fig. 7B). Intermolecular bonds are likely to occur using both the hydroxylysine and cysteine residues of the N-terminal nonhelical regions and those in the triple-helical cross-link site of two 28-nm overlapping antiparallel molecules. The triple-helical domains of the al(1V) and the a2(IV) chains are terminated by nonhelical regions, respectively 13 and 12 residues long, which define the border of the 7 S domain and provide the molecule with a flexible joint. This short segment allows the triple-helical molecules to form a bend that projects the major triple helix away from the 7 S domain, thus facilitating the formation of the network structure in the basement membrane. The adjacent triple-helical domains of both chains are characterized by a high content of proline and hydroxyproline residues. These proline- and hydroxyproline-richsequences form very stable triple helices and may stabilize the molecule in those regions that would otherwise be destabilized by the presence of neighboring nonhelical domains. Sequence analyses of the 7 S domain have been done (Glanville et al., 1985; Siebold et al., 1987), and a model based on hydrophobic interactions has been constructed. This model predicts close apposition of cross-linking sites and stronger binding between antiparallel segments than between parallel segments. In addition, a heterotrimer chain composition is favored over homotrimer assemblies (Siebold et al., 1987). These predictions are in excellent agreement with the experimental data and also suggest the role for the a2(IV) chain in the molecule. 2 . Supramolecular Organization a. 7 S Domain of Type ZV Collagen. Various models have been advanced to explain the organization of collagen IV molecules in basement membranes. On the basis of the absence of fibers in the basement membrane, such models proposed that the collagen was arranged between sheets of noncollagenous proteins or as a random mesh of molecules linked end to end through noncollagenous domains (Kefalides, 1973). A much more defined model has been obtained via the examination of the collagen IV molecule and of fragments derived from enzyme digests of basement membrane (Timpl et al., 1981). In these latter studies, most of the collagenous protein of the basement membrane was brought into solution after incubation with either pepsin or trypsin. A subsequent exposure to bacterial collagenase at 20°C was expected to destroy all collagenous protein but instead left behind a large component with a typical collagenous composition (Timpl et al., 1979b). This component formed a rather homogeneous boundary in the ultracentrifuge and had a sedimentation constant of 7 S. Material prepared in this manner is usually referred to as

BASEMENT MEMBRANE PROTEINS

17

7 S collagen and can be isolated from a variety of basement membranes (Risteli et al., 1980). Circular dichroism measurements showed that 7 S collagen contained triple-helical domains that showed thermal transitions at 48 and 70°C and were stabilized by disulfide bonds. Reduction of disulfide bonds lowered this thermal stability to 45°C and conferred collagenase sensitivity. A smaller form of 7 S collagen was also obtained utilizing collagenase digestion at 37°C (Risteli et al., 1980; Madri et al., 1983; Mayne et al., 1984). The nature and significance of the long and short forms of 7 S collagen became clear from examination of aggregates of collagen IV brought into solution by a brief digestion with pepsin (Fig. 8). Electron micrographs of

FIG.8. The network structure of type IV collagen as deduced from electron microscopy of fragments solubilized with proteases (Tirnpl et 01.. 1981). The top left micrograph shows the short form of the 7 S region. In the top right, a mere limited enzyme digestion allows a portion of the helical domain of each molecule to remain linked to the 7 S domain. The bottom left micrograph shows isolated dimeric NCI fragments and, at the lower right, dimeric triple helical material connected in the center by the NCI domain.

18

GEORGE R. MARTIN ET AL.

such digests revealed spider-like structures containing up to four thin, 370-nm long strands extending from a central rod-like domain (Kiihn er al., 1981; Timpl et al., 1981). The short form of 7 S collagen lacked these strands and consisted of the central rod structure. On the basis of the structure of the collagen IV molecule, it was concluded that the 7 S domain represented a site where four molecules overlapped by 28 nm, with two molecules in parallel and two molecules in an antiparallel orientation (Fig. 7B). The long form of 7 S collagen was an intermediate fragment consisting of the rod plus four short (30 nm) triple-helical extensions (Fig. 8). b. NCI Domain. Identification of the NCl domain, the C-terminal globule, as the other major cross-linking site of collagen IV was achieved in a similar manner. Electron microscopy of collagen IV solubilized by reduction or with acid from the EHS tumor matrix showed many dimeric structures, usually lacking the 7 S region but joined together through a shared globule (Timpl et al., 1981; Yurchenco and Furthmayr, 1984). Monomeric material terminated in a single, somewhat smaller globule (Fig. 3A). The NC1 complex can be isolated from a collagenase digest of various tissues and is predominantly a hexameric structure arising from the condensation of the NCl domains of six a chains of two collagen IV molecules (Weber et al., 1984). As discussed above, the NCI domains of the al(1V) and a2(IV) chains are highly homologous structures, particularly in the location and the size of disulfide-linked loops (Fig. 6). The loop structure is duplicated to create two highly homologous regions in each chain. The arrangement of these domains within the globule is not known, although crystals prepared from such material show in X-ray diffraction a high degree of symmetry (Timpl et al., 1985). The dimeric structure formed by the condensation of two NC1 domains is stabilized by disulfide bonds and by nonreducible bonds. c . Collagen ZV Network. Based on the observations discussed earlier, it was suggested that collagen IV molecules were arranged in a network with like ends of molecules in apposition (Timpl et al., 1981) (Fig. 8). This arrangement generates a very open structure that would also exhibit considerable tensile strength due to the presence of cross-links. In its most extended form, the distance from NC1 to the end of the 7 S domain would be about 390 nm and the distance from globule to globule about 780 nm. The network model describes how molecules are arranged in two dimensions, but it does not define how they are arranged in three dimensions. In its most open form, the molecules are in contact only at their ends. I n uitro experiments, however, provided evidence that the collagen IV molecules are also able to interact laterally with partial overlapping of their triple-helical domains (Yurchenco and Furthmayr, 1984).

BASEMENT MEMBRANE PROTEINS

19

When collagen IV prepared from the EHS tumor is incubated at 37°C it self-assembles, forming aggregates containing polygonal structures. These lateral associations are stabilized by the interaction between the domain NC1 and sites that occur along the triple helix and are separated from one another by about 100 nm (Tsilibary and Charonis, 1986). The additional possibility of lateral aggregation of the molecules would lead to a much more complex three-dimensional structure. Examination of type IV collagen produced by cells in tissue culture demonstrated in the cell layer the expected 7 S tetrameric and NCl dimeric structures discussed earlier. In contrast, the culture medium contained, in addition to monomeric collagen IV molecules, dimers and tetramers linked via their 7 S domains but no globule-linked dimers (Bachinger et af., 1982; Oberbaumer et al., 1982). It was also found that collagen IV molecules undergo a ready association via their 7 S domains in a concentration-dependent process (Yurchenco and Furthmayr, 1985). Upon longer incubation, the tetramers formed are stabilized by intramolecular disulfide bonds (Duncan et al., 1983). The formation of NCl-linked dimers and their stabilization by disulfide bonding is apparently a slower process, at least in vitro (Blumberg et al., 1986). The network structure has been observed by electron microscopy in Reichert’s membrane following treatment of tissue sections with plasmin (Inoue et al., 1983). This enzyme removes a major portion of the 3- to 8-nm-thick cords that constitute the major element in the basement membrane, leaving behind a network of fine filaments 1.5 to 2 nm in diameter. Presumably, the cords have a framework consisting of one or more collagen IV filaments that are coated with laminin and with other basement membrane components. A better understanding of the structure of collagen IV molecules explains some previous observations. The occurrence of the 7 S domain and nonhelical interruptions in the molecule can explain in part the heterogeneous mixture of collagenous components isolated from basement membrane, particularly when pepsin or other proteases are used to solubilize the protein. The molecules isolated from lathyritic basement membrane by acid extraction are truncated and lack the 7 S domain presumably due to cleavage by endogenous proteases. 3. Gene Structure and Evolution of Collagen ZV Genes Considerable progress has been made in studying the structure of collagen genes. The genes for the chains of collagens I, 11, 111, and IX have been isolated. The DNA coding for the major helical domains of collagens type I, 11, and I11 are divided into many exons of 54 bases each or of multiples of this number (Boedtker et al., 1983). Indeed, it has been

20

GEORGE R. MARTIN ET AL. TABLE1V Size of Helical Exons in the a2(1),al(lV), and a2(IV) Chain Genes al(1V)

a2(I)

Exon number

Exon size

Exon number

a2(IV)

Exon size

Exon number

Exon size

16b

64 I82 123

~~~

4 5

247(54)"

6

54 108 54 108 54 54

7 8 9 10 14

108

4 5 6 7 8 9 10 14

23(71)" 99 129 12 73 134 178 81

17 18

a The numbers in parentheses represent the number of bases in the helical portion of the fusion exon. * The location of these exons has not been determined, but they correspond to sequences from the center of the helical domain and are 5' to exon 14 in the al(1V) gene.

postulated that there was a primordial collagen gene of 54 bases coding for 18 amino acids consisting of six Gly-X-Y triplets and that the structure of this primordial gene has been duplicated and maintained (Yamada et al., 1980). This pattern, however, has been found only in the main triplehelical domains of the fiber-forming collagens. The gene for the al(1X) chain, for example, is smaller, and the portions coding for the helical domains are not divided into regularly sized exons (Lozano et al., 1985). The collagen IV gene also deviates from the pattern observed with the genes for the fiber-formingcollagens. The size of three helical exons in the a2(IV) chain (Kurkinen et al., 1985) and several helical exons in the al(1V) chain (Sakurai et al., 1986; Soininen et al., 1986a,b) have been determined by sequencing. None of these correspond to the 54-base pair pattern, and each has a unique size (see Table IV). Further, the initial and final codons of these exons are often split, in contrast to the genes for the chains of collagens 1-111. In addition, examination of the amino acid sequences of the al(1V) and a2(IV) chains show no homology with the chains of collagens 1-111 beyond that of the Gly-X-Y triplet characteristic of all collagens. Taken together the data show that there is little relation between collagen IV and other collagen types and that collagen IV genes evolved independently. Given that collagen IV occurs first during the initial development of the embryo (Leivo et al., 1980) and is present in invertebrates (i.e., hydra and sea urchins), it may have evolved prior to the interstitial collagens. The genes for cyl(IV) and a2(IV) chains have

BASEMENT MEMBRANE PROTEINS

21

been localized to a narrow segment of human chromosome 13 (Griffin et al., 1987), their locations indicating a close linkage. B. Laminin The strong reaction obtained in basement membranes with the periodic acid-Schiff stain is due to reaction with carbohydrate present in glycoproteins (Leblond et al., 1957). Subsequently, glycoproteins including some larger than M , 200K were solubilized with 8 M urea from lens capsule (Kefalides, 1972b). The probable importance of these glycoproteins to the structure of basement membranes was fully appreciated and most early structural models suggested that glycoproteins were part of the basement membrane structure. PYS cells were found to secrete material that was highly antigenic, and antibodies raised against these antigens reacted with authentic basement membranes. Johnson and Warfel(l976) used molecular-sieve chromatography to isolate a large noncollagenous protein(s) secreted by cultured PYS cells. They also found it in kidney extracts and showed that antibody to this material reacted with tissue basement membranes. Similarly, Hogan et al. (1980) as well as Howe and Solter (1980) showed that both cells derived from embryos and PYS cells secreted primarily two or three large proteins in culture. Chung et al. (1979) also studied a line of PYS-like cells that deposited an extracellular membrane, which, when freed of cellular elements and dissolved in detergent plus mercaptoethanol, was found to consist of two principal polypeptides called GPI (M, 320K) and GP2 (M, 230K). These glycoproteins were isolated by preparative electrophoresis and shown to be noncollagenous and distinct from fibronectin. Antibodies prepared against GP2 were found to react with basement membranes in kidney and some other organs. The nature of these components became clear after the isolation of laminin, a large glycoprotein ( M , 800-900K) that is a major constituent of basement membranes. Laminin was first isolated in its intact form from the EHS tumor (Timpl et al., 1979~).It is readily solubilized from this source with aqueous solutions (i.e., 0.5 M NaCl, pH 7.4) and can be purified to homogeneity by ion-exchange and molecular-sieve chromatography. In the absence of reducing agents, laminin migrates on electrophoresis as a single sharp band and sediments as a single sharp boundary (11 S) in the ultracentrifuge (Engel et al., 1981). Hydrodynamic studies indicate that laminin has an asymmetric and rather rigid structure. Circular dichroism revealed about 30% a helix and some 20% /3 structure in the molecule. Electrophoresis of laminin from the EHS tumor under reducing conditions resolved two polypeptide bands of similar intensity, the A (M, 400K)

22

GEORGE R. MARTIN ET AL.

and B (M, 220K)chains of laminin (Timpl et al., 1979~).Several lines of evidence indicated that the A and B chains were distinct and not, for example, related as precursor and product. The peptide maps obtained with the chains were different. No interconversion of one to the other was noted in pulse-chase experiments, and antibody to the B chain did not appear to react with the A chain (Chung et al., 1979;Cooper et al., 1981; Kurkinen et al., 1983;Howe and Dietzschold, 1983). Rotary shadowing and subsequent electron microscopy produced striking images of laminin (Engel et al., 1981). These showed a cross-shaped structure with one long (77nm) and three short (37nm) arms. Each short arm had two prominent globules, and the long arm terminated in a single large globule (Fig. 3b). Initial models suggested that three B chains constituted the short arms of laminin and one A chain constituted the long arm and that these chains were joined in a disulfide knot in the center of the cross. This model was supported by the appearance of the molecule, by the ratio of A to B chains in laminin prepared from the EHS tumor, and by the report that thrombin cleaved the long arm (i.e., the A chain) of laminin in preparations examined by electron microscopy, leaving the threeshort-arms structure with B chains intact (Rao et al., 1982). More recently, this model has undergone substantial modification. First, it became apparent from biosynthetic studies on cells in culture that there were two distinct B chains, BI and B2 (Cooper et al., 1981),which were not well resolved in laminin prepared from the EHS tumor. Analysis of the stoichiometry of the chains produced in these systems suggested that the laminin molecule contained one A, one B1, and one B2 chain (Fig. 9). Studies with a monoclonal antibody specific for the A chain indicated that the A chain formed part of the short arm as well as the long arm of laminin (Palm et al., 1985). The assembly of the molecule appears to proceed in discrete steps, with the initial assembly of a Bl-B2 dimer linked by disulfide bonds to which an A chain is added (Morita et al., 1985;Peters et al., 1985).

I . Domains, Fragments, and Sequence Data Detailed analysis of fragments isolated from laminin following cleavage with proteases and amino acid sequence deduced from cDNA clones from both the B1 and B2 chains of laminin have led to a further refinement of the structure. Various portions of the molecule survive proteolysis and have been isolated and characterized (Fig. 10A). Fragment 1 (M,275K) appeared to consist of the central portion of the cross, lacking terminal globules and the long arm. It could be isolated in good yield following pepsin and other protease digestion (Ott et al., 1982).Fragment 1 showed a strong reaction with antibody to laminin, a result indicating that it con-

BASEMENT MEMBRANE PROTEINS

YIGSR

f

23

A CHAIN (Mr = 400,OOO)

Cell Attachment

82 CHAIN (Mr=ZO5,000)

B1 CHAIN iMr

Coiled Coil Neurite Outgrowth

@I

Heparin Binding

FIG.9. Schematic model for laminin showing the 3-chain structure (A, B1, and B2) and the projection of the three chains down the long arm of the molecule. The location of a pentapeptide (YIGSR) with cell attachment activity is indicated, as well as the region with neurite-promoting activity (Baron van Evercooren et a / . , 1982; Edgar er al., 1984).

tains a major portion of the antigenic activity of laminin, is strikingly enriched in cysteine, and retains the ability to react with cellular receptors (Rao et al., 1982; Timpl et al., 1983b; Aumailley et al., 1987). It lacked both a helix and /3 structure. The three-short-arms structure, a3, although less stable than fragment 1, was isolated from laminin treated with thrombin (Rao et al., 1982) or elastase (Ott et al., 1982). The long arm of laminin was particularly sensitive to proteolysis, and its removal was associated with the complete loss of a-helical structures (Ott et al., 1982). Two fragments were isolated from the long arm of laminin after limited cleavage by elastase (fragment 8) or after cleavage with trypsin in conjunction with endogenous protease (fragment 25K). Fragment 8 was localized to the long arm of laminin on the basis of its appearance in electron micrographs, which showed a rather rigid rod terminating in a single globule. Fragment 8 also bound to heparin and reacted with antibody to fragment 3, the globular domain at the end of the long arm. Fragment 8 has a high content of a helix, presumably in the rod (Paulsson et al., 1985a). It contains a high-affinity cell attachment site (Aumailley et al., 1987; Goodman et al., 1987) and is responsible for the stimulation of neurite outgrowth (Edgar et al., 1984; Engvall et al., 1986). Fragment 25K also had a high content of a helix and reacted with antibody to fragment 8 (Paulsson et al., 1985a). It was found to consist of two disulfide-linkedpeptides (Mr 12K),which on the basis of immunologi-

A

FRAGMENT 1 CELL BINDING

FRAGMENT 3 HEPARIN BINDING

0

-

FRAGMENT 8

“25K”

B

a3 CELL COLLAGEN

/ -

Limited Elostose

BINDING

v

Domains

Globiar Domains

m

n

Homologous CvsteineRich Repeats -./

*.*

#.#’

/’

o-Helical Coiled Coils ’.. \. x.

.‘

I

S

COOH

FIG.10. (A) Proteolytic fragments derived from laminin and activities found to be associated with them. (B)A domain model for the B1 chain of mouse laminin deduced from the nucleotide structure of cDNA clones. Domains I and I1 are largely helical and probably form a coiled-coil structure with a similar portion of the B2 chain. There are several possible carbohydrate attachment sites. These domains are separated by a region “a” containing six cysteines closely bunched, possibly involved in cross-linking to the 8 2 and A chains. Domains 111and V are cysteine-rich regions composed of repetitive segments of about 50 amino acids each. These domains may form the two rod-like elements within the short arm, whereas domains IV and VI are thought to form the visible globular structures.

BASEMENT MEMBRANE PROTEINS

25

cal cross-reaction and sequence data were identified as arising from the B1 and B2 chains of laminin. The sequence of each peptides showed a heptad repeat, characteristic of helices in proteins such as tropomyosin, whose chains are arranged in coiled-coil structures. Fragment 3 (M, 50K)possessed p structure, appeared globular in electron micrographs, and was found to bind to heparin. It was assumed to be the globular region at the end of the long arm of laminin. This site is one of the main heparin- and heparan sulfate-binding domains in laminin (Ott et al., 1982). Advances in isolating and identifying cDNA clones for laminin provided considerable additional information on the amino acid sequences at the carboxyl end of the B1 and B2 chains (Barlow et al., 1984). These analyses showed that at least 350 residues of the B1 chain and over 200 residues of the B2 chain had the a-helical heptad repeat and predicted that these chains would be aligned together, along the long arm of laminin in a coiled-coil a-helical structure. Taken together, these and other data indicate that the laminin molecule is composed of one A, one BI, and one B2 chain (Fig. 9). The carboxyl terminus of each B chain extends down the long arm of laminin in a coiled-coil structure while the remainder of each chain forms a short arm. The A chain of laminin could also participate in such a coiled-coil structure, although this has not been shown and would require the alignment of a comparable a-helical domain in the A chain. However, the A chain is presumed to form one of the short arms and to extend through the molecule to form the globule at the end of the long arm of laminin. More recently, the complete amino acid sequence of the B1 and B2 chains of laminin has been deduced from the nucleotide sequence of cDNA clones (Yamada et al., 1985; Sasaki et al., 1987; M. Sasaki and Y. Yamada, unpublished). Computer analysis of possible secondary structures (Fig. IOB) shows a diversity of domains, including two a-helical-rich domains (I and 11), two cysteine-rich regions with regular repeats (domains I11 and V) and two cysteine-poor-possibly the globular-regions (domains IV and VI) (Fig. IOB). These various domains have been correlated recently with distinct laminin fragments by Edman degradation (R. Deutzmann, unpublished), this study demonstrating that domains IV and VI correspond to the inner and outer globular domains within one short arm of laminin, respectively, and that domain I11 is a typical constituent of the cysteine-rich fragment 1 . 2. Isoforms Laminin composed of A, B1, and B2 chains appears to be the major form of laminin secreted by cells that produce and deposit basement

26

GEORGE R. MARTIN ET AL.

membranes. However, other isoforms probably exist. For example, Bla and Blb chain are observed after in uitro translation of B1 chain mRNA (Kurkinen et al., 1983). Since this mRNA was isolated by hybridization to a single cDNA clone for the B1 chain, the Bla and Blb chains must arise from similar mRNA perhaps created by differential splicing of the original transcript. Laminin from placenta contains an additional component, the M chain, whose size is intermediate between the A and B chains of laminin (Ohno et al., 1983). These chains could form heteropolymers with different molecular and biological activities. The chains of laminin are not synthesized synchronously in early mouse embryo cells. Only the B1 chain is synthesized in the oocyte (Cooper and MacQueen, 1983). No laminin chains are produced at the 2-cell stage; and at the 4- and 8-cell stage, both B1 and B2 chains are synthesized. The A, B1, and B2 chains appear together only at the 16-cell stage. Production of the three chains is associated with the extracellular appearance and deposition of basement membranes and the initiation of morphogenesis. Laminin consisting of A and B chains has also been identified in embryonic cells of Drosophilu (Fessler et a / . , 1984b) and sea urchins (McCarthy et ul., 1987). These proteins showed a cross-shaped structure like that of mouse laminin, but with a more extended long arm. Some cells, including cultured Schwann cells, produce only the B chains of laminin (Palm and Furcht, 1983; Cornbrooks et al., 1983; Dziadek et al., 1986a). In addition, some animal tissues appear to contain B chains or B chain mRNA but to lack significant amounts of A chain (Mohan and Spero, 1986; Kleinman et al., 1987). C . NidogenlEntactin In addition to the A and B chains of laminin, SDS-mercaptoethanol extracts of the membranous sacs produced by a line of PYS-like cells contained a novel protein (M,158K) into which 35S04was incorporated (Carlin et al., 1981). This component was isolated by preparative electrophoresis and characterized by amino acid analysis, carbohydrate content, and antigenic reactivity. Antibodies against this sulfated glycoprotein were observed to localize to the basement membrane zones of various tissues. Due to a close association with epithelial and endodermal cell surfaces, the protein was named entactin. Additional studies utilizing immunoelectron microscopy showed that entactin was present in the lamina densa and colocalized with collagen IV, laminin, and heparan sulfate proteoglycan (Laurie et al., 1984). A similar component was independently observed to be produced by parietal endoderm cells and to be incorporated into Reichert’s membrane (Hogan et al., 1982a.b).

BASEMENT MEMBRANE PROTEINS

27

Nidogen was initially isolated from the EHS tumor as a fragment (M, 80K) that appeared by electron microscopy as an 8- to 10-nm globule with a short tail (10-14 nm) (Timpl et al., 1983a). It received the name nidogen on the basis of its ability to aggregate into nest-like structures (Latin: nidus, “nest”). Immunofluorescence studies with antibody to nidogen showed that it occurred in a wide variety of basement membrane. Even larger forms of nidogen were found in tissue extracts by immunological reaction of electrophoresed material. Subsequent work suggested that nidogen was rather susceptible to proteolysis but that the intact form (M, 150K)can be isolated from a variety of basement membranes using concentrated guanidine solutions containing protease inhibitors (Dziadek and Timpl, 1985). Such a component (M,150K)was obtained from Reichert’s membrane, which was sulfated and also reacted with nidogen antibody (Paulsson et al., 1985b). The sulfate was shown to occur in the protein as tyrosine sulfate, a well-known posttranslational modification common to a number of secreted proteins. The intact nidogen molecule is characterized by a single N-terminal amino acid sequence and has the shape of a dumbbell (Fig. 3C), consisting of two globular domains of unique size connected by a 16-nm rod-like segment (Paulsson et al., 1986, 1987a). Nidogen is readily degraded to a series of well-defined fragments (Dziadek ef al., 1985a). Such data suggest that nidogen consists of three to five separate domains that lack substantial portions of (Y helix or p structure. Strong complexes ( K D , 1 nM) were observed between nidogen and laminin, an association allowing the isolation of the complex by molecular-sieve chromatography (Dziadek et al., 1985a; Paulsson et al., 1987a), and the dissociated components are able to reassociate. Chemical and electron microscopical studies demonstrated binding of one nidogen molecule in the complex to the center of the cross (fragment l) of laminin. Specific antibodies to either laminin or nidogen precipitate the other when both are present in solution. In several basement membranes, laminin and nidogen occur in nearly equimolar amounts. However, nidogen appears rather later than laminin in embryonic development (Dziadek and Timpl, 1985). Certain observations suggest that entactin and nidogen are either identical or closely related proteins. These include their similarity in size, their strong affinity for laminin, and the cross reaction of antibodies to the intact form of each (Carlin et al., 1983; Hogan ef al., 1980; Palm and Furcht, 1983; Paulsson et al., 1985b). More affirmative proof of the relationship between both proteins will depend on direct sequence comparisons. The functions of nidogedentactin are not known, although complex

28

GEORGE R. MARTIN ET AL.

formation with laminin may modulate some of the functions of laminin, such as its binding to cells and collagen IV (Dziadek et al., 1985a;Aumailley et al., 1987).

D . Heparan Sulfate Proteoglycans Current concepts suggest that every extracellular matrix contains one or more proteoglycans in addition to collagens and glycoproteins. Proteoglycans are often classified on the bases of the glycosaminoglycan chains that they bear and their tissue source. However, it is their core proteins that are the unique gene products (Hassell et al., 1986). It is now firmly established that all basement membranes contain a distinctive class of heparan sulfate proteoglycans. The first indication of the presence of proteoglycans in basement membranes came from studies using cationic probes, such as cationized ferritin or ruthenium red, which react specifically at low pH with the sulfate groups in glycosaminoglycans and reveal regular deposits along the basement membrane (Kanwar and Farquhar, 1979a; Gordon and Bernfield, 1980). Also, radioactive sulfate was found to be incorporated into macromolecules that were deposited in basement membrane (Hay and Meier, 1974). Subsequently, physiological and ultrastructural studies on the clearance of macromolecules through the kidney demonstrated that the basement membrane in the glomerulus acts as a selective filter, preventing the passage of macromolecules (Karnovsky, 1979; Farquhar, 1981). Further, the charge on the macromolecule is important in determining its clearance, since negatively charged macromolecules show a lower passage than those with neutral or positive charges. These results indicated that there was an anionic barrier in the basement membrane that was important in regulating the passage of proteins from blood to the urinary space. The anionic groups observed in basement membrane with cationic stains were identified as heparan sulfate chains on the basis of their sensitivities to heparitinase and to nitrous acid (Kanwar and Farquhar, 1979b) and by chemical analysis (Kanwar and Farquhar, 1979c; Parthasarathy and Spiro, 1982). Current concepts suggest that the heparan sulfate chains are linked through a common oligosaccharide terminating in xylose linked to serine residues on the protein core and that certain xylosides can act as initiators for the synthesis of heparan sulfate. In the absence of added xyloside, the synthesis of heparan sulfate is entirely dependent on the synthesis of a core protein. Heparan sulfate proteoglycans have been isolated from glomerular basement membranes, from the EHS tumor matrix, and from various cells producing basement membranes, including endotheiial cells and epi-

29

BASEMENT MEMBRANE PROTEINS

thelial cells. In general, these studies show a size diversity of heparan sulfate proteoglycans. It should be noted that proteoglycans are not readily resolved from one another by ion-exchange chromatography due to their high negative charge. Rather, resolution is obtained by differential extraction, by density gradient centrifugation, and by molecular-sieve chromatography. In addition, the protein portion of the molecules is rather sensitive to endogenous proteases, and it is necessary to use protease inhibitors to limit artifactual heterogeneity. Proteoglycans prepared from the EHS tumor matrix have been extensively studied and may serve as prototypes for other basement membrane proteoglycans. The EHS tumor contains two different heparan sulfate proteoglycans, which can be distinguished by their buoyant density, size, and solubility (Fujiwara et al., 1984; Hassell et al., 1985). The largest proteoglycan ( M , 650K), a low-density proteoglycan with about 70% of its mass in the form of protein, is firmly associated with other components of the matrix and requires denaturing solvents such as 7 M urea or 6 M guanidine for extraction. High-density proteoglycans, with less than 25% protein are readily extracted from the tumor and are smaller ( M , 130K). These proteoglycans have been purified by ion-exchange chromatography, molecular-sieve chromatography, and CsC1-gradient centrifugation and are highly immunogenic when injected into rabbits. The specific antibodies produced are directed toward the protein portion of the proteoglycan, reacting with the tumor matrix and with all basement membranes in normal tissues (Hassell et al., 1980, 1985; Dziadek et al., 1985b). Rotary-shadowing electron microscopy (Fig. 11) together with chemical and physical analyses permit detailed models for both forms of EHS tumor proteoglycan to be proposed. The low-density proteoglycan consists of an elongated protein core (length, 80 nm) with three heparan sulfate chains clustered at one end (Paulsson et al., 1987b). The protein core, when released by treatment with heparitinase, migrates in electrophoresis more slowly than does the laminin A chain with a M , 500K, although the possible retention of carbohydrate and the lack of adequate standards limit accurate estimates of its size. It consists of a single polypeptide chain folded into about six compact globular domains. The multidomain structure of the protein core was confirmed by protease digestion experiments, which released several large protein fragments together with a small segment containing the heparan sulfate chains (Ledbetter et al., 1987; Paulsson et al., 1987b). This study also indicated that the heparan sulfate attachment region is at one end of the molecule. One form of high-density proteoglycan was obtained from NaCl extracts of EHS tumor and appears as a star-shaped molecule (Fig. 11). The

-

30

GEORGE R. MARTIN ET AL.

’\ \

Low density PG

\

High density PG

%*\.

*\

---

- -----‘\

(__-- -$---

#0 0

-

s 0 0

\r’

0

0

/‘

0 0

0

8

0

’
400K) from cells that synthesize basement membranes. Pulse-chase experiments indicate conversion of this nascent polypeptide to the low-density proteoglycans with the subsequent appearance of a high-density proteoglycan (Ledbetter et al., 1985). The simplest explanation for these observations is that heparan sulfate chains are added to the nascent protein to form the low-density proteoglycan, which is then converted to the high-density proteoglycan by proteolysis. Whether such processes account for the formation of all forms of high-density proteoglycan remains to be established. Heparan sulfate proteoglycans isolated from the basement membranes of certain tissues and from cultured cells are clearly different in size. For example, most studies on glomerular basement membrane (Kanwar et al., 1981, 1984; Stow et al., 1983) indicate that its heparan sulfate proteoglycan is a small, high-density form (M,130K) with four heparan sulfate chains (M,26K). Similar material has been obtained from kidney labeled in situ with sulfate and from glomeruli labeled in culture and was localized to the lamina lucida of glomerular basement membranes by antibody staining (Stow et al., 1985a). On the other hand, Spiro and Parthasarathy (1984) reported the isolation of a low-density heparan sulfate proteoglycan (M,200K, 70% protein) from glomerular basement membranes; and lens capsule also contains a larger form. The glomerular proteoglycan 14K) clustered in one appeared to contain four heparan sulfate chains (M, portion of the molecule. Also Oohira et al. (1983) reported that endothelial cells produced and deposited a large heparan sulfate proteoglycan (Mr 400K) that reacts with antibody to the EHS tumor proteoglycans. These basement membrane types of proteoglycans differ, however, from cellbound heparan sulfate proteoglycans (Hook et al., 1984) as shown by antibody staining of tissues and cells (Dziadek et al., 1985b; Stow et al., 1985b). It is difficult at present to reconcile these conflicting results, even in a simple model system such as the EHS tumor. However, since all tissue basement membranes react with antibodies prepared to the EHS tumor proteoglycan, it seems likely that they contain related species even though real differences may exist between the proteoglycans in different tissues. Such molecules could be separate gene products, arise from differential splicing of mRNA from the same gene, or be formed by specific

32

GEORGE R. MARTIN ET AL.

proteolytic cleavage of a low-density proteoglycan. Additional heterogeneity may arise due to differences in the size of the heparan sulfate chains. For example, the proteoglycan from Reichert’s membrane in contrast to that from the EHS tumor shows strong binding to antithrombin. Such binding, as shown previously for heparin, involves sulfation at 3-OH sites as found in the proteoglycans from Reichert’s membrane but not in the proteoglycans from the EHS tumor (Pejlar et al., 1987).

E. Calcium-Binding Proteins A few recent studies indicate that divalent cations, most likely calcium, are important for maintaining basement membrane structure, a conclusion suggesting the presence of several cation-binding proteins. Initially, studies on the heat-induced polymerization of laminin showed it to be increased in the presence of calcium and other divalent cations and arrested at the level of small oligomers by chelating agents (Yurchenco et al., 1985). Likewise, chelating agents increase the extraction of lamininnidogen complexes from the EHS tumor, a result indicating that calcium mediates its binding to the matrix (Paulsson et al., 1987a). It was also directly shown that laminin is in fact a calcium-binding protein, but the localization and number of binding sites and their affinities are not yet known. Another small calcium-binding protein, BM-40, was isolated from 6 M guanidine extracts of the EHS tumor (Dziadek et al., 1986b).Immunohistology showed it to be a component of the tumor matrix and of Reichert’s membrane. It was also identified as a constituent of other tissues (i.e., lens capsule) by immunoassay, even though it did not react immunohistologically. Later studies showed that BM-40 is efficiently extracted from the EHS tumor in physiological buffers containing EDTA (Mann et al., 1987). Partial amino acid sequence analysis indicates the identity of BM40 with bone osteonectin (Bolander et al., 1988) and with SPARC, a parietal endoderm protein (Mason et al., 1986a). Together the data demonstrate that this calcium-binding protein is a ubiquitous constituent of extracellular matrices, including most if not all basement membranes (Dziadek et al., 1986b; Mason et al., 1986b; Young et al., 1986). Complete sequence analysis based on cDNA was achieved for osteonectin and SPARC and indicated a disulfide-bonded, four-domain structure for the protein (Fig. 12). Remarkable features of the structure are a cluster of 16 glutamic acid residues at the N terminus and an EFhand domain located between the last two cysteine residues, both regions having the potential for calcium-binding (Bolander et al., 1988; Engel et al., 1987). Studies with BM-40 demonstrated a reversible change in ct helix from 30 to 20% upon removal of calcium. The data indicate the

BASEMENT MEMBRANE PROTEINS

33

EF dornai n I

BGlu

II I

Glu-rich domoin

domoin

FIG. 12. Predicted domain structure of the small calcium-binding protein BM-4010~teonectin/SPARC. Numbering is according to the sequence of SPARC (Mason et al.. 1986a). but omits the signal peptide. A dashed line denotes predicted a helix, C identifies cysteine residues, and + indicates clusters of basic residues. A potential calcium-binding domain of the EF type is noted as well as a cluster of glutamic acids. Modified from Engel ef al. (1987).

cooperative binding of several calcium ions with K D in the micromolar range (J. Engel, personal communication). No a helix is evident, but calcium binding with K D = 0.3 p M has been found for bone osteonectin (Romberg et al., 1985). Cells contain a large variety of calcium-binding proteins, with the K D for cation-binding falling within the 10- to 1OO-nM range of intracellular calcium concentration (Kretsinger, 1980). Limited changes in calcium concentration can therefore modulate binding behavior of these proteins, changes like those occurring during muscle contraction or transmission of secondary messages. The calcium concentration within basement membranes is not known, but it could be the same as that of extracellular fluids (0.1-1 mM). If this assumption is correct, BM-40 should be present in the extracellular matrix exclusively in calcium-associated form. It could serve there as a calcium-sequestering protein; or, alternatively, calcium may induce conformational changes that regulate its binding to other matrix proteins. Support for such speculations will depend on the better understanding of calcium binding by laminin and by BM-40 and the characterization of further calcium-binding proteins possibly present in basement membrane. F . Other Basement Membrane Components Collagen VII forms the anchoring fibrils in the reticular lamina that link the basement membrane to underlying connective tissue (Burgeson et al., 1985; Sakai et al., 1986). These attachment fibers are particularly abundant along the epidermal-dermal basement membrane, in the amnion, in lung, and in other sites where basement membranes are under tension. The fibers of collagen VII form a centrosymmetrically banded structure

34

GEORGE R. MARTIN ET AL.

about 800 nm in length. This distance apparently represents the length of two molecules each 460 nm arranged in a antiparallel fashion with an overlap of 60 nm. The anchoring fibrils form an extended network underneath the lamina densa and connect by their C-terminal domains to the lamina densa, with anchoring plaques being abundant in the reticular lamina (Keene et al., 1987). The plaques contain type IV collagen, a finding suggesting that it may interact with collagen VII. Anchoring fibrils and collagen VII are absent from the skin of certain patients with the recessive dystrophic variety of epidermolysis bullosa, a severe blistering disease (Goldsmith and Briggaman, 1983). 1 . Fibronectin While fibronectin is usually listed as a component of basement membranes, it is present in much larger amounts elsewhere and may be primarily an adventitious constituent of basement membrane. Fibronectin (M,450K) contains two similar or identical chains that are each 60 nm long and are linked at their carboxyl ends by disulfide bonds (Yamada, 1983). Each chain contains a number of discrete domains with distinct binding sites for cellular receptors, collagen, fibrin, DNA, heparan sulfate, and so on. Fibronectin exists as both plasma- and matrix/cell-associated forms whose structures show minor differences that arise by differential splicing of the transcript of the fibronectin gene (Yamada, 1983; Hynes, 1985). The entire amino acid sequence of the cellular form has been deduced from the nucleotide sequence of human cDNA clones (Kornblihtt et al., 1985) and by sequencing bovine plasma fibronectin (Skorstengaard et al., 1986). Immunofluorescent studies with antibody to fibronectin show the protein to be localized in the basement membrane zone. At the ultrastructural level, fibronectin is observed to be concentrated in structures in the reticular zone as well as being directly associated with the lamina densa (Madri et ul., 1980; Martinez-Hernandez et ul., 1981; Courtoy et ul., 1982; Semoff et al., 1982; Laurie et al., 1983; Fleischmajer and Timpl, 1984). Most studies suggest that fibronectin is not a ubiquitous component of all basement membranes or necessarily synthesized by the same cells that produce collagen IV and laminin. It may be acquired from the circulation and from nonepithelial cells. Fibronectin can bind to both collagen IV and heparan sulfate proteoglycan and could be localized to the lamina densa in this fashion. Studies on developing tissues suggest that the highest levels of fibronectin are associated with the newly deposited basement membranes and could play a role in their deposition as well as allowing fibroblastic cells to bind to basement membranes (Stenman and Vaheri, 1978; Wartiovaara et ul., 1979).

BASEMENT MEMBRANE PROTEINS

35

2 . Chondroitin Sulfate Proteoglycans Chondroitin sulfate was identified in embryonic basement membrane on the basis of studies showing the susceptibility of sulfate-labeled material to chondroitinase and a reaction with monoclonal antibodies (Couchman et al., 1984; Paulsson et al., 1985b). Similarly, Reichert’s membrane, endothelial L2, PYS cells, EHS tumor cells, and isolated glomeruli produce variable amounts of these proteoglycan (Oohira et al., 1983; Stow et al., 1983; Oldberg et al., 1981; Tyree et al., 1984; Hassell et al., 1980). The molecule produced by L2 cells (M, 40K) contains a small protein core with a unique amino acid sequence (Bourdon et al., 1985). It appears that in such systems, little of the chondroitin sulfate proteoglycan is deposited in the matrix. 3. Amyloid P Component This protein (M, 230K) is an acute-phase protein produced in response to either trauma or toxic factors (Pepys and Baltz, 1983). Amyloid P was also observed by histology in glomerular and certain vascular basement membranes. It appears to be bound to type IV collagen, since it is brought into solution with collagenase (Dyck et al., 1980). Amyloid P was suggested to form the basotubule structures observed in some basement membranes (Inoue et al., 1983; Inoue and Leblond, 1985). 4 . Acetylcholinesterase This enzyme is concentrated in the synaptic clefts between nerve ending and muscle. These are cholinergic synapses, and acetylcholinesterase inactivates acetylcholine released by the nerve to allow muscular relaxation. A considerable proportion of this acetylcholinesterase is bound to the basement membrane in the cleft separating the muscle surface from the nerve process. Some studies suggest that the basement membrane contains the asymmetric form of acetylcholine esterase, which contains a collagenous tail (McMahan et al., 1978; Inestrosa et al., 1982; Dreyfus et al., 1983). Binding studies show that this form has a rather high affinity for heparan sulfate proteoglycan, and its binding to the basement membrane could be through this interaction, since this enzyme does not bind to laminin, collagen IV, or fibronectin (Vigny et al., 1983; Grassi et al., 1983).

5 . Bullous Pemphigoid Antigen This protein is limited to basement membranes beneath squamous epithelia such as the epidermal, urethral, bladder, bronchial, and gall bladder basement membranes. It was detected using autoantibodies in the sera of

36

GEORGE R. MARTIN ET AL.

patients with bullous pemphigoid, a dermatological disorder characterized by multiple blistering of the skin. These blisters develop due to the deposition of the autoantibody in the epidermal basement membranes, a process that initiates an inflammatory reaction and leads to the separation of the epidermis from the dermis at the level of the basement membrane (Lever, 1979; Patel et al., 1983). Bullous pemphigoid antigen is synthesized in culture by epidermal cells and has been identified as a disulfide230K) (Stanley et al., 1981, 1982). It linked oligomer of a unique chain (M, has an affinity for the surface of epidermal cells and is the first component of the epidermal basement membrane to be deposited by epidermal cells during wound healing. 6 . Goodpasture Antigen The occurrence of this component was also discovered due to the presence of autoantibodies in patients with a rare form of glomerulonephritis. Such patients present with a syndrome of hematuria associated with pulmonary hemorrhage, which may progress rapidly and lead to the complete loss of kidney function. In this disorder, basement membranes in the glomeruli and lung contain linear deposits of IgG, whose presence elicits inflammatory reactions that lead to the loss of tissue function. Such antibodies can be eluted from the affected kidneys after their removal and are also detected in the blood of these patients (Wilson and Dixon, 1981; Peters et al., 1982). These autoantibodies react with the kidney basement membranes, but not, for example, with those in skin. While the Goodpasture antigen was originally thought to reside only in lung and kidney, it may also occur in smaller amount or in a cryptic form in other basement membranes (Wieslander and Heinegard, 1985). The antigen is solubilized by collagenase and copurifies with the NCl domain of type IV collagen (Wieslander et al., 1984a,b;Timpl et al., 1985). The epitope appears to be partially cryptic, and a stronger reaction with autoantibodies is obtained after dissociation of the hexameric NCl structure into subunits (Wieslander et al., 1985). However, not all of the subunits possess the epitope, and the precise relationship of the NC1 globule to the Goodpasture antigen remains to be established. It is possible that the intact antigen is a unique collagen IV chain (Butkowski et al., 1985, 1987).

IV. SELF-ASSEMBLY AND INTERACTION BETWEEN COMPONENTS Self-assembly processes are usually invoked to explain the deposition of collagenous matrices, since they form outside the cell. Self-assembly in the stroma is based on the ability of interstitial collagen types 1-111 to

BASEMENT MEMBRANE PROTEINS

37

spontaneously align in an ordered manner. These particular collagens are secreted as soluble precursors, the procollagens 1-111, which are converted to collagen molecules by the enzymatic excision of propeptides. This modification reduces the solubility of these proteins and encourages their lateral association, thereby giving rise to the rapid formation of fibers at physiological pH and temperature. A somewhat different pattern is observed with collagen IV. It is incorporated into the matrix without proteolytic modification and its deposition is not associated with a change from a soluble to an insoluble form. However, spontaneous interactions are observed between molecules that may be involved in the deposition of basement membrane. Monomeric preparations of intact collagen IV can be obtained from the media used to culture cells that produce the protein. These monomers of collagen IV, when incubated under physiological conditions, particularly in the presence of a glutathione redox system, form dimeric, trimeric, and tetrameric aggregates. Association only occurs at the amino terminus of the molecule, without any globular interactions. No requirement for cation was found, nor did laminin accelerate the process (Duncan et af., 1983). The data suggest that the formation of aggregates of collagen IV through their 7 S domains can occur spontaneously in a stepwise fashion up to the tetrameric level. The collagen IV extracted from lathyritic tissue under reducing conditions (Kleinman et af., 1982) is predominantly composed of dimers, with the molecules linked at their globular ends. When incubated in physiological solvents at 25"C, tetrameric, hexameric, and octameric aggregates are formed that resemble the aggregates of collagen IV brought into solution by limited digestion of basement membrane with proteases (Yurchenco and Furthmayr, 1984). When solutions of the dimeric preparation of collagen IV are incubated at 37"C, a more condensed and regular lattice network is observed. This material consisted of the aggregate structures formed at 25°C but two or three triple helices were laterally associated to form polygonal structures. The globular domains of the dimers are arranged on the vertices of the polygon in a regular fashion and are separated by an average distance of 170 nm (Yurchenco and Furthmayr, 1984). More recent studies suggest that dimeric globules isolated by collagenase treatment of collagen IV bind at specific sites along the helix (Tsilibary and Charonis, 1986). In summary, these observations suggest that purified collagen IV can form amino-terminal interactions, such as those occurring in situ, but not the globular (NCl) interactions. The open network formed by the association of like ends of collagen IV molecules can undergo additional associations determined by the affinity of dimeric NC 1 globules

38

GEORGE R. MARTIN ET AL.

for sites along the triple helix. Such a condensed structure, with the other component of basement membrane arrayed on it, could generate the cords observed in the lamina densa. A . Laminin Laminin has also been observed to polymerize in uitro. Polymerization is observed under physiological conditions at 37°C above a critical concentration (100 pg/ml), requires calcium ions, and is reversible at low temperatures (Yurchenco et al., 1985). Examination of the laminin aggregates suggests that the interactions occur through the terminal globules on the arms of laminin. Denatured laminin does not undergo the polymerization reaction. Antibodies to the end of the long arm of laminin block the polymerization of laminin (Charonis et al., 1986).A similar calcium-mediated polymerization is also observed with the laminin-nidogen complex (Paulsson et al., 1987a).

B. Multiple Interactions Collagen IV has been found to bind to both laminin and heparan sulfate proteoglycan, whereas laminin binds both the nidogen and heparan sulfate proteoglycan. These observations suggest that multiple interactions of a specific nature could be important in determining both the composition and the deposition of basement membranes. Collagen IV binds about an equal amount of laminin (Woodley et al., 1983).The initial interactions between the two appears to involve a globular portion of a short arm of laminin binding to a site on the collagen IV molecule about 80-140 nm from the NC1 domain (Charonis et al., 1985; Rao et al., 1985; Laurie et al., 1986). Subsequently, the laminin appears to collapse onto the collagen IV molecule. The short-arm fragment of laminin, which lacks the long arm, is also able to bind to type IV collagen, whereas the P1 fragment lacking globules does not (Rao et al., 1982). The binding affinity of laminin for collagen IV has been reported to be on the order of KD = lo7,and denaturation of either protein destroys their interaction (Woodley et al., 1983). Others have not observed interactions in solution or saturable binding (Charonis et al., 1985). Both the large and small forms of the heparan sulfate proteoglycans bind to laminin and to collagen IV (Woodley et al., 1983; Fujiwara et al., 1984). Binding of these proteoglycans occur at the globule on the end of the long arm of laminin, presumably due to electrostatic forces, since binding is reversed by relatively low levels of NaCl (Ott et al., 1982). Binding of heparan sulfate proteoglycans to collagen IV has been studied

BASEMENT MEMBRANE PROTEINS

39

both by affinity chromatography and by electron microscopy. A major binding site for proteoglycan binding has been identified at about 200 nm from the NCl domain (Laurie et al., 1986). Fibronectin has also been found to bind to collagen IV. In this case, binding is stronger to the denatured protein than to the native form, a binding pattern also seen with other collagen types (Woodley et al., 1983). Binding of fibronectin to collagen IV appears to involve a site on the collagen molecule about 200 nm from the NC1 domain and a region at the end of a fibronectin chain (Laurie et al., 1986). Even though the laminin and fibronectin binding sites are distinct on collagen IV, the binding of laminin blocks any binding of fibronectin but not vice versa. Presumably, this interference occurs for steric reasons due to the condensation of laminin on the collagen IV molecule (Terranova et al., 1986a). Such data indicate that the association of the major components of basement membrane-collagen IV, laminin, heparan sulfate proteoglycans, and nidogen-are specific and occur through a limited number of binding sites. Direct evidence that such interactions occur between mixtures of these components has been obtained by combining the purified proteins as well as by studying the interaction of the materials from unfractionated extracts (Kleinman et al., 1983, 1986). These studies showed that mixtures of collagen IV, laminin, and the large heparan sulfate proteoglycan precipitate at 37°C in the ratio 1 : 1 : 0.15. The stoichiometry observed in this interaction and the presence of a limited number of binding sites on each molecule are consistent with their ability to form defined complexes. Similar interactions were observed when extracts of the EHS tumor matrix, which contain laminin, nidogen, heparan sulfate proteoglycans, and a variety of other proteins, were examined (Kleinman et al., 1986). Examination of the extract by molecular-sieve chromatography showed that the majority of the laminin in the extract was present in an aggregate with nidogen and heparan sulfate proteoglycan. By electron microscopy, the aggregate was observed to contain a central core of proteoglycan to which was attached several outstretched laminin molecules. Nidogen was not visualized, possibly because it is much smaller than the other molecules. Other data indicated that laminin-nidogen complexes are particularly stable (Paulsson et al., 1987a)and that tissues contain equal amounts of these proteins (Dziadek and Timpl, 1985). These findings suggest the ubiquitous existence of such complexes in basement membranes. Supplementation of the extract with collagen IV leads to the rapid assembly of certain components in the extract into a solid gel composed of an interconnected network of lamellar structures resembling basement membranes (Kleinman et al., 1986). The major components of the gel

40

GEORGE R. MARTIN ET AL.

were identified as equal amounts of collagen IV, laminin, and nidogen, plus smaller amounts of proteoglycan. In summary, both collagen IV and laminin show the ability to selfassemble. In addition, the various components of basement membrane have an affinity for one another that involves binding to specific sites. These interactions generate rather defined aggregates in solution and lead to the deposition of the components in a gel-like form whose ultrastructure resembles in some details authentic basement membranes. Such multiple interactions would be expected to be stronger than single interactions and may account for the codistributions of these components in basement membranes.

V. BIOLOGICALASPECTS A . Structural Functions

Basement membranes are the primary extracellular matrix of epithelial tissues. As such, basement membranes are presumed to provide physical stability and thus maintain tissue shape and integrity. The physical characteristics of the basement membranes have not been adequately studied but are expected to exhibit much greater elasticity than other collagenous matrices due to the end-to-end arrangement of molecules. Such a network structure would also facilitate the passage of fluids through capillary and glomerular walls. However, basement membranes in the kidney, in capillaries, and possibly in other sites, exhibit the ability to restrict the passage of negatively charged proteins. This barrier in the basement membrane is formed of the heparan sulfate chains of the proteoglycan, which are arranged along its surfaces (Farquhar, 1981). In embryogenesis, proteoglycan appears to be absent from the basement membranes in those regions of developing glands that are undergoing rapid growth and branching (Bernfield et al., 1984). It has been suggested that the absence of proteoglycans from these regions allows macromolecules, such as growth factors, to pass through the basement membrane and stimulate the growth and morphogenesis of the tissue on the other side. Indeed, an important function of basement membranes in the mature individual may be to prevent biologically active mediators produced by the separated tissues from reaching each other. In diabetes, basement membranes may also become defective and leak serum proteins, a pathology resulting in degenerative changes in kidney, eye, and blood vessels. Additionally, basement membranes in a number of sites, including kidney, capillary, and nerve, become grossly thickened with increased collagen IV but reduced proteoglycan contents. Two gen-

BASEMENT MEMBRANE PROTEINS

41

era1 concepts have been advanced to explain these changes (Brownlee and Cerami, 1981; Rohrbach et al., 1983; Tarsio et al., 1987). The basement membrane produced in the diabetic has a reduced proteoglycan content and therefore is functionally altered. Additionally, an increased nonenzymatic glycosylation of basement membrane proteins occurs in diabetes due to the elevated blood glucose and interferes with the binding of the components to one another, most strongly altering proteoglycan binding (Tarseo et al., 1987). The fact that the thickened basement membranes in diabetes are located in sites where quantities of fluid are filtered points to the involvement of compensatory mechanisms underlying the increased production of basement membrane proteins.

B . Cellular Receptors for Collagen IV and Laminin Many cells attach to basement membranes through cellular receptors that are specific for various components of this matrix. Diverse mechanisms undoubtedly exist, since some cells bind preferentially to collagen IV, others to laminin, and others to the two proteins together (Terranova et al., 1980; Aumailley and Timpl, 1986). Colligin ( M , 47K), a collagen IV binding protein that also binds gelatin, has been isolated from endodermal cell membranes and could be a collagen receptor (Kurkinen et al., 1984; Hughes et al., 1987). Much more is known about the interaction of cells with laminin. A highto 4 X receptor for laminin has been found on affinity (KD= 1 x many cells including myoblasts, tumor cells, and macrophages (Lesot et al., 1983; Rao et al., 1983; Malinoff and Wicha, 1983). The laminin receptor (M, 67K) is solubilized by detergent and has all the characteristics of an integral membrane protein. A partial amino acid sequence of the receptor has been deduced from the nucleotide sequence of cDNA clones. These show a possible transmembrane sequence and suggest that this receptor has substantial cytoplasmic and extracellular domains (Wewer et al., 1986). A small peptide from the B1 chain of laminin has been identified as a major binding site for the laminin receptor (Graf et al., 1987). To identify this sequence as the active site, synthetic peptides (20-mers) were prepared to sequences in the various domains of the B1 chain and used as immunogens to induce the production of specific antibodies. The antibody to the peptide from domain 111, a cysteine-rich domain of homologous repeats, inhibited cell attachment, although the peptide itself did not exhibit activity. Additional peptides were prepared from neighboring sequences and a nonapeptide CDPGYIGSR was found to support cell attachment, to be chemotactic, and to elute the laminin receptor from a column of immobilized laminin. The pentapeptide YIGSR is the smallest

42

GEORGE R. MARTIN ET AL.

active sequence that will mediate cell attachment and receptor binding. On a molar basis, YIGSR shows only 0.5-1% of the activity of laminin, a result suggesting that other sequences or conformational factors are important. As expected, this peptide does not stimulate neurite outgrowth, since this activity has been localized to a different domain in laminin within or adjacent to the large globule at the end of the long arm (Edgar et al.. 1984). Presumably there is a neurite-specific receptor, although that has not been shown directly; and additional laminin receptors for other cells (Horwitz et al., 1985; Aumailley et al., 1987) may exist. Presumably these receptors react with other peptide sequences, as shown by inhibition of cell attachment to laminin by the peptide SDGR (Yamada and Kennedy, 1987), which resembles the inverted cell-binding sequence of fibronectin (Pierschbacher and Ruoslahti, 1984).

C. Turnover and Degradation Basement membranes are arrayed as thin but continuous sheets that form a significant physical barrier to the passage of cells. Current concepts suggest that degradative enzymes are required for turnover of the basement membrane and for passage of cells across it. These could include collagenases, heparitinases, and less specific enzymes. Given the cross-linked network that collagen IV forms in the basement membrane, one would expect that its removal is the critical step. Indeed, turnover studies show that collagen IV is more long lived than laminin and that heparan sulfate is the most rapidly replaced. Collagen IV is not attacked by the collagenases that attack stromal collagens, and a metalloproteinase that has been identified in the media of cultured tumor cells cleaves collagen IV molecules at a specific site (Liotta et al., 1979; Salo et al., 1983; Fessler et al., 1984a). The requirement for different collagenases to degrade fibrous versus basement membrane collagen is consistent with the role that basement membranes play in maintaining tissue architecture during the turnover of stromal tissue. In addition to the metalloproteinase IV, a variety of enzymes including elastase, pepsin, and chymotrypsin cleave collagen IV (Uitto et al., 1980; Mainardi et al., 1980). The importance of the various enzymes in the turnover and degradation of basement membranes has not been thoroughly examined. The ability of tumor cells to invade through basement membranes signifies their progression to malignant status (Liotta et al., 1986). As noted earlier, the collagen IV-cleaving metalloproteinase was originally discovered in the media used to culture metastatic tumor cells (Liotta et al., 1979). Binding of tumor cells to laminin, which precedes basement membrane invasion, results in a significant stimulation of protease release (Turpeenniemi-Hujanen et al., 1986). Current concepts on the process of

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tumor cell dissemination suggest that their ability to cross basement membranes is necessary for their invasive activity. Basement membrane-lined tissues and intact reconstituted basement membranes have been used to assess tumor cell invasiveness in in uitro assays (Terranova et al., 1986a,b; Albini et al., 1987). These studies show that most, if not all, metastatic cells are able to invade through basement membranes and that specific mechanisms are involved. Interestingly inhibitors of collagenase retard the invasion of tumor cells (Reich et al., 1988). This process represents a potential site to direct therapeutic agents.

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Sakurai, Y., Sullivan, M., and Yamada, Y. (1986). J. Biol. Chem. 261, 6654-6657. Salo, T., Liotta, L. A., and Tryggvason, K. (1983). J. Biol. Chem. 258, 3058-3063. Sasaki, M., Kato, S., Kohno, K., Martin, G. R., and Yarnada, Y. (1987). Proc. Natl. Acad. Sci. U.S.A. 04, 935-939. Schwarz, U., Schuppan, D., Oberbaurner, I., Glanville, R. W., Deutzmann, R., Timpl, R., and Kiihn, K. (1986). Eur. J. Biochem. 157, 49-56. Schwarz-Magdolen, U.,Oberbaumer, I., and Kiihn, K. (1986). FEBS Lett. 208, 203-207. Semoff, S . , Hogan, B. L. M., and Hopkins, C. R. (1982). EMBOJ. 1, 1171-1175. Shibata, S.. ed. (1985). Proc. Int. Symp. Basement Membr. Mishima, June 24-26. Siebold, B., Quian, R. G., Glanville, R. W., Hofman, H., Deutzrnann, R., and Kiihn, K. (1987). Eur. J . Biochem. 168,569-575. Skorstegaard, K., Jensen, M. S . , Sahl, P., Petersen, T. E., and Magnusson, S . (1986). Eur. J . Biochem. 161,441-453. Soininen, R., Tikka, L., Chow, L., Pihlajaniemi, T., Kurkinen, M., Prockop, D. J., Boyd, C. D., and Tryggvason, K. (1986a). Proc. Natl. Acad. Sci. U.S.A. 83, 1568-1572.

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Soininen, R., Chow, L., Kurkinen, M., Tryggvason, K., and Prockop, D. J. (1986b). E M B O J. 5, 2821-2823. Stanley, J. R., Hawley-Nelson, P., Yuspa, S. H., Shevach, E. M., and Katz, S. 1. (1981). Cell 24, 897-903. Stanley, J. R.,Hawley-Nelson, P., Yaar, M., Martin, G. R., and Katz, S. I. (1982). J . Invest. Dermatol. 78, 456-459. Stenman, S., and Vaheri, A. (1978). Exp. Med. 147, 1054-1064. Stow, J. L., Glasgow, E. F., Handley, C. F., and Hascall, V. C. (1983). Arch. Biochem. Biophys. 225,950-957. Stow, J. L., Sawada, H., and Farquhar, M. G. (1985a). Proc. Narl. Acad. Sci. U . S . A . 82, 3296-3300. Stow, J. L., Kjellen, L., Unger, E., Hook, M., and Farquhar, M. G. (1985b). J . Cell Biol. 100,975-980. Strickland, S . , Smith, K. K., and Marotti, K. R. (1980). Cell 21, 347-355. Tarsio, J. F., Reger, L. A., and Furcht, L. T. (1987). Biochemistry 26, 1014-1020. Terranova, V. P., Rohrbach, D. H.. and Martin, G . R. (1980). Cell 22, 719-726. Terranova, V. P., Hujanen, E. S., Loeb, D. M., Martin, G. R., Thornberg, L., and Glushko, V. (1986a). Proc. Natl. Acad. Sci. U . S . A . 83, 465-469. Terranova, V. P., Aumailley. M., Sultan, L. H., Martin, G. R., and Kleinman, H. K. (1986b). J. Cell. Physiol. 127, 473-479. Terranova, V. P., Hujanen, E. S., and Martin, G. R. (1986~).J. Natl. Cancer lnsr. 77,311316. Timpl, R., and Dziadek, M. (1986). l n t . Rev. Exp. Parhol. 29, 1-112. Timpl, R., and Martin, G. R. (1982). I n “Immunochemistry of the Extracellular Matrix” (H. Furthmayr, ed.), Vol. 11, pp. 119-150. CRC Press, Boca Raton, Florida. Timpl, R., Bruckner, P., and Fietzek, P. (1979a). Eur. J . Biochem. 95, 255-263. Timpl, R., Risteli, J., and Bachinger, H. P. (1979b). FEES Lerr. 101, 265-268. Timpl, R., Rohde, H., Gehron Robey, P., Rennard, S. I., Foidart, J.-M.. and Martin, G. R. (1979~).J. Biol. Chem. 254, 9933-9937. Timpl, R., Wiedemann, H., van Delden, V., Furthmayr, H., and Kiihn, K. (1981). Eur. J. Biochem. 1M,203-21 1. Timpl, R., Dziadek, M., Fujiwara, S., Nowack, H., and Wick, G. (1983a). Eur. J. Biochem. 137,455-465. Timpl, R., Johansson, S., van Delden, V., Oberbaumer, I., and Hook, M. (1983b). J. Biol. Chem. 258, 8922-8927. Timpl, R., Oberbaumer, I., von der Mark, H., Bode, W., Wick, G., Weber, S., and Engel, J. (1985). Ann. N. Y. Acad. Sci. 460, 58-72. Trueb, B., Grobli, B., Spiess, M., Odermatt, B. F., and Winterhalter, K. H. (1982). J. Biol. Chem. 257, 5239-5245. Tsilibary, E. C., and Charonis, A. S. (1986). J . Cell Biol. 103, 2467-2473. Turpeenniemi-Hujanen, T., Thorgeirsson, U. P., Rao, C. N., and Liotta, L. A. (1986). J . Biol. Chem. 261, 1883-1889. Tyree, B., Horigan, E. A., Klippenstein, D. L., and Hassell, J. R. (1984). Arch. Biochem. Biophys. 231, 328-335. Uitto, V. J., Schwartz, D., and Veis, A. (1980). Eur. J. Biochem. 105,409-417. Vigny, M., Martin, G. R., and Grotendorst, G. R. (1983). J . Biol. Chem. 258, 8794-8798. Von der Mark, K., and Kiihl, U. (1985). Biochim. Biophys. Acra 823, 147-160. Wartiovaara, J., Leivo, I., and Vaheri, A. (1979). Dev.Biol. 69, 247-257. Weber, S.. Engel, J., Wiedemann, H., Glanville, R. W., and Timpl, R. (1984). Eur. J. Biochem. 139,401-410.

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Wieslander, J., Barr, J. F., Butkowski, R. J., Edwards, S.J., Bygren, P., Heinegard, D., and Hudson, B. G. (1984b). Proc. Narl. Acad. Sci. U.S.A. 81, 3838-3842. Wieslander, J., Langeveld, J., Butkowski, R., Jodlowski, M., Noelken, M., and Hudson, B. G. (1985). J . Eiol. Chem. 260, 8564-8570. Wilson, C. B., and Dixon. F. J. (1981). I n “The Kidney” (B. M. Brenner and F. C. Pector, eds.), pp. 1237-1352. Saunders, Philadelphia. Woodley, D. T., Rao, C. N.,Hassell, J. R., Liotta, L. A,, Martin, G. R., and Kleinman, H. K. (1983). Eiochim. Biophys. Acra 761, 278-283. Yamada. K. M. (1983). Annu. Rev. Biochem. 52,761-799. Yamada, K. M., and Kennedy, D. W. (1987). J. Cell. Physiol. WO, 21-28. Yamada, Y., Avvedimento, V. E., Mudryj, M., Ohkubo, H., Vogeli, G., Irani, M., Pastan, I., and de Crombrugghe, B. (1980). Cell 22, 887-892. Yamada, Y., Sasaki, N., Kohno, K., Kleinman, H. K., Kato, S.,and Martin, G. R. (1985). In “Basement Membranes” (S.Shibata, ed.), pp. 139-146. Elsevier, Amsterdam. Young, M.F., Bolander, M. E., Day, A. A., Ramis, C. I., Gehron Robey, P., Yamada, Y., and Termine, J. D. (1986). Nucleic Acids Res. 14, 4483-4497. Yurchenco, P. D., and Furthmayr, H. (1984). Biochemistry 23, 1839-1850. Yurchenco, P. D., and Furthmayr, H. (1985). Ann. N.Y. Acad. Sci. 460, 530-533. Yurchenco, P. D., Tsilibary, E. C., Charonis, A. S.,and Furthmayr, H. (1985). J. Eiol. Chem. 260, 7636-7644.

DESIGN OF PEPTIDES AND PROTEINS By WILLIAM F. DEGRADO Contnl R o n r c h 8nd Dovolopmmt Dop.rtmant, E. 1. du Pont do ISlmours 6 Compmy, Incorpratd Exporlmontal Station, Wllmlngton. W m a r o 19898

.............................. ......................... A. Conformational Properties of the Commonly Occurring Amino Acids. . . . B. Properties of Some Conformationally Constrained Amino Acids . . . . . . C. Cyclic Peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Design of Analogs of the Pentapeptide Enkephalin . . . . . . . . . . . . 111. Design of Medium-Sized Peptides . . . . . . . . . . . . . . . . . . . . . A. Factors Stabilizing the Formation of Secondary Structures in Aqueous Solution . . . . . . . . . . . . . . . . . . . . B. Enhancing the Helical Potential of Natural Peptides . . . . . . . . . . . C. Design of Peptides That Form Amphiphilic Secondary Structures . . . . . IV. ProteinDesign. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Why Design Proteins? . . . . . . . . . . . . . . . . . . . . . . . . B. a-Helical Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . C. fi Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. Introduction.

11. Design of Small Peptides

51 53 55

57 61 61 68 68 75 80

101 101 102 114 117 118

I. INTRODUCTION Recent dramatic advances in methodologies for the synthesis, modification, and analysis of peptides have increased the ease with which novel sequences can be prepared. This development has opened a number of new possibilities for studying peptide and protein structure/function relationships (Oxender and Fox, 1987). For instance, the techniques of peptide synthesis and site-directed mutagenesis provide convenient methods for introducing systematic changes into a native protein sequence, thereby allowing one to evaluate how one or more side chains contribute to the physical and biological properties of a protein. Furthermore, as our understanding of the determinants of peptide and protein structure expands, it should be increasingly possible to design peptides and proteins with predetermined structures and properties. Initially, this approach should be useful for understanding natural peptides and proteins, for it critically tests our understanding of these systems; but ultimately it also holds the promise of providing entirely novel substances with unprecedented properties. 51 ADVANCES IN PROTEIN CHEMISTRY, Vol. 39

Copyright Q 1988 by Academic Press, Inc. All rights of reproductionin any form resewed.

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WILLIAM F. DEGRADO

This article will summarize some cptemporary approaches to the design of structurally defined peptides and proteins with special emphasis being given to how this approach has helped to elucidate the structural basis for the function of a variety of natural molecules. When in their proper biological milieu, peptides and proteins assume fairly well-defined conformations, which are responsible for their biological and physical properties. The mechanisms by which these molecules adopt defined three-dimensionalstructures depends on their size and chemical compositions. With few exceptions (Shoemaker et al., 1985, 1987a,b) short to medium-sized peptides (fewer than 30-50 residues) tend to be unordered in dilute aqueous solutions; that is, they adopt a large number of different conformations that are in dynamic equilibrium. However, peptides of this size adopt defined conformations when they bind to their receptors (e.g., membranes, proteins), which often have preorganized structures. Thus, short and medium-sized peptides tend to adopt conformations that are complementary to those of their receptors. In contrast, proteins tend to adopt fairly well defined three-dimensional structures, even in the absence of stabilizing interactions with other molecules or molecular surfaces. The intramolecular interactions involved in the stabilization of protein structures are weak and tend to be of the same type as those used by peptides in their intermolecular interactions with receptors. Thus, a major difference in the conformational properties of peptides and those of proteins is that proteins are large enough to contain sufficient loci for intramolecular, noncovalent interactions to overcome the unfavorable conformational entropy associated with adopting a defined three-dimensional structure. There are a variety of strategies and approaches for protein and peptide design. Those adopted for a given problem will depend on the size and molecular characteristics of the desired target molecule. If one wishes to create a model for a small peptide (e.g., the pentapeptide hormone enkephalin), then it might be feasible to design an analog of this molecule that is locked into a biologically active conformation. In this case, a conformation (or set of conformations) that promotes a productive interaction with a receptor is (or are) reinforced by introducing conformational constraints including macrocyclization and addition of alkyl groups that sterically restrict the conformational space available to the peptide chain. For peptides of longer chain lengths, it is not possible to introduce sufficient conformational constraints to restrict the peptide to just one or a few sets of conformations. In these cases, a different type of approach often produces impressive results. One postulates a hypothetical structure for the receptor-bound conformation of a given peptide, and then one designs an analog of this peptide that has minimal sequence homology to the

DESIGN OF PEPTIDES AND PROTEINS

53

parent peptide but quintessentially embodies the conformational and molecular characteristics thought to be important for biological activity. Finally, the design of proteins that adopt predetermined three-dimensional structures is perhaps the most ambitious goal, and one that has been attacked only recently. The large size and hence large number of conformational possibilities for even a small protein of 100 residues reaches astronomical proportions (Creighton, 1984). Nevertheless, by carefully considering the structures of natural proteins and by judiciously applying computational and graphical techniques in conjunction with physical models, it appears possible to achieve this goal. This review will highlight each of these three approaches to peptide and protein design. 11. DESIGNOF SMALL PEPTIDES

The design of conformationally constrained peptides is a powerful method for elucidating the biologically active conformation of a peptide when it is bound to its receptor (for reviews, see Hruby, 1982, 1984, 1985a,b; Freidinger and Veber, 1984; Struthers et al., 1984). In solution, small peptides tend to adopt large ensembles of conformations, and the biologically active conformation might only be rarely populated (Hruby , 1982, 1984, 1985a,b). Therefore, the solution conformations of a peptide deduced by experimental or theoretical methods, although interesting in establishing the dynamic range of conformations available to a given peptide, are not necessarily of relevance to the receptor-bound conformation. For instance, the conformations of Leu-enkephalin have been investigated by X-ray spectroscopy, NMR, conformational calculations, and fluorescence spectroscopy (reviewed in Schiller, 1984). The picture emerging from these techniques is that the predominant backbone conformation of this flexible molecule is highly dependent on its environment. Figure 1 illustrates two of the H-bonding patterns observed for Metenkephalin observed in crystal structures (Smith and Griffin, 1978; Stezowski et al., 1985; Griffin et al., 1986). Some of the observed conformations for Leu-enkephalin such as the extended /3 strands (Camerman et al., 1983; Griffin et al., 1986) do not appear to be consistent with the receptor-bound conformations inferred from studies with cyclic peptides (Schiller, 1984). On the other hand, some of the other experimentally and theoretically derived conformations, particularly those involving turns, are broadly consistent with studies with conformationally constrained peptides (Schiller, 1984). Thus, the synthesis of conformationally constrained peptides used in conjunction with theoretical and experimental methods of conformational analysis provides a powerful method for determining which (if any) of the many conformations observed in solution

54

WILLIAM F. DEGRADO

0

N-H

H

Met/tiofJ Phe 0

H

FIG.1. Small peptides can assume multiple conformational states in the solid state. The hydrogen-bonding patterns for two crystalline forms of Met-enkephalin (A and B) and a potent derivative of Met-enkephalin as determined by X-ray crystallography are illustrated (Smith and Griffin,1978; Stezowski et al., 1985; Grimn er al., 1986). The conformationsare antiparallel /3 pleated sheet (A), type I' @ turn (B),and type 11' /3 turn (C).

are responsible for binding and activating a receptor. In addition, conformationally constrained peptides might also be expected to bind more tightly to their receptors, because their binding should occur with a smaller decrease in the conformational entropy than the binding of an unconstrained analog. Finally, different receptor types appear to recognize different conformations of the same hormones (Schiller and DiMaio, 1982). Therefore, conformationally restricted peptides can have marked selectivities for binding to a certain receptor class. An important first step in the design of conformationally constrained peptides is the delineation of the residues that are important for activity. This goal is generally accomplished by first synthesizing a set of analogs that vary from the parent peptide by having shorter chain lengths, establishing the minimal chain lengths for complete and partial activities. Once a minimal chain length has been established, each side chain is systemati-

DESIGN OF PEPTIDES AND PROTEINS

55

cally varied to determine the importance of charge, steric bulk, hydrophobicity, aromaticity, and chirality at each position. These studies, although somewhat tedious, are essential for establishing the positions at which substituents might be introduced to constrain the conformation of the peptide. Also, after evaluation of the properties of a large set of analogs, it is often possible to postulate hypotheses concerning the conformational features involved in binding to the receptor. These hypotheses can be tested by preparing conformationally constrained analogs. A . Conformational Properties of the Commonly Occurring Amino Acids

In a discussion of the effects of conformational constraints on the overall configuration of a peptide, the conformation versus potential energy maps introduced by Ramachandran (Ramachandran et al., 1963) are useful (Fig. 2). The peptide bond generally adopts a trans configuration and deviates only slightly from planarity (Cantor and Schimmel, 1980). Therefore, to a first approximation, only two angles need to be considered to define the backbone configuration of a peptide chain: @, which defines the torsional angle about the Nu-C, bond, and V, which defines the angle about the C,-C (carbonyl) bond. Figure 2A illustrates the calculated conformational energy versus @, V angle diagram for N-acetylg1ycine-N'methylamide (Zimmerman et al., 1977; Scheraga, 1968). Even for this least hindered of amino acids, much of the conformational space is not allowed due to unfavorable overlaps of neighboring atoms. Figure 2B illustrates the effect that addition of a single methyl group has on the available conformational space (Zimmerman et al., 1977). Much of the conformational space available to glycine is not allowed for alanine, and the conformations corresponding to structural themes such as a helices and /3 sheets fall near local minima in the potential surface. The plots for the other common L-amino acids are similar, with the exception of proline. Before proceeding to a discussion of unusual, conformationally constrained amino acids, it is useful to discuss this residue (Cantor and Schimmel, 1980). The angle @ of proline is fixed at approximately -60" due to the constraints of the pyrrolidone ring. The values allowed for the corresponding V angle are approximately -55" and 130". The @ = -60", V = -55" set of angles leads to a partial reversal in the direction of the chain and explains the occurrence of proline at bends and the beginning of helices. The N-alkyl group of proline also sterically restricts the conformation of the residue preceding it in sequence, thereby making it difficult for that residue to adopt an a-helical conformation. This is in large part the origin of proline's tendency to break helices; the lack of an amide NH

56

WILLIAM F. DEGRADO

A

.

I I

-180

-120

-60

0

I20

60

I80

+ (drgrrrr) I 00

I20

B

60

Ti

-*

o

0

-60

-I

-120-

-la0

-I20

-60

I

(A

I

0

60

I20

1 180

C

480

IM

€4

0

M

20

B

*

lb)

120

b0

0

M

110

Id0

Id*q'*.sl

FIG.2. Conformational energy contour map of N-acetyl-Gly-N'-methylamide (A), Nacetyl-Ala-N'-methylamide(B) (taken from Zimmerman et al., 19771, and N-acetyl-a-Aib-

DESIGN OF PEPTIDES AND PROTEINS

57

group also is an important factor. The /3 sheet and left-handed a-helical conformations remain accessible for the residue preceding proline in the sequence.

B. Properties of Some Con&ormationallyConstrained Amino Acids A large number of amino acid and dipeptide derivatives with built-in conformational constraints have been synthesized and incorporated into biologically active peptides (reviewed in Hruby, 1982). In this review we shall consider only those modifications that appear to be generally applicable and for which the amino acids are either commercially available or can be prepared relatively easily. 1 . C,-Methylamino Acids One of the most successful methods of constraining the conformation of an amino acid unit is to replace the hydrogen atom at the a-carbon with a methyl group to give a dialkylamino acid (reviewed in Prasad and Balaram, 1984;Toniolo et al., 1983; Bosch et al., 1984). Figure 2C illustrates the conformational energy map for Ca-methyl derivative of alanine, N acetyl-C, ,C,-dimethylglycine-”-methylamine (Ac-Aib-NMe, where Aib refers to a-aminoisobutyric acid), as a prototype for the C,-methylamino acids (Paterson et al., 1981). The result of the addition of the a-methyl group to alanine is similar to the effect of adding a methyl group onto glycine. About 50% of the plot is accessible to glycine (Fig. 2A); addition of a single methyl group decreases the accessible area to 16% for alanine (Fig. 2B), whereas only a few percent remains accessible for the Aib residue (Fig. 2C). The region allowed for the Aib residue is particularly useful; it includes both the left-handed and right-handed a and 310helices. If the residues adjoining the Aib residue are chiral, a given helical sense will be favored: left-handed for D-residues and right-handed for L-residues. Thus, the addition of one or several Aib residues should be a useful method to promote either a turn or a helical conformation. Depending on the surrounding sequence, one can obtain type I, 1’,111, or 111’ conformations (Rose et al., 1985); or, if no helix-breaking residues are present, a helix of either sense can be engineered. Indeed, crystallographic and N‘methylamide (C) (taken from Paterson er al., 1981). Locations of minima are indicated by the filled circles. The contour lines in A and B are labeled with energy in kcalhol above the minimum-energy point. The contour lines in C are drawn at I , 3,5, 10, and 15 kcalhol above the 10” grid point of lowest energy for each map. The left graph in C was calculated using asymmetric bond lengths and angles for the dimethyl substituent as determined by X-ray crystallography. In the right graph, standard symmetrical, tetrahedral geometries were used.

58

WILLIAM F. DEGRADO

NMR investigations of Aib-containing peptides have confirmed this expectation (Prasad and Balaram, 1984; Bosch et al., 1984, 1985a,b; Fox and Richards, 1982; Kokkinidis et al., 1986; Bavaso et al., 1986; Karle et al., 1986). Alamethicin and related antibiotics contain multiple Aib residues in their sequences, and the crystal structures of a large number of fragments of these peptides have been determined to a high degree of resolution. As reviewed previously (Prasad and Balaram, 1984),the Q, and Y angles of Aib residues virtually always occur in the allowed region of the Q,, q plot (Fig. 2C). Long peptides tend to occur in 3,o- or a-helical conformation, whereas short Aib-containing peptides generally adopt type I or I11 turns, although type I1 turns are also occasionally observed. Clearly, Ca-methylation is a particularly useful method of dictating a desired range of @, q values for a given residue in a peptide chain. Methodology that should have broad applicability is appearing for the synthesis of &methyl derivatives of amino acids other than alanine. In particular, the chiral auxiliary approach of Schollkopf (1983), which involves the alkylation of Meerwein salts of lactim ethers of diketopiperazines, is promising. The “self-reproduction of chirality” approach of Seebach (Seebach and Fadel, 1985; Seebach et al., 1983, 1985) is even more attractive for the synthesis of chiral dialkyl glycine derivatives. In this method (Seebach and Fadel, 1985), an optically active amino acid is condensed with pivaldehyde, a reaction giving a cis-1,3-oxazolidin-5-one; the corresponding chiral enolate is then alkylated with methyl iodide, a reaction giving, after hydrolysis, the Ca-methylamino acid in high yield and diastereomeric purity. Such methods-elegant in their conceptual and experimental simplicity-should open the way to the widespread use of Ca-methylaminoacids as conformational constraints.

2. Na-MethylaminoAcids There are a variety of other conformational constraints that can be introduced into amino acids, but none appear to give the same degree of predictability or to have been studied as extensively as Ca-methylation. N,-methyl groups occur frequently in peptide antibiotics (Ovchinnikov and Ivanov, 1975)and have been introduced into peptides, occasionally to test the possibility of backbone hydrogen bonding (Sugano et al., 1974). The conformational implications of this transformation are complex and extend far beyond a simple change in hydrogen-bonding ability. Calculations (Tonelli, 1976; Manavalan and Momany, 1980) show that an Namethyl group has a major effect on the conformation of both the residue to which it is attached (the ith residue) and the residue preceding it in the sequence (the i - 1 th residue). For both residues, when the peptide bond preceding the N-methyl group is in a trans conformation, the right-handed

DESIGN OF PEPTIDES AND PROTEINS

59

a-helical portion of the Q,, Vr plot is high in energy, as assessed with the model compounds N-acetyl-N-methylalanine-N'-methylamide (NNMA) and N-acetylalanine-N'-dimethylamide,and the p portion of the plot is sharpened somewhat, giving a minimum at Q, = - 135", P = 75" (Manavalan and Momany, 1980). In addition, the left-handed a-helical portion of the plot represents the second lowest conformer for the ith residue, whereas for the i - 1th residue the CS(extended) conformation is second lowest in energy. N-Methylamino acids, like proline, often adopt cis as well as trans conformations about the peptide bond. Conformational calculations and NMR studies on sarcosylsarcosine (Howard et al., 1973) show that the two forms are nearly isoenthalpic, the cis isomer being less favored by 0.6 kcal/mol in DzO. The trans form of NNMA is calculated to be more stable than the cis form by approximately 4.5 kcal/mol (Manavalan and Momany, 1980). The lowest energy conformer for the cis form of NNMA has Q, and P angles of approximately - 140" and 70°, respectively. The effect of the introduction of N-methylamino acids into peptides has been experimentally addressed in a series of investigations by Vitoux et al. (1981, 1986; Aubry et al., 1981). The model peptide pivaloyl-L-prolylN-methyl-L-alanyl-N-methylamideadopts a cis peptide bond in dimethyl sulfoxide (DMSO), CCld, and in the crystalline state, with a type VI p turn centered between the two amino acids (for a review on turns, see Rose et al., 1985). In contrast, a type I1 p turn with all trans peptide bonds is adopted by the corresponding peptide lacking a methyl group at the amide nitrogen of alanine (Vitoux et al., 1981). For the diastereotopic the trans peptide pivaloyl-L-prolyl-N-methyl-~-alanyl-N-methylam~de, form is most stable, and a type I1 turn is formed in the crystal structure of the anhydrous form of this compound (Aubry et al., 1981). A monohydrate of this peptide was also examined; it had a similar backbone conformation (all trans peptide bonds), although minor adjustments in backbone angles allowed a bridging hydrogen-bonding water molecule to be inserted between the pivaloyl CO group and the C-terminal N-methylamide NH group (Aubry et al., 1981). More recent work (Vitoux et al., 1986) has focused on peptides of the general form Piv-X-Me-Y-NHMe (where X = Gly, L-Ala, L-Pro, and Y = Gly, and L- or D-Ala, Leu, or Phe). When the configurations of X and Y were both L, the peptide tended to adopt a type VI turn with a cis peptide bond, whereas the heterochiral pairs tended to adopt a type I1 turn with all trans peptide bonds. These conclusions were also consistent with the known conformations of more complex peptides that contain N-methylamino acids (Vitoux et al., 1986). For glycine-containing residues, the behavior was somewhat more complex; both cis and trans forms were populated.

60

WILLIAM F. DEGRADO

3. D-Amino Acids Another change that is commonly made in peptides is the reversal of the chirality of one or more amino acid residues (reviewed in Rose et al., 1985). This is a particularly popular modification, because protected Damino acids are commercially available, and the resulting analogs, if active, would have enhanced stabilities to enzymatic degradation. The chirality of the amino acids in the central two positions (i + 1 and i + 2) of a turn have a profound effect on the type of turn that is formed. If the central two residues are both of the L configuration, a type I turn is often formed. If the residue at position i + 1 is L and that at position i + 2 is D (an L, D pair) then a type I1 turn is stabilized, while a D, L pair at the central position will stabilize a type 11' turn (Rose et al., 1985). For this reason, type I1 turns are often referred to as L, D turns and type 11' turns as D, L turns. It is interesting to note that a type I turn composed of all L-amino acids has an overall topography that is similar to that for a type 11' with a D, L pair at the i + 1 and i + 2 positions (Rose et al., 1985). The backbone atoms in these turns tend to lie in a plane, and, because H-bonded turns are cyclic structures, they can be considered to have substituents in either axial or equatorial positions. Type I and 11' turns differ significantly only in the @ and V angles about the second residue. For the L, L pair, these are -6O", -30" (type I), and +60", - 120" for the D, L pair (type 11'). The result is that the side chains project from roughly the same position in both types of turns if the chirality of the second amino acid is reversed in going from a type I to a type 11' turn. Thus, if a type I turn is found in a native all+ peptide, substitution of a D-amino acid at position 2 of the turn might lead to an active analog. An enhancement of activity might even be possible as the hydrogen bond formed at a type 11' is tighter and more linear than that formed in a type I turn. In fact, substitution of a D- for an L-amino acid at position 8 of somatostatin gives rise to an analog with enhanced potency (Arison et al., 1978; Veber et al., 1981). In this case, NMR studies on this peptide and related analogs containing a D-residue at position 8 provide strong support for the formation of a type 11' turn at this position (Veber, 1981). 4 . a,P-Unsaturated Amino Acids Yet another substitution that restricts the conformational freedom of amino acids is the introduction of a double bond at the C, and C, atoms. In addition to the obvious effect that a,p-unsaturation has on the sidechain mobility, this modification also affects the conformation of the backbone. The effects of C,-Cp unsaturation have been explored experi-

DESIGN OF PEPTIDES AND PROTEINS

61

mentally and by semiempirical potential energy calculations (Aj6 et al., 1980). They are strongly dependent on the presence of alkyl groups in the y position and the configuration of this substituent about the double bond. More recently, Bach and Gierasch (1986) have focused on the conformational preferences of Z-dehydrophenylalanine (AZPhe)when incorporated into peptides. NMR of linear and cyclic peptides showed that this amino acid may readily be incorporated into the i or i + 2 positions of p turns, or the i + 1 position of a y turn. The preference for AzPheto occupy the i + 2 position of /? turns has been confirmed by Singh et al. (1987) and Chauhan et al. (1987) in their studies of N-acetylated derivatives of tripeptides in which AZPheoccupies the central position. The overall conformational impact of this residue was similar to that observed for D-Phe. Interestingly, dehydrophenylalaninewas found to have a rigidifying effect on the conformations of flexible peptides, thereby encouraging linear peptides to adopt turn conformations. C . Cyclic Peptides Another method of constraining the peptide backbone, perhaps even more drastic than incorporating unusual amino acids, is the introduction of covalent cross-links. Macrocyclization is often accomplished by forming an amide bond between the peptide N and C termini, between a side chain and the N or C terminus, or between two side chains. Disulfides can also be introduced, but they may be reduced in uiuo. The conformational entropy of a cyclic peptide is lower than its linear counterpart. Thus, adoption of a specific conformation may occur with a smaller decrease in entropy for a cyclic analog than for an acyclic analog, thereby making the free energy for the process more favorable. The literature concerning cyclic peptides is extensive and has been exhaustively reviewed (Rose et al., 1985; Ovchinnikov and Ivanov, 1975; Kessler, 1982). D . Design of Analogs of the Pentapeptide Enkephalin The design of conformationally constrained peptides has helped to elucidate the structural bases for the activities of a number of important biologically active peptides, including somatostatin (Veber, 1981), oxytocin (Hruby, 1985b), and melanotropin (Lebl et al., 1984). Here, we shall consider the design of analogs of the pentapeptides Leu- and Metenkephalin (Tyr-Gly-Gly-Phe-Leu and Tyr-Gly-Gly-Phe-Met)as a recent example of this approach. An extraordinarily large number of analogs of these peptides have been synthesized, so the functional groups required for activity are well known (Morley, 1980). One of the most intriguing questions concerning the enkephalins is how they can compete with compounds such as morphine for binding to opiate receptors. Apparently the

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enkephalins adopt bioactive conformations in which the appropriate functional groups are positioned such that their topography mimics that of morphine. Ultimately, it should be possible to elucidate the essential conformational and molecular features of this structure by synthesizing conformationally constrained peptides. However, this has been complicated by the considerable flexibility of the enkephalins as well as by the fact that nonequivalent conformations of peptides can nevertheless have side chains in nearly equivalent orientations. Thus, when a series of peptides are examined, they may not share a single bioactive conformation. Nevertheless, in the last several years, the study of conformationally constrained peptides has contributed much to our understanding of the structural and dynamic requirements for binding and activation of opiate receptors. For example, it has been shown that receptor subtypes recognize distinct conformational states of enkephalins. In the following section we shall review the recent findings in this rapidly advancing field. 1 . Enkephalins Bearing Conformationally Constrained Amino Acids

The enkephalins are highly flexible molecules, and this flexibility has hampered attempts to elucidate their bioactive conformers. In particular, the two consecutive glycyl residues provide the enkephalins with even more potential conformational states than a typical pentapeptide composed of all L-amino acids. This flexibility is apparent in Fig. 1, which illustrates several conformations of enkephalins in the solid state. A large number of conformations for enkephalins dissolved in various solvents has also been proposed on the basis of the NMR of enkephalins in various solvents (reviewed in Schiller, 1984). Computational studies have also underscored the large number of potential conformations. Most of the NMR and computational studies show structures involving a p turn center between the Gly-Gly or the Gly-Phe bond to be favored (Schiller, 1984). Therefore, in an effort to limit the conformational space available to the native molecule, a large number of analogs bearing various conformational constraints have been prepared and evaluated. An early discovery was that substitution of a D-Ala residue at position 2 of the sequence led to an enhancement of activity (Roemer et al., 1977), a finding suggesting that a turn might be centered at the D-Ala-Gly bond. To further stabilize this turn, a set of molecules was synthesized in which Aib residues either singly or in a pair replaced the Gly-Gly bond in enkephalin (Sudha and Balaram, 1983). It was found that substitution of an Aib residue for the first Gly residue gave rise to highly active analogs: both the Aib-Gly and Aib-Aib peptides showed potent in uiuo activities (see also Gorin et al., 1980). In contrast, the Gly-Aib analog showed much lower, but still significant, activity. Unfortunately, no in uitro studies were reported, so it is difficult to assess the extent to which an increased resistance to proteoly-

DESIGN OF PEPTIDES AND PROTEINS

63

sis influenced these results. The conformations of the Aib-containing enkephalins and fragments thereof were investigated by CD (Sudha and Balaram, 1981),NMR (Sudha and Balaram, 1983),and X-ray crystallography (Prasad et al., 1983). It was shown that replacement of the Gly with Aib residues caused the peptides to adopt conformations more ordered than those of the parent compound (Sudha and Balaram, 1981). NMR studies of the peptides in dimethyl sulfoxide and chloroform (Sudha and Balaram, 1983) showed that the Aib-Gly peptide adopted a p turn centered about the Aib-Gly peptide bond, whereas the Gly-Aib peptide adopted a p turn centered about the Aib-Phe bond. The Aib-Aib peptide adopted a 3lo-helicalconformation with two consecutive turns centered at the Aib-Aib and Aib-Phe bonds. This conformation is also seen in the crystal structure of Boc-Aib-Aib-Phe-Met-CONH2(Prasad et al., 1983). These results led the authors to suggest that the enkephaiins must adopt folded conformations, possibly with a turn centered at the Gly-Gly bond. The authors of these studies note that stabilization of this turn conformation by hydrogen bonding must not be essential, because analogs with Nmethyl-Phe or Met at positions 4 and 5 are known to be highly active (Sudha and Balaram, 1983). Along similar lines, Stammer and co-workers (Shimohigashi and Stammer, 1982a,b; Shirnohigashi et al., 1982) have investigated the effects of substituting the glycyl residues in enkephalin with dehydroalanine. Substitution at position 2 (Shimohigashi and Stammer, 1982a)gives an analog that is nearly equipotent to Leu-enkephalin in the 6 receptor binding assay but is more than twice as potent in the p receptor assay. Thus, the AAla2 peptide binds to p receptors nearly as well as to 6 receptors, having a ratio of 0.45 for the association constants for binding to the two receptors. (Enkephalin favors binding to the 6 receptor by a factor of approximately 5 . ) AAla3-Leu-enkephalin(Shimohigashi and Stammer, 1982b) shows a marked decrease in its affinity for the 6 receptor (4% as active), but only a 20% decrease in its affinity for the p receptor. Thus, the ratio of the association constant for the p versus the 6 receptors is approximately 1.O for this peptide. It would be of interest to determine whether these intriguing differences in receptor affinities are a result of a modification of the conformational properties or whether they are a result of the increased hydrophobicities of the analogs. The same researchers have also incorporated A'Phe into the 4-position of Leu-enkephalin (Shimohigashi et al., 1982). The resulting peptide is a highly potent, A-selective binder.

2 . Cyclic Enkephalins The studies discussed in the preceding sections demonstrate both the potential and the limitations associated with the use of conformationally restricted amino acids for elucidating bioactive conformations. Even

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WILLIAM F. DEGRADO

FIG. 3. Structure of a cyclic enkephalin analog designed by Schiller and co-workers (DiMaio ef al., 1982).

though the conformations of the substituted amino acids are restricted, the overall chain still has a great deal of flexibility. If the possible configurations of the side chains are also considered, the problem is increased by orders of magnitude. In principle, it should be possible to combine more than one conformational constraint into a single peptide, but the resulting peptides still may have a large number of low-energy conformational states. Therefore, more recent work has focused on the introduction of even more extreme conformational constraints-in the form of macrocyclization-into enkephalins. Operating under the assumption that enkephalins adopt folded turn-like conformations at their receptors, Schiller and co-workers designed a series of cyclic enkephalins in which the 2-position was occupied by a D-amino acid bearing an alkylamino side chain (DiMaio et al., 1982): Cyclization was achieved by forming an amide bond between this side chain amino group and the C-terminal acarboxylate (Fig. 3). The prototype cyclic analog, Tyr-cyclo[N4-~-DbuGly-Phe-Leu] (Dbu = 2,4-diaminobutanoic acid) was an extraordinarily p selective ligand. Relative to Leu-enkephalin, it was 17 times more potent in the guinea pig ileum (GPJ, a tissue rich in p receptors) assay and 7-fold less effective in the mouse vas deferens (MVD, a tissue rich in 6 receptors) assay. The conformational properties of the ring could be modulated by adjusting the number of methylene groups in the side chain at the 2position. Four peptides were synthesized with one to four methylene groups. Relative to Leu-enkephalin, each had 5- to 50-fold higher activities in the GPI assay, and up to 40-fold lower potencies in the MVD assay. It was found that as the number of methylenes was increased from one to four, the selectivity ratio (the ratio of the potency in the GPI versus the MVD assays) increased, reaching a value of 29 for the analog with four methylene groups.

DESIGN OF PEPTIDES AND PROTEINS

65

The receptor selectivity of the cyclic peptides could be only partially explained in terms of their binding affinities for the p and 6 receptors. Each of the cyclic analogs had about the same binding affinity for the p receptor as Leu-enkephalin, although they had markedly poorer affinities for the 6 receptor. The ratio of the association constants for the p versus the 6 receptors reached a maximum of 8 when n = 2. Thus, the high degree of activity of the cyclic analogs in the GPI versus MVD assay systems had two causes: (I) the peptides bound rather more poorly to 6 receptors than to p receptors; and (2) they also had greater efficacy at the p receptor than did Leu-enkephalin. (That is, at a given degree of receptor-site saturation, the cyclic enkephalins produced a larger response than did Leu-enkephalin.)A reasonable explanation of these data is that multiple conformational forms of Leu-enkephalin bind to the p receptor but only one or a small subset of these bound conformational forms is responsible for signal transduction (DiMaio et al., 1982). Cyclization preferentially stabilizes the conformations that are important for signal transduction. Also consistent with this view are the results obtained with acyclic correlates of the above cyclic peptides (DiMaio et al., 1982; Schiller and DiMaio, 1982). These peptides are related to their cyclic analogs by a reductive cleavage of the ring and hence differ in chemical composition by the addition of only two hydrogen atoms. The prototype for this series is Tyr-Abu-Gly-Phe-Leu-CONHz (Abu = 2-aminobutanoic acid). These peptides have biological properties similar to those of Leu-enkephalinamide and are nonselective for p versus 6 receptors, both in binding and activity assays. Thus, the enhanced efficacy of the cyclic analogs must be a consequence solely of their conformational properties. In an extension of the above approach, partially retro-inverso analogs were synof the prototype peptide, Tyr-cyclo[N4-~-Dbu-Gly-Phe-Leu], thesized and evaluated (Berman et al., 1983). [A retro-inverso peptide (Goodman and Chorev, 1979) differs from its parent peptide by having the direction of its peptide bonds reversed and the chirality of each amino acid residue reversed. Nevertheless, the overall stereochemical relationships of the side chains remains roughly unchanged; C,-CO-NH-CL is converted to C,-NH-CO-CL.] These peptides contain a reversal of the peptide bond at two or more centers centered about the Phe-Leu bond. The cyclic retro-inverso analogs maintained the high p selectivity and efficacy of the parent compound. Thus, the orientation of the side chains must be similar in the retro-inverso analogs, and the backbone atoms in the modified region must serve primarily as a structural framework for positioning these side chains in proper juxtaposition. The conformations of several retro-inverso enkephalin analogs have been examined by a combination of computational and NMR techniques (Mammi et al., 1985;

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WILLIAM F. DEGRADO

Hassan and Goodman, 1986; Mammi and Goodman, 1986). Molecular dynamics and systematic search methods were used to search conformational space to obtain starting conformations for energy minimization. Several low-lying backbone and side-chain conformations were found for each of the analogs. In comparison with the linear enkephalins, the cyclic analogs had relatively few low-energy conformers. However, they still appeared to be partially flexible in the 10-ps molecular dynamics simulations. Their side chains were rather variable in conformation, but their main chains also showed multiple transitions between hydrogen-bonding patterns and regions of the @, 9 plot. The conformations of the peptides were then evaluated by standard NMR techniques. The interpretation of the probable positions of hydrogen bonds was aided by examination of the low-energy conformations of the peptides. Each of the peptides was found to adopt preferred conformations in dimethyl sulfoxide, and some appeared to adopt the same preferred conformation in water as well. The peptides all had one or two hydrogen bonds within the ring structure, and the parent compound had two C7 hydrogen bonds in dimethyl sulfoxide. By searching for structural commonalities among the low-energy conformations of this class of cyclic peptides, it may be possible to surmise some structural requirements for binding to the opiate p receptor. In addition, it should now be possible to design peptides with even greater conformational restrictions by incorporating conformationally constrained amino acids into cyclic enkephalin analogs. A second class of cyclic enkephalins consists of a set of analogs that contain intramolecular disulfide bonds. The prototype of this series is Tyr-cyclo(D-Cys-Gly-Phe-Cys)-CONH2. This peptide (Schiller et al., 1981), and the corresponding peptide with two D-CYSresidues, were 38and 73-fold more active than Met-enkephalin in the GPI assay. Again, this enhanced potency was only partially due to more potent binding to the p receptor; the D, L and D, D compounds bound 2- and 3.5-fold more tightly to the p receptor than did Met-enkephalin. Interestingly, these peptides failed to show substantial specificity for the p receptor. The ratios of the association constants for the p versus the 6 receptors were approximately 2.5 for both peptides. Thus, the cystine-bridged enkephalins were less selective than the methylene-bridged analogs, even when the same number of atoms was involved in the ring. This is presumably due to the increased rigidity of the latter class of cyclic peptides. The methylenebridged enkephalins contain one more rigid, planar peptide bond in their ring structures than do the disulfide-bridged enkephalins. In an effort to decrease the flexibility of the disulfide-bridged enkephalins, penicillamine residues (C, , CB-dimethylcysteine,Pen) have been introduced in place of the cysteine residues. The D-Pen2-D-CysSpeptide exhibited substantial 6 receptor activities (Mosberg et al., 1982). Even

DESIGN OF PEPTIDES AND PROTEINS

67

more impressive effects were obtained when both cysteine residues were replaced by penicillamine (Mosberg et al., 1983). The D-Pen2-D-PenS not only reenkephalin analog [Tyr-cyclo(D-Pen-Gly-Phe-~-Pen)-COOHl tained the high potency of the D-CYS~-D-CYS~ analog in the MVD assay but also displayed a high selectivity for 6 receptors. The 6 receptor selectivity ratio was 3200 in a bioassay (MVD versus GPI) and 370 in a binding assay. More recently, conformationally restricted amino acids have been introduced into the D-PenZ-D-PenScyclic analog in an effort to further restrict the conformation of the peptide (Hruby et al., 1985). Substitution of N-methyl-Phe in the 4-position leads to a compound with reduced activity in both the MVD and GPI assays and with lower 6 selectivity than the parent compound. However, replacing Phe4with tetrahydroisoquinoline carboxylate gives rise to an analog with very high 6 selectivity. These results led the authors to suggest that the Phe4side chain prefers to be in a gauche (-) conformation when bound to the 6 receptor. Conformational analysis of the disulfide-linked cyclic enkephalins should lead to an understanding of the conformational differences between 6- and p-selective ligands. NMR analysis of Pen-containing analogs has shown that they indeed have more rigid conformations than the corresponding Cys-containing analog (Hruby et al., 1985; Mosberg and Schiller, 1984). However, no specific conformations have been proposed yet for these peptides. A third class of cyclic enkephalins are those in which cyclization is achieved by amide bond formation between two side chains (Schiller et al., 1985). The morphiceptin analog, Tyr-cyclo(D-Orn-Phe-Asp)-CONHz, binds selectively to p receptors with a binding selectivity ratio of 213. The corresponding peptide Tyr-cyclo(D-Lys-Phe-Glu)-CONH2 is far less selective, binding with a selectivity ratio of 3.0. The loss in selectivity has been explained in terms of an increase in flexibility when the ring is expanded from 13 to 15 atoms. The antiparallel cyclodimer of the D-0rn2Asp4peptide was also examined and found to be nonselective in its receptor binding preferences. Thus, the selectivity of the monomeric cyclic peptide was probably due to its conformational properties. In conclusion, studies with conformationally constrained analogs of enkephalins have provided important information concerning the relationship between the conformations and the bioactivity of the enkephalins. Clearly, a folded, turnlike conformation is recognized by the p and 6 receptors. The primary purpose of the amide main chain appears to be to position the side chains in proper juxtaposition for interaction with receptors. Further, the 6 and p receptors appear to recognize different conformational forms of enkephalin. By synthesizing and analyzing the conformations of increasingly rigid cyclic enkephalin analogs, it should be possible to determine what arrangement of side chains is recognized by

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the p and 6 receptors. Also, it should be possible to determine whether some flexibility in the enkephalin molecules is required for their activities. PEPTIDES 111. DESIGNOF MEDIUM-SIZED The design of medium-sized peptides is beset with many of the same problems encountered in the design of small peptides. Medium-sized peptides are also very flexible and tend to lack defined conformations at room temperature in aqueous solutions. An additional problem in designing medium-sized peptides is the increase in chain length. This increases the time required for synthesis and purification, while at the same time exponentially increasing the number of possible variants for a given sequence. With a peptide of approximately five residues, it is feasible to try all possible amino acids at a given position and to ultimately introduce conformational constraints that greatly restrict the peptide in solution. The same approach is simply not practical for peptides of greater than approximately 20 residues. Fortunately, simple secondary structures (ahelices, /3 sheets) figure largely in the bioactive conformations of peptides of this length (Kaiser and Kkzdy, 1983, 1984). This fact suggests a rational and experimentally feasible approach to designing medium-sized peptides. Proceeding from a hypothesis for the bioactive conformation of a peptide (which includes the locations of secondary structures), peptides that should favor formation of this hypothetical structure are designed. The resulting analogs are still quite flexible but should more favorably interact with their receptors, if the guiding hypothesis is correct. It has been proposed that both a helices and /3 sheets are involved in the bioactive conformations of medium-sized peptides (Kaiser and KCzdy , 1984). However, a-helical secondary structure have been more clearly implicated in the conformations of a variety of peptides, including apolipoproteins (Kaiser and Kezdy, 1983, 1984), toxins (DeGrado et al., 1981), hormones (Kaiser and Kkzdy , 1983, 1984), and calmodulin-binding peptides (DeGrado et al., 1985). Also, the techniques for prediction and analysis of a helices are more advanced than those for /3 sheets. Therefore, the following section will primarily highlight the design of a-helical peptides. A . Factors Stabilizing the Formation of Secondary Structures in Aqueous Solution

I . Short-Range Interactions Several approaches have been adopted for elucidating the factors involved in helix formation and stability. Perhaps the simplest approaches of these are the statistical approaches as typified by the Chou-Fasman

DESIGN OF PEPTIDES AND PROTEINS

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method (Chou and Fasman, 1978). In this method the frequencies of occurrence of a given amino acid in helical versus nonhelical structures are determined by analysis of a large number of protein crystal structures. Residues that frequently occur in helical conformations are reasonably assumed to favor helix formation. An alternate experimental approach is the host-guest method of Scheraga (1978). This method attempts to determine the extent to which a given amino acid stabilizes or destabilizes the helical conformation by examining amino acid copolymers. A random copolymer of the amino acid of interest (the guest) and a helix-favoring amino acid (the host) is formed, with the host amino acid in large molar excess. The thermally induced helix-to-coil transitions of the host polymer with and without the guest amino acid are then evaluated to investigate the perturbational effect of the guest. The data are analyzed using the Zimm-Bragg formalism, which includes parameters for both helix initiation (u)and helix propagation (s). The parameter u can be interpreted as an equilibrium constant for formation of the first turn of the a helix from a random coil polymer. This parameter is unfavorable and is on the order of The parameter s corresponds to the equilibrium constant for addition of a single residue to an already initiated helix. The values of s calculated from experimental data of host-guest copolymers range from 0.6 for Gly to 1.32 for protonated Glu. There is a good qualitative agreement between the Zimm-Bragg s values that obtained and the ChouFasman parameters for helix formation (Scheraga, 1978; Chou and Fasman, 1978; Sueki et al., 19841, a result suggesting that both parameters are measures of the intrinsic conformational preferences of individual amino acids. 2 . Medium-Range Interactions Host-guest studies of random copolymers suggest that peptides of 10 to 30 residues should show very little helix formation in dilute aqueous solution, even when these peptides are composed of helix-favoring amino acids. This prediction is generally true for amino acid homopolymers, but recent work with peptides of defined sequence has shown that sequencedependent side chain-side chain or side chain-main chain interactions, which are not included in the Zimm-Bragg formalism, can have a dramatic influence on the stabilities of isolated helices in solution. This outcome has been most clearly shown in studies (Bierzynski et al., 1982; Kim and Baldwin, 1984; Shoemaker et al., 1985, 1987a) of the N-terminal a helix of ribonuclease A, which spans residues 3-13 in the crystal structure of this enzyme. As predicted from the Zimm-Bragg model, peptides corresponding to this sequence have little helical structure in water at room temperature, as assessed by NMR (Rico et al., 1986) or CD spectroscopy (Bier-

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WILLIAM F. DEGRADO

zynski et al., 1982; Brown and Klee, 1971). However, they deviate from this model at lower temperatures; near 0°C in aqueous buffers at neutral pH, a peptide spanning the N-terminal 13 residues of the ribonuclease sequence (C-peptide, residues 1-13 terminating with a homoserine lactone) is approximately 30% helical (Bierzynski et al., 1982).Clearly, there must be specific interactions that provide the extra stabilizing force required for helix formation. The pH dependence of helix formation for this peptide indicates that two groups with apparent pK, values of 3.3 and 6.5 are important for helix stability. Initially it was hypothesized that there was a stabilizing salt bridge formed between the side chains of Glu-9 and His-12, which are located slightly more than a single a-helical turn apart, (Bierzynski et al., 1982); subsequent systematic studies with synthetic peptides disproved this hypothesis. It appears instead that the ionizing groups are Glu-2 and His-12, residues that are located at opposite ends of the helix. Their location suggests that they might serve to interact favorably with the ahelical dipole (Shoemaker et d., 1985, 1987a). In an a helix the amide NH groups all point approximately toward the N terminus of the helix, and the C=O groups point in the opposite direction, an arrangement giving rise to a macrodipole (Hol et al., 1981; Sheridan et al., 1982). Negatively charged residues at the N terminus of C-peptide and positively charged residues at the C terminus appear to interact favorably with the helix dipole and favor helix formation (Shoemaker et al., 1985, 1987a; Blagdon and Goodman, 1975). The salt dependence of helix formation for various analogs of C-peptide is also consistent with the helical dipole model (Shoemaker et al., 1985, 1987a). Also consistent with’thisview are studies in which the charge of the N-terminal residue is systematically varied in the following series: +2 (Lys); + 1 (Ala); 0 (acetyl-Ala); - 1 (succinylAla). The extent of helix formation increased steadily as the charge was varied from +2 to - 1 (Table I, compounds I-IV). Similarly, neutralization of the negative charge at the C terminus by conversion of the carboxylate to a carboxamide or homoserine lactone, also favors helix formation (Shoemaker et al., 1985, 1987a). Two other types of specific side-chain interactions have been proposed to stabilize the a helix formed by C-peptide (Shoemaker et al., 1987b). A salt bridge between Glu-2 and Arg-10 has been detected in the crystal structure of ribonuclease A (Wlodawer and Sjolin, 1983) as well as in Cpeptide in aqueous solution (Rico et al., 1986). This salt bridge also fixes the N-terminal boundary of the helix between Glu-2 and Thr-3. It is not sterically possible to make this ion pair if Glu-2 is part of the helix. Synthetic studies in which either Glu-2 or Arg-10 is replaced by Ala have provided support for the importance of this interaction in stabilizing the a-

71

DESIGN OF PEFTIDES AND PROTEINS

TABLEI Correlation of Association Constant and Thermostability of Semisynthetic RNase S with Peptide Helix Content'

Peptide

Peptide ellipticity (3°C) (deg cm2 dmol-I)

S-peptide (1-15) S-peptide (1-19) V (Lysl, Gin")' I (Lysl, Glu") I1 (Alal, Glull) VI (SucAla', Gln1I) I11 (AcAla', Glull) IV (SucAlaI, Glull)

2,500 3,500 8,200 11,100 12,800 13,000 15,800 16,200

K, (35°C) (lo6 M - 9 0.865 1.05 2.19 4.99 6.32 4.03 6.45 10.00

f 0.03

f 0.12 2 0.41

1.30 1.09 f 1.0s 2 2.29 f 2.13 f f

Tmb ("C) 48.1 45.9 41.5 49.3 50.4 48.8 51.1 51.9

f 0.9 f 0.7 2 0.2 f 0.2 f 0.1 f 0.4

From Mitchinson and Baldwin (1986); all values refer to 0.10 M NaCI, pH 7.30. Suc and Ac refer to a-N-succinyl and a-N-acetyl groups, respectively. T, measured at an RNase S concentration between 100 and 200 p M with 30% mole excess peptide. Compounds I-IV correspond to S-peptide 1-1s with Leu substituted for Glu at position 9, and other changes as indicated in parentheses.

helical conformation of C-peptide. However, the analysis is not simple, due to the dual role played by the Glu at position 2. This residue appears to be important for interacting with the helical dipole as well as forming a salt bridge to Arg-10. The protonated form of His-12 might also play a dud role in stabilizing helix formation. In addition to interacting favorably with the helix dipole, this residue might form an aromatic interaction (Blundell et al., 1986; Burley and Petsko, 1985) with Phe-8 in solution (Shoemaker et al., 1987b). Preliminary evidence suggests that this interaction is stronger for the protonated than for the nonprotonated form of His-12 (Shoemaker et al., 1987b). There are probably other specific sidechain interactions that have yet to be identified. Although the magnitude of the stabilizing side-chain interactions are small (they tend to account for a factor of 2 to 3), they work in concert to make helix formation by Cpeptide only slightly unfavorable. 3. Long-Range Interactions The studies mentioned above have shown how short-range and medium-range interactions can stabilize helix formation in isolated peptides. Also important for structural stability are long-range interactions between the residues within a secondary structural unit and groups not included in that unit. In some cases such long-range interactions play a decisive role in determining which secondary structure is formed (DeGrado and Lear, 1985). Several types of attractive forces, including hydrogen bonding,

WILLIAM F. DEGRADO

72

electrostatic interactions, and hydrophobic forces, are known to be involved in long-range interactions (Creighton, 1984). Hydrophobic forces (Tanford, 1980) in particular appear to be important for stabilizing secondary structures in globular proteins (Kauzmann, 1959; Janin, 1979), peptide hormone-hormone receptor complexes (Kaiser and Kkzdy, 1983), and apolipoprotein-phospholipid complexes (Kaiser and Kezdy, 1984). a Helices and antiparallel /3 sheets often lie along the surface of globular proteins (Richardson, 1981) and are hence amphiphilic (Eisenberg et al., 1982a); their solvent-exposedfaces are more hydrophilic than their opposite faces, which are in contact with the apolar interior of the protein. A similar amphiphilic disposition of hydrophobic and hydrophilic groups has been envisioned for peptides that bind to extrinsic apolar surfaces, such as phospholipid membranes and hormone receptors (Kaiser and Kkzdy, 1983, 1984). Amphiphilic secondary structures show periodic distributions of hydrophilic and hydrophobic amino acids in their sequences; the repeat period for this distribution matches that of the secondary structure (Eisenberg et al., 1984). Hydrophobic residues repeat approximately every two residues in an amphiphilic /3 sheet and every three to four residues in an amphiphilic a helix. Upon interacting with an apolar interface, the peptide adopts a secondary structure corresponding to its hydrophobic period, providing a low-energy conformation in which the hydrophobic residues are maximally dehydrated and the hydrophilic residues are maximally hydrated. This is shown schematically in Fig. 4. Thus, hydration forces play an important role in determining the conformations of amphiphilic secondary structures. Hydrophobic periodicity can have such a large influence on the formation of secondary structure that it dominates over short- and mediumrange interactions (DeGrado and Lear, 1985). This has been demonstrated recently, using a set of designed peptides with leucyl and lysyl residues in identical ratios but with different hydrophobic periodicities. peptide 1 peptide 2 peptide 3

FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu), FMOC-(Leu-Lys-Lys-Leu-Leu-Lys-Leu)2 FMOC-(Leu-Lys-Leu-Lys-Leu-Lys-Leu),

Chou-Fasman parameters (Chou and Fasman, 1978) were identical for each of these peptides, as were the Zimm-Bragg s values (Scheraga, 1978) for helix formation. The peptides differ only in their hydrophobic periodicities (3.5 for peptides 1 and 2; 2.0 for peptide 3) and chain lengths, thus allowing the effect of these parameters on peptide conformation to be investigated while keeping short-range interactions approximately constant. All three of these peptides adopted random conformations in very dilute, aqueous solutions, but peptides 2 and 3 could be induced to form

DESIGN OF PEF’TIDES AND PROTEINS

73

-

PERIODICITY 3.5

s+

*

PERIODICITY- 2.0

HYDROPHOBIC PERIODICITY (PRIMARY STRUCTURE)

APOLAR + (INTERFACE) SURFACE

INDUCED SECONDARY STRUCTURE

FIG.4. Schematic illustration of how hydrophobic periodicity can influence secondary structure formation. In this illustration, the closed circles symbolize apolar residues and open circles symbolize polar residues. In dilute, aqueous solution, the peptides lack a single defined conformation. However, in the presence of an apolar-water interface, they adopt a secondary structure that maximizes the interactions of the apolar groups with the apolar medium and the polar groups with water. Taken from DeGrado and Lear (1985).

defined secondary structures in the presence of an apolar-water interface. Peptide 2 was strongly surface-active and formed stable monolayers at the air-water interface [see Adamson, (1976) for a review on methods for studying molecules absorbed to the air-water interface]. Surface pressure-area isotherms were analyzed according to the equation ?r(A - Ao) = kT/nDP, where n is the surface pressure at a given area, A (expressed in Az per amino acid residue), DP is the number of residues per peptide, k is Boltzmann’s constant, and T is temperature in degrees Kelvin. Linear regression gives Ao, the limiting cross-sectional molecular area, and n , the degree of aggregation of the peptide at the air-water interface, which should be close to unity for an ideal surface gas. Peptide 2 appeared to form monomers or dimers at the air-water interface, and its molecular cross-sectional area (16 A2/amino acid residue) was consistent with a helical conformation. Compressed monolayers were transferred to solid, planar surfaces and examined by CD spectroscopy and attenuated total

74

WILLIAM F. DEGRADO

reflectance infrared spectroscopy. Both techniques indicated that the peptides were in predominantly a-helical conformations in the transferred monolayers. At high peptide concentration, peptide 2 also formed helical tetramers in aqueous salt-containing solutions. Thus, an extrinsic hydrophobic surface (air) or the hydrophobic surface of a neighboring, selfassociated helix or helices could serve to stabilize helix formation. The 7-residue peptide 3, with a hydrophobic period of 2.0, was also highly surface active and appeared to form p sheets at the air-water interface. Analysis of surface pressure-area isotherms for this peptide indicated a high degree of self-association (n = 30), a result suggesting that it might be forming extended, intermolecularly hydrogen-bonded p sheets. The observed cross-sectional surface areas (20 &residue) were consistent with this interpretation, and CD and IR spectroscopy of transferred monolayers of this peptide together indicated that the peptides were in an antiparallel p conformation. Peptide 3 also aggregated in aqueous salt-containing solution to form p sheets. In contrast, the 7-residue peptide 1, with a hydrophobic repeat of 3.5, showed very low surface activity. CD data showed that it failed to adopt an ordered configuration in aqueous solution under a variety of conditions. The above data illustrate the crucial role that hydrophobic periodicity plays in determining secondary structure formation. These data also illustrate the effect of chain length on secondary structure formation. Helix formation required a 14-residue segment, whereas p sheet formation was achieved with only 7 residues. This result is in agreement with studies of amino acid homopolymers of defined lengths. Under forcing conditions (low dielectric solvents that strongly favor secondary structure formation), the critical chain lengths for a helix and p sheet formation are approximately 8-13 residues and 4-6 residues, respectively (Narita et al., 1984; Mutter, 1985). Thus, chain length is another extremely important consideration in the design of secondary structures. The systematic studies of peptides with defined hydrophobic periodicities described above are also consistent with earlier results obtained from studies of amino acid copolymers of high molecular weight ( lo00 0.4 Lys Leu Leu Lys Leu Gly 0.2 Lys Leu Leu Lys Leu Gly

The data are taken from Cox et al. (1985) (peptides 1-3) and from DeGrado et al. (1985) (peptides

4, 5).

FMOC, 0-N-Fluorenylmethyloxycarbonyl.

CBP2 (Table 11), corresponded to peptides 1 and 2 of Section III,A,3; and the third peptide, CBP3, was a negatively charged analog of CBP2. It was hypothesized that if helix formation were indeed important for high affinity binding ( K D < 100 a), then CBPl, which was too short to form a very stable a helix, should bind rather weakly. On the other hand, CBP2 readily formed helices in the presence of apolar-water interfaces, so it should bind to calmodulin much more tightly than CBPl did. CBP3 was designed to test the electrostatic requirements for binding; it was expected to have conformational properties very similar to those of CBP2. In accordance with these predictions, CBPl and CBP2 were found to bind to calmodulin with approximately 0.2 pM and 3 nM dissociation constants, respectively. CBP3 failed to bind calmodulin at all, even at micromolar concentration, a result that demonstrated that electrostatic interactions must play an important role in recognition. However, polylysine binds very poorly to calmodulin, an observation indicating that electrostatic interactions alone are not responsible for binding. Taken together, these data strongly suggest that a basic, amphiphilic a helix is an important structural feature for the binding of many peptides to calmodulin. The synthetic modeling approach described above is largely conceptual in nature and is based entirely on a guiding structural hypothesis concerning the peptide alone. In this approach one begins with the simplifying assumption that the receptor protein is entirely indifferent to the fine details of the topography of the helical surface of the peptide and that only the overall distribution of charge, hydrophobicity, and amphiphilicity is important. Clearly, this is a gross approximation; that this approach is so successful is somewhat surprising and requires an explanation. In considering the interaction of calmodulin with CBP2, it is important to consider the flexibility of the side chains on the surface of the peptide and protein.

DESIGN OF PEFTIDES AND PROTEINS

89

The main chain atoms also possess a certain degree of flexibility. Thus, the peptide and protein can adjust their surface topographies to maximize their mutual complementarity. However, there are limits to the degree to which peptides can vary their conformations, and it seems unlikely that a regularly repeating structure such as that of CBP2 could assume a conformation that is perfectly complementary to a surface as irregular as a protein-water interface. Therefore, the sequence of CBP2 is almost certainly not optimally designed for affinity or specificity. In addition to not having an optimal fit with calmodulin, it is probably too amphiphilic in its design. For instance, CBP2 also binds to and disrupts a variety of membranes (W. F. DeGrado, unpublished results). In contrast, the target enzymes of calmodulin tend to be highly specific in their interactions with this regulatory protein. The design of peptides with improved affinities and specificities for calmodulin requires a consideration of the structure of calmodulin as well as that of the peptide. Recently, the crystal structure of calmodulin has been solved, using data that extends to 3.0 8, (Babu et al., 1985). At this level of resolution, the positions of the calcium-binding sites and secondary structures could easily be discerned, but the exact positions of side chains will remain ambiguous until the structure has been refined to higher resolution. Prior to the publication of the crystallographic structure, two related structures were predicted for calmodulin on the basis of its sequence homology with two proteins of known three-dimensional structures, intestinal calcium-binding protein and parvalbumin (O’Neil and DeGrado, 1985). Because of the high degree of sequence homology between parvalbumin or intestinal calcium-binding protein and calmodulin, the models were found to be qualitatively similar to the crystal structure of calmodulin. An examination of the surface of these models resulted in the identification of a likely site for binding amphiphilic peptides. This site was primarily composed of residues near the C terminus of calmodulin and contained a hydrophobic patch that was flanked by several acidic residues. With computer graphics it was possible to “dock” CBP2 onto this site in such a way that the apolar side of the helix contacted the hydrophobic surface. Further analysis suggested that the affinity of CBP2 might be enhanced by adding a lysine residue near its N terminus on the otherwise uninterrupted hydrophobic side of the helix. Also, it appeared that a tryptophanyl residue might be well accommodated near the N terminus of the sequence. These and other considerations (DeGrado e? al., 1985) led to the design of CBP4 and CBP5 (Table 11). Both peptides had a tryptophan in the 3-position of their sequences, and they varied only in the sequence of their N terminal dipeptides. CBP4 had the N terminal sequence Lys-Leu, which placed the lysine on the

90

WILLIAM F. DEGRADO

hydrophilic side of the helix, while peptide 6 had the N-terminal sequence Leu-Lys, which placed the lysine on the hydrophobic side of the helix. CBP4 was found to bind calmodulin with a 0.4-nM dissociation constant, a finding indicating that adding a tryptophan to CBP2 and increasing its chain length indeed improved its affinity for calmodulin. CBP5 bound even tighter, with a dissociation constant of 0.2 nM. Thus, interrupting the perfect amphiphilicity of CBP4 led to an analog that bound more tightly to calmodulin. These data demonstrate how second-order conformational considerations can lead to the design of peptides with higher specificity and affinity for their receptors. While these studies with model peptides were in progress, other groups were involved in elucidating the calmodulin-binding sites of two related target enzymes, smooth muscle and skeletal muscle myosin light-chain kinase (MLCK) (Lucas et al., 1986; Blumenthal et al., 1985; Klevit et al., 1985; Edelman et al., 1985). Analysis of CNBr digests for each of these enzymes gave rise to a single peptide for each protein that could bind calmodulin with extremely high affinity. The peptides derived from the smooth muscle (Lucas et al., 1986) and skeletal muscle enzymes (Blumenthal et al., 1985) were 20 and 27 residues in length, respectively, and each lies near the C terminus of these enzymes. CD studies showed that when calmodulin and either of these two peptides were mixed in a 1 : 1 molar ratio, the helicity of the complexes was greater than that for the sum of the two individual components (Lucas et al., 1986; Klevit et al., 1985), an outcome that was consistent with the hypothesis that these peptides form helices when they bind to calmodulin. Studies involving limited proteolysis of native skeletal muscle MLCK have provided evidence that strongly suggests that the peptide isolated from the CNBr digest is involved in binding calmodulin in the native enzyme (Edelman et al., 1985). Thus, these peptides appear to make up much if not all of the calmodulin-binding domains of the enzymes. Each of the natural peptides derived from MLCK contains a 16-residue segment that conforms to the structural paradigm derived from the studies of model amphiphilic peptides. When aligned as in Fig. 8, hydrophobic and basic residues tend to occupy invariant positions in their sequences. As illustrated in the helical net diagram (Fig. 9),all three peptides display a potential apolar ridge. This ridge is formed by hydrophobic residues that occur at positions i - 3, i , i + 3 over a 10-residue stretch of the chain. This repeat has been found in many helix-helix packings (Chothia, 1984). Preliminary data from a number of laboratories suggests that the calmodulin-binding domains of several other enzymes might contain basic, amphiphilic a helices. The catalytic subunit ( y subunit) of phosphorylase b kinase is known to bind extremely tightly to calmodulin (the 6 subunit;

DESIGN OF PEEIDES AND PROTEINS

91

Rel. Skeletal Muscle MLCK 342361

T a b &.. 1986

Lys-Argdrg-Trp~ys-Lys-AsnPhe-lle-Ab -ValSer-Ab-Ala-Asn- Arg-Phe-Lys-Lys -Ile

Smwlh Muscle MLCK peplide

Lucas&..

Arg .Arg- Lys-Trp-Gln-Lys-Thr .Gly-H-Ala-Val-Arg-Ala- Ile- Gty-Arg-LeuSer SerSer

8-subnil 01 p II Cam Kinaseb97-316)

Bennen 8 Kennedy, 1987

Arg.Arg-Lys.Leu-Lys-Gly.Ab- Ila-LeuThr-Thr-Me1 Leu-Ala-Thr-Arg- Asn-Phe Ser Val

Unknown Calrrcdukn

Skeb I Hahn. 1987

Arg Arg-Lys-Leu-Lys-Ala-Ab-VaCLys-Ala-Val-Val-Ala-Ser Ser-Arg.Leu.Gly.Ser- Ah

1986

KiMW

CBP5

DeGrado &.,

1985

LeuLysTrp-Lys-Lys-LeuLeuLysLeuLeuLys Lys Leu-Leu Lys Leu Gty t

t

t

Hb

t

t

H b t HbHb

Hb

t

Hb

FIG.8. Amino acid sequences of the model calmodulin-bindingpeptide, CBPS, and of the proposed calmodulin-binding domains of four calmodulin-dependent kinases. The + symbols on the bottom row indicate positions where a positively charged residue occurs in at least half of the aligned sequences, the Hb symbol refers to positions generally occupied by hydrophobic residues.

Kee and Graves, 1986). A computer-assisted examination of the sequence of the y subunit revealed there is a very basic 21-residue segment with a high helical hydrophobic moment (Eisenberg et al., 1982a,b) near the C terminus of this protein (DeGrado et al., 1987a). A synthetic replicate of this segment binds calmodulin with approximately a 10-nM dissociation constant, a result indicating that this region might constitute a portion of the calmodulin-binding domain of the y subunit.

FIG.9. Helical net diagram (Crick, 1953) of a model calmodulin-bindingpeptide and the putative calmodulin-binding domains of two forms of myosin light-chain kinase (MLCK). The sequences are drawn together on a single helical net and are taken from (clockwise from left) the model peptide described by DeGrado et al. (1985), skeletal muscle MLCK peptide 342-359 (Edelman et al., 19851, and the N-terminal 18 residues of a peptide derived from smooth muscle MLCK (Lucas er al., 1986). The amino acids in the sequences are given in single letter codes. Positions that are hydrophobic in all three sequences are indicated by shading.

92

WILLIAM F. DEGRADO

The sequence of the p subunit of brain type I1 Ca*+/calmodulin-dependent protein kinase has been recently determined by molecular cloning (Fig. 8) (Bennett and Kennedy, 1987). The sequence of this enzyme begins with a region of approximately 300 residues that has a high degree of homology to a variety of protein kinases, including kinases that are not regulated by calmodulin. This region of the protein contains the active site residues and hence represents the catalytic portion of the protein. Immediately downstream (toward the C terminus) to this sequence is a short segment that is capable of forming a basic, amphiphilic a helix and is proposed to form this enzyme’s calmodulin-binding domain (Bennett and Kennedy, 1987). The calmodulin-binding domain of skeletal muscle MLCK is also downstream to the putative catalytic portion of the enzyme. Very recently, the partial sequence of another calmodulin-binding brain protein was determined by a novel genetic method (Sikela and Hahn, 1987). Mouse brain cDNA was cloned into a vector that expresses the inserted DNA as a fusion protein, and the resulting clones were selected for their ability to produce calmodulin-binding proteins. The partial sequence of a protein that shows some homology to the p subunit of brain type I1 Ca2+/calmodulin-dependentprotein kinase was obtained by this procedure. Restriction enzyme mapping experiments indicated that the calmodulin-binding segment of this protein was contained within a 39residue segment; a 20-residue segment within this region had a high potential to form a basic, amphiphilic a helix, and it had a high degree of homology with the proposed calmodulin-binding domain of the p subunit of brain type I1 Ca2+/calmodulin-dependentprotein kinase (Fig. 8). A major limitation of the above studies of calmodulin-peptide interactions was that spectral evidence to support helix formation was limited to predictive algorithms and measurements of the difference in the circular dichroism of peptides and calmodulin in free solution and the CD in 1 : 1 complexes. Interpretation of such experiments was severely limited by the fact that calmodulin probably undergoes conformational changes upon binding peptides (Klevit et al., 1985). One elegant NMR study has been reported on a complex of melittin and bacterial-derived perdeuterated calmodulin; the results were consistent with helix formation by the peptide in the complex (Seeholzer et al., 1986). To obtain additional evidence in support of helix formation, O’Neil et al. (1987) adopted a novel approach that may be applicable to a number of other systems involving peptide-protein interactions. In a set of analogs of CBPS, the tryptophan was systematically moved from its original position at residue 3 to every other possible position in the peptide chain. The fluorescence properties of tryptophan are markedly dependent on environment and can provide information concerning the solvent accessibil-

DESIGN OF PEPTIDES AND PROTEINS

93

ity, rigidity, and polarity of the immediate vicinity of the indole moiety. As tryptophan residues become increasingly less solvent-accessible in the interiors of proteins, they tend to have lower Stern-Volmer constants for acrylamide quenching of the tryptophan fluorescence, higher anisotropies (indicative of a larger degree of immobilization), and emission maxima that are increasingly shifted more toward the blue relative to those of tryptophan in water (Lakowicz, 1983). CBP5, which has a tryptophan at position 3 on the hydrophobic side of the helix, was found to have a low accessibility to acrylamide, a high anisotropy , and a highly blue-shifted emission maximum (DeGrado et al., 1985). Figure 10 illustrates the results that might be expected when tryptophan is moved to other positions, assuming that the peptides bind to calmodulin in a helical conformation with their apolar residues contacting the hydrophobic surface of calmodulin. If the fluorescence properties of the entire set of tryptophan-containing peptides are considered as a function of the position of the tryptophan in the sequence, then the fluorescence properties should be periodic and have a repeat period that matches that of an a helix. This was indeed found to be the case (O’Neil et al., 1987). The peptides bound to calmodulin with dissociation constants ranging from 0.2 nit4 (for the Trp-3 peptide) to 3.0 nit4 (for the Trp-10, Trp-14, and Trp15 peptides). Therefore, the perturbational effect due to the introduction of the tryptophan was small when compared with the total binding energy, which ranged from approximately - 12 to - 13 kcal/mol. Significantly,the affinity was the highest when tryptophan was placed in position 3, as in the original design of CBPS. The Stern-Volmer quenching constants, the anisotropies, and the emission maxima of the full set of peptides were periodic with respect to the position of the tryptophan in the sequence. Fourier analysis of the data gave a period of approximately 3.3, in good agreement with the value of 3.6 expected for an a helix. Figure 1 1 illustrates a normalized average of each of these parameters as a function of the tryptophan position. The dashed curve is a theoretical curve calculated with the parameters obtained from the best fit of a sine wave (3.3 residue period) to the data. The deviation of the data from the dashed curve is greater than can be explained by experimental error and arises from the fact that the peptides do not bind to a planar interface that varies smoothly in its dielectric, rigidity, and solvent accessibility. This property might also explain the deviation of the observed repeat of 3.3 residues from the ideal value of 3.6 residues for an a helix. Thus, the deviations of the experimental points from the smooth curve provide information concerning the fine structure of the peptide-binding site. For instance, data points on the upper portion of the curve in Fig. 1 1 correspond to residues on the apolar side of the

94

WILLIAM F. DEGRADO

FIG.10. Highly schematic representation of the orientation of several tryptophan-containing peptides with respect to calmodulin. (A) With tryptophan in position 1, the indole is located on the hydrophilic side of the helix and is exposed to solvent. Peptides with tryptophan on this face of the helix should exhibit emission maxima near that of indole in water (-350 nm), a small anisotropy, and a high accessibility for acrylamide quenching. (B) In position 2, the tryptophan is partially exposed at the interface between the peptide and calmodulin. Peptides with a tryptophan in this location should have fluorescence properties that are intermediate between example A and C. (C) The tryptophan is on the hydrophobic side of the helix and is almost entirely buried. The emission maximum should be strongly blue-shifted, the anisotropy should be large, and the accessibility to acrylamide quenching low. Taken from O’Neil ef al. (1987).

helix, which presumably directly contact the surface of calmodulin. The affinities of the peptides for calmodulin relative to a peptide lacking tryptophan are given above each of the local maxima. There is a correspondence between the height of each of the local maxima and the tightness of the interaction for the corresponding derivative. This correspondence

DESIGN OF PEPTIDES AND PROTEINS

0.3' 0

I

I

I

2

4

6

I

I

I

8 10 12 TRP POSITION

95

I

14

16

FIG.11. Variation of the fluorescence properties of a set of tryptophan-containing peptides as a function of the position of the tryptophan in their sequence. The parameterfAvE describes the degree of rigidity and hydrophobicity of the tryptophan's environment; it is based on emission maximum, anisotropy, and accessibility to acrylamide. When the values for each of these parameters were similar to those expected for indole in water, a value near 0 was assigned tof, whereas values up to 1.O were assigned as the fluorescence parameters more closely resembled those observed in very rigid and apolar environments such as the interior of a protein or ethylene glycol at -60°C (Lakowicz, 1983). The values offcalcu~ . dotted curve was generated lated for each parameter were then averaged to give f A ~ The by fitting a sine wave to the data (period = 3.3 residues). Taken from O'Neil et al. (1987).

indicates that the tryptophan can increase the affinity of the peptide for calmodulin only if there is a hydrophobic pocket on the surface of calmodulin that can accommodate the indole moiety of the tryptophan side chain. Furthermore, the data in Fig. 11 suggest several ways in which the sequence of peptide 6 could be changed to further improve the affinity and specificity of the peptide for calmodulin. Residue 12 is a lysine in peptide 6, and yet it appears to be experiencing a hydrophobic environment when bound to calmodulin (Fig. 11). Also, residue 14 is a leucine in peptide 6, and yet it is experiencing a very hydrophilic environment. In the aligned sequences of the calmodulin-bindingdomains shown in Fig. 8, the positions homologous to residue 12 and 14 of peptide 6 are generally occupied by hydrophobic and hydrophilic amino acids, respectively. These differences between the natural and designed peptides are likely to have two consequences: the natural peptides are less amphiphilic than the designed peptide and therefore the natural peptides are less likely to bind nondiscriminately to hydrophobic surfaces, and the surface topographies of the natural peptides are likely to be more complementary to the surface of calmodulin. The aforementioned results demonstrate the synergism between studying natural and model peptides. The use of designed model peptides al-

96

WILLIAM F. DEGRADO

lowed the identification of a basic, amphiphilic a helix as a structural feature that was important for binding. Subsequent spectroscopic studies as well as studies with natural peptides increased the understanding of the peptide-protein interface. This knowledge allowed the simple amphiphilic a-helical model to be refined to include the contribution of residues that decrease the overall amphiphilicity of the structure but increase the specificity of the peptides for calmodulin. Within several years, the structures of several calmodulin-peptide complexes should be known in a reasonable degree of detail. In the near future, the use of calmodulin-binding peptides bearing the photoaffinity amino acid p-benzoylphenylalanine should aid in the determination of those residues on calmodulin that are involved in peptide binding (Kauer et al., 1986). Data from such experiments can be evaluated in light of the known crystal structure of calmodulin to provide a reasonable estimation of the conformational features of the complex. Also, crystallographic (Tanaka et al., 1985) and NMR studies on the interaction of peptides with calmodulin (Klevit et al., 1985; Seeholzer et al., 1986) have been initiated and should provide much higher resolution structures. The availability of structural details for peptide-calmodulin complexes should make them attractive model systems for studying the interaction of amphiphilic peptides with their receptors. 3. Peptide Hormones The design of model peptides has recently been applied to elucidating the structural features involved in peptide hormone-receptor interactions. A first step in designing a model for a medium-sized peptide hormone is to assign a structural and functional role to each portion of a peptide sequence. This process is aided considerably if structure-activity relationships have been established from synthetic and phylogenetic studies. As described previously (Kaiser and KCzdy, 1984; Taylor and Kaiser, 1986; Schwyzer, 1982), the amino acid sequences of many medium-sized peptide hormones can be divided into two or more distinct domains on the basis of the tolerance of these domains to changes in their sequences. Often, there is a single domain (referred to here as the specificity element) that has a highly conserved sequence. Synthetic modifications in this domain lead to hormones with decreased potency, and, in some cases, peptides spanning just the specificity element are active as agonists or antagonists. The specificity elements appear to be directly and intimately involved in interactions with the receptor. The remaining portion or portions of the structure show more sequence variation and will be referred to here as auxiliary sequences. They appear to play a more structural role and serve to decrease the susceptibility of the hormone to enzymatic

97

DESIGN OF PEPTIDES AND PROTEINS

attack, to improve its binding to a receptor, and to modulate the affinity of the hormone for its receptor or receptors. The next step in the design of a model peptide hormone involves replacing the auxiliary sequences with structurally similar surrogate sequences. In general, it is safest to change one structural region at a time, but the changes within that region should be radical enough to allow unambiguous conclusions to be drawn from the experiments. Finally, the properties of the analogs are evaluated, new hypotheses are formulated, and new model peptides designed. The modeling process proceeds in an incremental manner until a peptide with the desired properties has been produced. The encompassing contributions of Kaiser and co-workers in the design of analogs of p-endorphin serve to illustrate this approach. Human p-endorphin is a 31-residue opioid peptide that is biosynthetically derived from a larger precursor, proopiomelanocortin (Hollt, 1983). It has been postulated (Taylor and Kaiser, 1986) to be composed of three modular units (Fig. 12). The N-terminal pentapeptide forms the specificity element and is identical to Met-enkephalin; the C-terminal 18 residues are postulated to form an a helix; and the 8-residue segment between these two modules is proposed to serve as a flexible linking sequence. The enkephalin portion is thought to be directly involved in binding to opiate receptors, while the two auxiliary sequences are thought to be responsible for modulating the receptor subtype specificity and for stabilizing the hormone against proteoly sis. Taylor, Kaiser, and their co-workers (Taylor and Kaiser, 1986) prepared a series of model peptides that were designed to test the role of each of the postulated modules in p-endorphin. Figure 13 illustrates a-helical net diagrams of the proposed helical auxiliary sequence of p-endorphin. The hydrophobic residues in this structure cover half the surface of the helix, twisting around the structure in a clockwise manner. [It has also been noted that if this sequence were to form a 7r helix rather than an a

*& Specficity Element

Flexible Linking Sequence

5

1

10

H2N-Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-

a-Helical Auxiliary 1

f

15

20

25

30

Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-lle-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu

FIG.12. Amino acid sequence and structural assignments for human /3-endorphin (Taylor and Kaiser, 1986).

WILLIAM F. DEGRADO

98

P-Endorphin

P-2 13-31

P-1 13-31

13-31

P-3 13-31

P-4 13-31

FIG. 13. Helical net diagrams (Crick, 1953) of the proposed helical region of human p-endorphin and analogs thereof (Taylor and Kaiser, 1986).

helix, the hydrophobic residues would lie along one face of the helix and generate a substantially more amphiphilic structure (Taylor and Kaiser, 1986). At present, there are no direct, experimental data to support this hypothesis.] The first two model peptides were designed to maximize the amphiphilicity of the helical auxiliary when in an a-helical conformation. In the first model, p-1, residues 20-31 were replaced with a model sequence containing just three different types of residues: leucine, lysine, and glutamine

DESIGN OF PEF'TIDES AND PROTEINS

99

(Taylor et al., 1981). These residues were arranged so that the hydrophobic patch of the proposed a helix would lie parallel to the length of the ahelical axis throughout the entire length of the structure rather than spiraling around the helix (Fig. 13). In p-2, the sequence of the entire helical auxiliary was replaced with a model sequence (Fig. 13), and the flexible linking sequence was replaced by a model sequence composed of repeating Ser-Gly dipeptidyl units (Taylor et al., 1982). In p-3, the helical auxiliary was replaced with a model peptide in which the hydrophobic residues spiral in a clockwise manner around the helix (Fig. 13) as in the native sequence (Taylor et al., 1983). Finally, 0-4 has the same amino acid composition as p-2, but the residues in the helical auxiliary portion of the sequence have been arranged so that the helix is not amphiphilic and does not contain an extended hydrophobic patch (Fig. 13) (Blanc et al., 1983). The pharmacological and physical properties of p-1 to p-4 have been characterized in considerable detail (Taylor and Kaiser, 1986). The evaluation of the pharmacological data is complicated by the fact that each of the peptides contains the pentapeptide Met-enkephalin as their specificity element. To a large extent, this pentapeptide sequence controls the pharmacological behavior of the peptides, and many of the properties of Metenkephalin and p-endorphin are similar. However, there are also several important differences that distinguish Met-enkephalin from p-endorphin (Taylor and Kaiser, 1986). Met-enkephalin is considerably more susceptible to enzymatic degradation by enkephalinases and aminopeptidases. This finding might account for the fact that Met-enkephalin is much less active than p-endorphin as an analgesic when administered intracerebrally. In uitro, p-endorphin binds to p receptors about as tightly as it does to 6 receptors, and it has a very high potency in the rat vas deferens (RVD)assay, whereas Met-enkephalin binds preferentially to 6 receptors and has a much lower activity in the R V D assay. The following discussion of the properties of the model peptides will focus on those properties that differentiate p-endorphin from Met-enkephalin. Each of the model peptides including the nonamphiphilic analog p-4, bound the p receptor with high affinity (Taylor and Kaiser, 1986). The considerable potency of p-4 is supportive of other work that suggests that amphiphilic a helix formation is not obligatory for potent p receptor binding (Blake et al., 1981). In contrast, /3-4 was inactive in the R V D assay, while the other three model peptides were potent agonists (Taylor and Kaiser, 1986). Interestingly, the model that had the highest homology to p-endorphin, p-1, was most potent in this assay, with a potency approximately the same as p-endorphin. The peptides p-1,p-2, and p-3 were more stable than p-endorphin toward enzymatic attack in the rat brain homogenate and smooth muscle assays, whereas the nonamphiphilic pep-

100

WILLIAM F. DEGRADO

tide p-4 was rapidly degraded in these assays. Thus, an amphiphilic helical secondary structure appears to be important for enzymatic stability and in uitro activity in the RVD assay. Although peptides p-1 and p-2 differ from p-3 in the disposition of their hydrophobic residues along the putative helix, they have very similar properties in the above assays. This observation raises the question as to whether there is any functional significance to the fact that the hydrophobic patch runs diagonally along the helical net diagram in p-endorphin (Fig. 13). A comparison of the pharmacokinetic and in uiuo properties of peptides p-1 to p-4 suggests that the shape of the hydrophobic domain is indeed important for fine-tuning of the properties of this hormone (Taylor and Kaiser, 1986). For instance, of these four analogs, only p-3 has analgesic activity when administered by intracerebralventricular injection with an activity nearly half that of human p-endorphin in this assay. It is possible that amphiphilicity is indeed important for in uiuo activity, but peptides p-1 and p-2 are too amphiphilic and hence bind indiscriminately to cell surfaces. This nonspecific absorption might retard or prevent diffusion to the receptors from the site of injection. Even the active analog, p-3, required approximately five to ten times longer than human p-endorphin to produce its maximal effect, a finding suggesting that diffusion of this analog to the site of action was slower than for the native hormone. These differences in the pharmacokinetic effects were also seen in the RVD assay where the very amphiphilic peptides p-1 and p-2 required much longer than p-3 or p-endorphin to achieve their maximal effects. Recently, it has been shown that the auxiliary sequences in p-endorphin can be replaced by segments composed entirely of amino acids other than the 20 commonly occurring residues. In one analog of p-endorphin, the postulated a-helical segment was replaced by a segment composed entirely of D-amino acids (Blanc and Kaiser, 1984). This peptide was capable of forming left-handed a helices in helix-inducing solvents such as 50% aqueous trifluoroethanol, but it had biological and physical properties very similar to those of peptide p-3. In a second analog, the flexible linking sequence that spans residues 6-12 was replaced with four units of the y-amino acid, y-amino, y-hydroxymethylbutyric acid (Rajashekhar and Kaiser, 1986). This peptide also had physical and biological properties that were similar to those of p-3, a result indicating that residues 6- 12 of p-endorphin served as a flexible linking sequence joining the helical auxiliary with the specificity element. The aforementioned synthetic modeling approach, which was so successfully applied to studying the structural properties of p-endorphin, appears to be general and has been applied to the study of several other peptide hormones and hormone-releasing factors including glucagon

DESIGN OF PEPTIDES AND PROTEINS

101

(Musso et al., 1983, 1984), vasoactive intestinal peptide (G. Musso, personal communication),growth hormone-releasingfactor (Tou et al., 1986; Velicelebi et al., 1986), and calcitonin (Moe et al., 1983; Moe and Kaiser, 1985). In each of these cases, highly active analogs were produced by replacing a portion of the peptide sequence with a model amphiphilic (Yhelical sequence that had minimal homology to the parent hormone. IV. PROTEIN DESIGN A . Why Design Proteins?

The final topic of this review will be the de nouo design of proteins. While there exist ample precedents for the design of small peptides, the same does not hold for the design of proteins. There are two fundamental reasons for this: Until very recently the synthetic challenge associated with assembling a polypeptide chain the size of a protein was considerable, and it is only with the advent of recombinant DNA that this has become routine. In addition, our understanding of the mechanisms by which proteins adopt their tertiary structures is at a rather primitive state. Therefore, the design of functional proteins must begin with a consideration of the protein folding problem. In contrast to peptides that assume conformations complementary to those of their receptors, proteins are large enough to fold back upon themselves and adopt stable conformations in aqueous solution. When studied on an individual basis, these structures appear to be almost hopelessly complex, and indeed it is not currently possible to predict de nouo the structures of natural proteins from their amino acid sequences. However, the systematic study of the three-dimensional structures of large numbers of proteins has led to the recognition of some structural features that are common to a variety of proteins and has provided fundamental insights into the protein folding process (for reviews, see Levitt and Chothia, 1976; Richards, 1977; Richardson, 1981; Salemme, 1983; Chothia, 1984). Regularly repeating secondary structures make up large portions of proteins, and there is a high degree of order in the packing of individual elements of secondary structures. Elements of secondary structure tend to form layers that pack against neighboring layers of secondary structures in a nonrandom manner (Richardson, 1981). Thus, there are preferred angles for helix-helix, helix-sheet, and sheet-sheet packings (Chothia, 1984). The preferred crossover angles occur because there are a limited number of ways to closely pack side chains at the interface between secondary structures, as can be shown by construction of simple three-dimensional models of proteins (Salemme, 1983; Ohlen-

102

WILLIAM F. DEGRADO

dorf et al., 1987; Chothia, 1984) or by energy calculations (Salemme, 1983; Chou et al., 1984, 1985, 1986). Furthermore, there appear to be a finite number of higher level structural folding patterns found in the domains of proteins (Levitt and Chothia, 1976; Richardson, 1981). Thus, our understanding of the protein folding problem, although far from complete, has advanced far enough to provide solhe fundamental rules and principles from which to construct proteins. At present, the primary benefit to be derived from designing proteins is that it critically tests and advances our understanding of the principles governing protein stability and folding. Physical organic chemists have long relied on the construction of model compounds as an important tool for elucidating reaction mechanisms, similarly peptide chemists have found model peptides useful for addressing conformational questions. The construction of model proteins should likewise further our understanding of the kinetics, dynamics, and thermodynamics associated with protein folding. With this focus in mind, the following section will review some preliminary attempts to design such model proteins. A second potential benefit that might be derived from the construction of artificial proteins is that it may eventually be possible to design proteins with novel catalytic, pharmaceutical, or fibrous properties. Along these lines there have already been occasional reports of having achieved the design of peptides or proteins with specific binding and/or catalytic properties. These include a DDT-binding peptide (Moser et al., 1983,1985). an enkephalin-binding peptide (Kullmann, 1984), and a model for ribonuclease (Moser et al., 1983; Gutte et al., 1979; Jaenicke et al., 1980). The intended folding patterns for these peptides and proteins were not strictly modeled after entire domains of known proteins, but do incorporate some supersecondary structures known to occur in proteins. To date, the structural properties of these designed peptides have not been fully characterized. B. a-Helical Proteins The preceding sections reviewed some of the considerable progress that has recently been made in the design of peptides that form a helices in amphiphilic environments. A logical extension of this work is the design of proteins that are formed by the coalescence of two or more a helices. The study of amphiphilic peptides suggested that this should indeed be possible. For instance, melittin aggregates in solution to form ahelical tetramers, and crystallographic analysis of the tetramer showed that it had many of the attributes normally associated with folded proteins: Most of the apolar side chains were closely packed in the interior of the structure, and the helical crossing angles were typical of those found

DESIGN OF PEFTIDES AND PROTEINS

103

in folded proteins (Terwillinger and Eisenberg, 1982b). This finding raises the possibility of designing amphiphilic a-helical peptides that would selfassemble into helical proteins of predetermined geometries. 1. Coiled Coils

The simplest of a-helical proteins are the two-stranded coiled coils found in such fibrous proteins as the keratins and tropomyosin. In this structure, two a helices coil about one another with a left-handed superhelical twist (reviewed in Talbot and Hodges, 1982; Cohen and Parry, 1986). The left-handed superhelical twisting arises from the nonintegral repeat of the a helix, which makes it impossible to pack straight a helices over extended distances with a regular, repeating interaction pattern. One way in which a helices can favorably interact is referred to as “knobsinto-holes” packing (Crick, 1953) (Figs. 14A and 15); a side chain on one a helix packs between residues at positions i , i + 3, i + 4, and i + 7 of a neighboring helix. For two straight a helices, this interaction pattern can extend over several helical turns if the a helices are inclined with respect to one another by about 18” (Fig. 14A), although the helices will eventually diverge from one another. However, if the a helices wrap about one another with a left-handed sense, they remain at a constant distance from one another irrespective of their lengths, and their interaction pattern becomes integrally periodic, repeating every seven residues. This interaction pattern is also reflected in the amino acid sequences of proteins such as tropomyosin, which can be shown to be composed of a basic seven-residue sequence that is repeated 40 times without intermption (Hodges et al., 1972; McLachlan and Stewart, 1975). Hydrophobic residues almost invariably occupy the second and fifth positions of the heptad and are presumably directed toward the major axis of the superhelix, where they serve to stabilize the structure by hydrophobic effects (Fig. 15). The charged side chains are also nonrandomly distributed and are believed to form interhelical ion pairs that further stabilize the structure (Talbot and Hodges, 1982). Hodges and co-workers have designed a series of model peptides (Talbot and Hodges, 1982; Lau et al., 1984) containing the structural features envisioned to be important for stabilizingthe two-stranded coiled coil. To evaluate the role of chain length in stabilizing the coiled-coil conformation, a series of peptides were prepared containing from one to five copies of the model heptapeptide illustrated in Fig. 15 (Lau et al., 1984). CD spectroscopy indicated that, as the number of heptad units increased, the helicity and stability of the structures increased in concert; the peptides containing one or two heptad repeats showed no helical structure in aqueous solution, whereas the remaining peptides showed

I04

WILLIAM F. DEGRADO

FIG.14. Two types of helix-helix packing that give rise to nearly antiparallel crossover angles. The side-chain interaction pattern for a pair of a helices can be conveniently represented by drawing helical nets that describe the superposition of the two helices (Crick, 1953). The “knobs into holes” packing of Crick (1953) (A), and the “ridges into grooves” packing of Chothia et al. (1977, 1981) (B) give rise to similar interhelical packing angles, although the side chains are packed somewhat differently in the two models. Drawing by F. R. Salemme and P. Weber.

increasing amounts of helicity as the number of heptads was increased from three to five. Size exclusion chromatography and sedimentation equilibrium ultracentrifugation showed that the peptides with four or five heptameric repeats formed dimers in solution. Thermal denaturation studies indicated that these peptides were even more stable than carboxymethylated tropomyosin; the temperatures required to decrease the magnitude of their ellipticities by 30% of the value measured at 93°C were 62, 74, and 37”C, respectively. Presumably tropomyosin is thermally less stable than the model peptides because it contains a number of hydrophilic groups or alanines at the positions occupied by leucine in the model peptides (Lau et al., 1984). The role of electrostatic interactions in stabilizing the two-stranded

DESIGN OF PEPTIDES AND PROTEINS

105

POTENTIAL ELECTROSTATIC INTERACTIONS

HYDROPHOBIC CORE LlU

LlU

u POTENTIAL ELECTROSTATIC INTERACTIONS

FIG.15. A single heptad repeat of an idealized model for coiled-coil proteins (Talbot and Hodges, 1982; Hodges ef al., 1981). The primary driving force for formation of the structure arises from the interdigitation of the apolar leucyl side chains. In addition, electrostatic interactions between the oppositely charged residues on neighboring helices may also contribute to the stability of the parallel form of the coiled coil (Hodges ef al., 1981).

coiled coil remains ambiguous. The a-helical dimer formed by the peptide containing five heptad units was found to have greater thermal stability at pH 2.5 than at pH 7.0, and the ionic strength dependence of the stability was the opposite of what would be predicted if interhelical salt bridges contributed largely to the stabilization of the structure (Lau et al., 1984). In the tropomyosin coiled coil, the a helices run parallel to one another and in register (Talbot and Hodges, 1982). Experiments with disulfide cross-linked model peptides suggested that the same orientation of the helices was maintained in the aforementioned model peptides (Hodges er al., 1981). A peptide of the sequence A-Bs-L~s(A = Lys-Cys-Ala-Gluformed a covalent diLeu-Glu-G1y ; B = Lys-Leu-Glu-Ala-Leu-Glu-Gly) mer upon air oxidation; the covalent dimer had properties that were very similar to those of the corresponding reduced peptide; their CD spectra were nearly identical, and the shapes of their thermal unfolding curves were also similar. However, the midpoint of the thermal unfolding curve was 63°C higher for the oxidized peptide, a result indicating that it formed a more stable structure. Presumably the additional stabilization arose from a decrease in the conformational entropy of the unfolded form of the oxidized peptide from that of the reduced peptide (Kauzmann, 1959).

2. Four-Helix Bundles The study of models of fibrous proteins has provided a number of principles that might be applied to the design of globular proteins. In

106

WILLIAM F. DEGRADO

particular, the use of simplified sequences and amino acids that strongly favor the formation of a given secondary structure should allow design of simple, highly stable proteins. However, the design of globular proteins is fundamentally a far more complex problem. While fibrous proteins such as coiled coils have highly symmetrical structures with relatively short periodicities, globular proteins are far less simple, showing less symmetry. One possible exception is the four-helix bundle class of proteins (Fig. 16), which includes myohemerythrin, apoferritin, tobacco mosaic virus coat protein, and cytochrome c’ (Weber and Salemme, 1980;Richardson, 1981). As pointed out by Weber and Salemme (1980;Ohlendorf et al., 1987),if the directionality of the helices is ignored, then the helices are related by a pseudo 4-fold rotational axis that runs down the center of the structure. As in two-stranded coiled coils, neighboring helices cross at approximately 18” angles in the four-helix bundle structures (Weber and Salemme, 1980). However, there is a fundamental difference between the arrangement of the a helices in the coiled coils and that of the helices in the four-helix bundle structures. In the coiled-coil structures, the helices remain at a constant distance from one another because of the strong lefthanded supercoiling, whereas in the four-helix bundle structures, the a helices diverge from a point of closest approach, an arrangement giving rise to a cavity at the base of the structures (Weber and Salemme, 1980; Ohlendorf et al., 1987). This cavity provides a binding site for prosthetic groups in many of the four-helix bundles. The high structural simplicity and functional diversity of the four-helix bundle makes it an attractive target for the study of protein folding as well as the eventual design of synthetic binding sites. Various models have been proposed to account for the high frequency of occurrence of the interhelical packing angle observed in four-helix bundle proteins (Chothia, 1984;Chothia et al., 1977,1981;Richmond and Richards, 1978; Weber and Salemme, 1980; Ohlendorf et al., 1987). Chothia and co-workers proposed that interhelical side-chain packing interactions are the dominant factor influencing the interhelical packing angles. These authors suggested that the packing could more closely be described as “ridges into grooves” rather than “knobs into holes” as observed in coiled-coils. In “ridges into grooves” packing, residues at positions i - 4, i, and i + 4 appear to form a ridge that packs against residues at positions j - 3, j, and j + 3 on a neighboring helix (Fig. 14B). This defines the packing angle between neighboring helices and allows tight packing of the apolar side chains, thereby driving the folding process. Ohlendorf et al. (1987;Weber and Salemme, 1980)have pointed out that this is probably an oversimplification, as a detailed comparison of the known four-helix bundle proteins showed unexpected variability in pack-

DESIGN OF PEPTIDES AND PROTEINS

Myohemerythrin

107

Cytochrome bSG2

FIG.16. Some examples of the four-helix bundle folding motif. Taken from Richardson (1981).

ing interactions. These authors failed to find one single packing interaction that optimally described all naturally occurring bundles, but nevertheless found that “independent of detailed pairwise interactions between helices relatively inclined at 18”, square arrays of four helices with this interaction angle tend naturally to produce structures where all pairwise helix interactions are symmetry related and those of individual residues

108

WILLIAM F. DEGRADO

pseudoequivalent” (Ohlendorf et al., 1987). Electrostatic interactions between helical macrodipoles (Sheridan et al., 1982) have also been proposed to increase the stability of four-helix bundles. Finally the loops between helices, although variable in length and sequence, nevertheless serve to break the helices and direct the formation of hairpin loops (Ohlendorf et al., 1987). Recently, an attempt at the de n o w design of a four-helix bundle was initiated (Eisenberg et al., 1986; Ho and DeGrado, 1987). In this designed protein, the pseudo 2,2,2 symmetry found in natural four-helix bundle proteins was idealized. This simplified the modeling process; with a highly symmetrical structure, it was only necessary to design a single a helix that upon application of a symmetry operator would provide a tightly packed, protein-like structure. In addition, it allowed the modeling process to be approached in an experimental, incremental manner, as described in Fig. 17. The first step of this approach involved the design of a peptide that could self-assemble into a helical tetramer composed of four identical monomers (Fig. 17a). The stability of the aggregate relative to that of the monomeric peptides could be assessed from the monomer-to-tetramer equilibrium constant. The availability of a quantitative parameter describing the stability of the aggregate allowed the evaluation of alternate designs. Subsequent to optimizing the a-helical sequence, designed loops were inserted between two identical helical sequences, and the stability of the resulting peptide evaluated (Fig. 17b). Finally, the entire four-helix bundle was constructed from four identical helical sequences and three identical loop sequences. The design of the helical sequences (Fig. 18) was first accomplished with physical models and later refined by computer graphics (Eisenberg et al., 1986; Ho and DeGrado, 1987). Models of four 16-residue helices were arranged so that their side chains could interact in a manner similar to that described by Chothia (1984; Chothia et al., 1977, 1981). Leucine side chains were placed at positions that projected into the interior of the structure, while glutamate and lysine side chains were placed at positions projecting toward the exterior of the structure. Glycine residues were placed at the N and C termini to help break the helix and to contribute to the formation of a hairpin loop. A helical net of the resulting structure, ala,is illustrated in Fig. 18. Subsequent refinement of the model for the tetramer (Ho and DeGrado, 1987) led to the design of a l b (Fig. 18); it appeared that the Leu side chain at position 11 was excessively exposed to solvent, while the Glu side chain at position 13 was partially buried. Consequently, a second peptide, a l b , was designed in which Leu was changed to Lys at position 11 and Glu was changed to Leu at position 13.

DESIGN OF PEPTIDES AND PROTEINS

109

Y

T

D

T C

FIG. 17. An incremental approach to the design of a four-helix bundle protein (Hoand DeGrado, 1987). (a) The sequence of a peptide is first optimized for forming a very stable tetramer of a helices. The stability of the tetramer can be assessed from the dissociation constant for the cooperative monomer-to-tetramer equilibrium. (b) Two optimized helical sequences are then connected in a head-to-tail manner by a single loop. The loop sequence can be optimized by evaluating a series of alternate designs. (c) Finally, the entire four-helix bundle structure can be constructed from four optimized helices and three optimized loops.

In addition, the Lys side chain at position 2 and the a-carboxylate at the C terminus were expected to interact unfavorably with the helical macrodipole (Shoemaker ef al., 1987a)and were therefore converted to a Glu and an a-carboxamide, respectively (Fig. 18). The assembly of the peptides into tetramers was assessed by size exclusion chromatography and by analyzing the concentration dependence of their CD spectra (Fig. 19). At low concentrations, the peptides were

110

WILLIAM F. DEGRADO

A

B 01

al0

Ac-Glu-Leu-Leu-Lys-Lys-Leu-Lsu-Glu-Glu-Leu-Lys-G1y-COOH Ac-Gly-Lys -Leu-Glu-Glu-Leu-Leu -Lys-Lys-Leu-Leu-Glu-Glu-Leu-Lys-Gl~COOH Ac-Gly-Glu-Leu-Glu-Glu-Leu-Leu-Lys-Lys-Leu-Lys-Glu-Leu-Leu-Lys-Gly.CONH~

FIG. 18. Helical net diagrams (A) and amino acid sequences (B) of a,,q a r and alb.In the helical nets the hydrophobic residues are circled, and potential salt bridges between the side chains of residues spaced at positions i 2 3 or residues at i 2 4 are indicated by solid and dashed lines, respectively.

monomeric and had low helical contents; in concentrated solutions, however, they formed a-helical aggregates. These conformational changes were reflected in the CD spectra of the peptides. The concentration dependence of the CD spectra were extremely well described by a highly cooperative monomer-to-tetramer equilibrium (Ho and DeGrado, 1987). Analysis of these curves provided the stabilities of the tetramer (RT In KD),and the approximate helical contents of the monomeric and tetrameric forms of the peptides. These parameters are listed in Table 111for a l a and (rib, as well as for a1 ,a 12-residue fragment of alaisolated as a byproduct that arose during the synthesis of a l a (Eisenberg et al., 1986). The helical contents of the monomeric forms of these peptides were dependent on their chain lengths. The 12-residue peptide was approximately 15% helical, whereas the 16-residue peptides were approximately 30% helical-a value that is unusually high when compared with that for other monomeric peptides in aqueous solution at room temperature (Section 111,A). Thus, ataand (Ylb were probably capable of forming helices at a rather low energetic cost. The helical content of the tetramers formed by all three peptides was approximately 70%, a value consistent with the proposed four-helix bundle structure. The stabilities of the tetramers depended both on chain

DESIGN OF PEPTIDES AND PROTEINS

111

1:

li

4

E

U \ N

E 12

0 U

T

P x

9

N

m"

-n = 3 --- n.6

6

12

9

FIG.19. Concentration dependence of the ellipticity at 222 nm of ala.The lines are computer-generated, theoretical curves describing various monomer-to-nmer equilibria. The top panel shows the monomer-tetramer equilibrium, the middle panel shows monomertrimer and monomer-hexamer equilbria, and the bottom panel shows monomer-dimer and monomer-octamer equilibria.

TABLE111 Amino Acid Sequences and Free Energies of Tetramerization or Dimerization of Synthetic Peprides" Peptide alb

aIAb aIBb

amc

Sequence

RTIIIK* (kcal/mol)

A C - G ~ U - L ~ U - L ~ U - L ~ S - L ~ S - L ~ U - L ~ U - G I U - G I U - L ~ U - L ~ S - G I-11.14 Y-C~H Ac-Gly-Lys-Leu-Glu-Glu-Leu-Leu-Lys-Lys-LLys-Gly-COOH - 19 Ac-Gly-Glu-Leu-Glu-Glu-Leu-Leu-Lys-Lys-Leu-Lys-GIu-Leu-Leu-Lys-GIy-CONHz-22 Ac-Gly-Glu-Leu-Glu-Glu-Leu-Leu-Lys-Lys-Leu-Lys-Glu-Leu-Leu-Lys-Gly-~~~g-~g - 13

Gly-Glu-Leu-Glu-Glu-Leu-Leu-Lys-Lys-Leu-Lys-Glu-Leu-Leu-Lys-Gly-CONHz

Data taken from H o and DeGrado (1987). Monomer-tetramer equilibria. Monomer-dimer equilibrium.

-em

x 10-4

(monomer) 0.55 0.87

0.94 -0.9

-em

x 10-4

(tetramer) 2.17 2.12

2.30 2.10

DESIGN OF PEF'TIDES AND PROTEINS

113

length and on sequence, increasing in the order at 46, 47, 49

Voss, T., 12, 18, 45, 46 Votano, J. R., 167, 186 Vyas, N. K.,167, 188

W Wada, A., 129, 189 Wadsii, I., 207, 208, 209, 210, 211, 232, 233

Wagner, C. W.. 41, 48 Wakamatsu, K.,84, 122 Walkinshaw, M. D., 126, 157, 162, 187 Wallqvist, A,, 164, 187 Walsh, K. A., 86, 90, 91, 118, 119, 123 Walter, R., 169, 189 Wang, A. H.-J., 131, 144, 167, 186, 189 Wang, S. Y., 12, 43 Warashina, A., 227, 234 Warfel, J., 21, 45

Warshel, A., 134, 189 Wartiovaara, J., 20, 34, 46, 49 Wattemon, D. M., 81, 90, 91, 121 Weast, R. C., 210, 234 Weber, P. C., 104, 106, 122, 124 Weber, S., 18, 27, 47, 49 Weber, T., 58, 123 Weinhold, F., 142, 188 Weiss, B., 86, 122 Wemmer, D. E.,90, 92, 96, 121 Wen, W. Y.. 217, 232 Werness, P. G . , 33, 48 Weston, Ch., 226, 232 Wetlaufer, D. E., 195, 234 Waver, U. M.,2, 10, 41, 42, 44, 46, 49

Whelan, J., 2, 48 Wicha, M. S., 41, 47 Wick, C., 18, 27, 49 Wider, G., 77, 118 Wiedemann, H., 10, 11, 12, 16, 17, 18, 19, 29, 30. 31, 38, 45, 46, 47, 49 Wieslander, J., 36, 43, 49 Wight, T. N., 10, 31, 35, 47 Wilcock, R.J., 215, 234 Wilcox, W., 119 Wilczek, J., 29, 35, 45 Wilhelm, E., 212, 215, 234 Wilhoit, R. C., 208, 210, 215, 234 Wilkinson, A. J., 145, 189 Williams, J. G., 32, 47 Wilson, C. B., 36, 50 Winter, C., 145, 174, 189 Winter, W., 57, 58, 118 Winterhalter, K. H.,11, 47, 49 Wirth, P., 41, 49 Wisdom. B. J., 36, 43 Wistow, G. J.. 161, 186, 189 Wittnchieber, E., 102, 120 Wlodawer, A., 70, 124, 174, 189 Wodak, 195, 232 Wolfe, H.R.,Jr., 68, 86, 88, 89, 91, 93, 119, 120, 122

Wolfenden, R.,193, 234 Wollmer, A:, 102, 120 Woodley, D. T., 38, 39, 50 Woodward, C. H., 174. 189 Wutrich, K.,77, 118 Wyckoff, H. W., 75, 76, 122 Wyman, J., 194, 211, 232, 234

AUTHOR INDEX

Y Yaar, M., 36, 48 Yamada, K. M., 2, 20, 26, 32, 34, 42, 43, 46, 48, 50 Yamada, T., 237, 238 Yamada, Y., 25, 41, 45, 50 Yamakura, F., 150, 188 Yamamura, H. I., 66, 67, 121 Yasuhara, T.,84, 122 Yasunobu, K. T., 162, 188 Yoon, J. W., 41, 48 York, E. J., 52, 69, 70, 71, 109, 123, 135, 188 Yoshida, M., 85. 121

253

Yotauhashi, K., 236, 237, 238 Young, M. E, 32, 43, 50 Yurchenco, P. D.,12, 18, 19, 29, 32, 37, 38, 44, 47, 50

Yuspa, S. H., 36, 48

2 Zettergren, J. G., 11, 47 Z h f u , M. H., 193, 234 Zimmerman, S. S., 55, 56, 124 Zipp. A., 204, 226, 234 Zoller, M., 117, 120 Zwolinslri, B. J., 208, 210, 215, 234

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A Acetic acid, dimerization energy and geometry, electmtatic effects, 180

N-Acetyl-a-Aib-N’-methylamide, conformational energy contour map, 56

N-Acetyl-Ala-N‘-methylamide,

conformational energy contour map, 56

N-Acetyl-Cly-N’-methylamide,

conformational energy contour map, 56 Acetylcholinesterase, basement membranes, 7, 35 a-Helical peptides, binding to phospholipid surfaces, design, 81-85 a-Helical proteins, 102-103, see also a-Helix formation coiled coils, 103-105 four-helix bundles, 105-114 a-Helix formation, see also a-Helical proteins long-range interactions, 71-75 hydrophobic forces, 72 hydrophobic periodicity, 72-74 medium-range interactions, 69-71 C-peptide, 70 N-terminal a-helii of ribonuclease A, 69-70 protonated form of His-12, 71 salt dependence, 70 side chain-side chain interactions, 69-70 short-range interactions, 68-69 Chou-Fasman method, 69 host-guest method, 69 AMBER all-atom force field, 131 Amino-aromatic interactions, 173-176

Amino acid sequences a],~ I A ,~ I B and , ~ P B ,112 calmodulin-binding domains of calmodulin-dependentkinases, 91 calmoddin-binding peptide CBP5, 91 collagen IV al(1V) and aZ(IV) chains, 12-16 human &endorphin, 97 laminin in basement membranes, 25 Amino acids aromatic, weakly polar, 151-156 aromatic side chains, electronic quadrupole moments, 139-140 conformational properties, 55-57 conformationally constrained, mlvphalins bearing, 62-63 naturally occurring, partial charges for all atoms, table, 132-133 cY,&unsaturated, properties, 60-61 D-Amino acids, properties, 60 Amino-terminal domain, collagen IV, 13, 15-16 Amphiphilic secondary structures calmodulin-binding peptides, 85-96 membrane-binding peptides, 81-85 peptide hormones, 96-101 Amyloid P component, basement membranes, 35 Analphylatoxin C3a, enhancement of helical potential, 78-80 Apolipoprotein A-1, models, design, 81-82 Apomyoglobin, intrinsic viscosity, temperature dependence, 230 Aromatic-aromatic interactions, 162-173 strength of, 182 Atomic mdtipoles, estimation, 128 Average structure, 183 255

256

SUBJECT INDEX

B

molecular structure, stereodrawing, 144

Backbone configuration, @ and angles, 55 Basement membranes acetylcholinesterase, 7, 35 amyloid P component, 35 BM-40, 7, 32-33 bullous pemphigoid antigen, 7, 35-36 calcium-binding proteins, 32-33 chondroitin sulfate proteoglycans, 35 collagen IV, 7-11 interactions, 38 isolation methods, 9 molecular structure, 11-16 receptors, 41-42 self-assembly, 36-38 entactin, 7, 26 fibronectin, 34 interactions, 39 Goodpature antigen, 36 heparan sulfate proteoglycans, 7, 28-32 interactions, 38-39 interactions between components, 38-40 intrineic components, table, 7 laminin, 7, 21-22 A and B chains, 22 B1 chain domain model, 24 domains, 22-25 fragments, 22-25 isoforms, 25-26 isolation and preparation, 21 receptors, 41-42 self-assembly, 38 sequence data, 25 3-chain structure, model, 23 morphology, 3-6 nidogen, 7, 21-28 interactions, 39 self-assemblyof components, 36-38 structural functions, 40-41 turnover and degradation, 42-43 ultrastructure, 3-6 Benzene rings, interacting, van der Waals stereodrawing, 163 N-Benzoyl-5’0-tertbutyldimethylsilyl-2’ deoxyadenosine monohydrate,

0 Proteins, design, 114-115 BM-40 calcium-binding protein, 7,32-33 Bombohins, amphiphilic a-helii formation, 84 Botabellin, design, 116-117 Brookhaven Protein Data Bank, hydrogen-bonding patterns in proteins, 147-150 Buffon’s needle problem, 154 Bullous pemphigoid antigen, basement membranes, 7, 35-36

C C3a analphylatoxin, enhancement of helical potential, 78-80 Ca’+/calmodulin-dependent protein kinase (brain type II), 0-subunit sequence, 92 Calcium-binding proteins, basement membranes, 32-33 Calmodulin, aromatic-aromatic interactions, 164-165 Calmodulin-binding peptides CBPl, CBP2, CBPS, CBP4, and CBP5, 87-90 design, 85-96 model, helical net diagram, 91 myosin light-chain kinase, 90 Cambridge Crystallographic Data Base hydrogen bonding to sp-and spJ-hybridized oxygen atom, 146 interactions between oxygen atoms and phenyl rings, 157 intermolecular interactions between phenylalanine phenyl rings, 163-164 Carbon, as hydrogen donor atom, hydrogen bonds with, 143-145 Carbonate dehydratase, thermodynamic parameters of denaturation, table, 197 Carboxyl-carboxylate interactions, 136 Carboxypeptidase A, mobile tyrosine residue, aromatic-aromatic interactions, 166 Cecropin A, amphiphilic a-helix formation, 85

257

SUBJEcr INDEX Charge-charge interactions, 128-130 distance dependence, 130 potential energy of interaction. 130 salt bridges, 129 strength of, 182 Charge-dipole interactions, 134-137 a-helii stabilization. 135 distance dependence, 137 potential energy due to, 136 Charge-quadrupole interactions, 139-140 Charge distribution, molecular, see Molecular charge distribution CHARMM all-atom force field, 131 Chi-square test, 155 2 , 2 - b ~ ~ ~ o p ~ l ) - l , l , binding peptide, artificial, 115-116 Chondroitin sulfate proteoglycans, basement membranes, 35 Chou-Fasman method, 69 Chymotrypsin, collagen IV-cleaving, 42 a-Chymotrypsin, thermodynamic parameters of denaturation, table, 197 y-Chymotrypsin, mobile tyrosine residue, aromatic-aromatic interactions, 166 Coiled-coil proteins, design, 103-105 Collagen IV, basement membranes, 7-11 d(IV) and aZ(IV) chains, 11 amino acid sequence, 12-16 domains, 11-16 genes, size of helical exons, table, 20 loop structures in NCI domain, 13 NCI domain, 18 nontripeptide sections, 14 7 S domain, 13, 15-18 electron micrograph, 8 genes, structure and evolution, 19-21 isolation methods, 9 network arrangement, 18-19 receptors, 41-42 s e l f - m b l y , 36-38 Collagenous proteins, types, table, 9 Compact reference state, 221 Compact state, nonpolar substances, 224-225

Conformational constraints, peptide design and, 52-53 Conformational energy contour maps N-acetyl-a-Aib-N'-methylamide, 56

N-acetyl-Ala-N'-methylamide,56 N-acetyl-Gly-N'-methylamide,56

Conservation, amino-aromatic interactions, 174 Crabrolin, amphiphilic a-helix formation, 84 Cross-links, covalent, introduction in peptides, 61 y-Crystallin, sulfur-aromatic interactions, 161 Cyclic enkephalins. 63-68 amide bond formation between two side chains, 67 analogs containing intramolecular &sulfide bonds, 66-67 receptor selectivity, 65 l Cyclic - ~ ~peptides, o ~ e -design, 61 Cytachme c, thermodynamic parameters of denaturation, table, 197

D DDT-binding peptide, artificial, 115-116 Deoxyhemoglobin A amino-aromatic interactions, 174, 176 aromatic-aromatic interactions, 166-168 oxygen-aromatic interactions, 158, 160 Deoxyribonucleic acid, polar interactions and, 172 Dipole-dipole interactions, 137-138 angular dependence, 138 hydrogen bond, 138 Dipole-quadrupole interactions, 139-140

Dipoles, electronic a-helix dipoles in proteins, 135 formation, 135 of a peptide unit, 135 Dispersion interactions, in proteins, 140-141

Dissolution, nonpolar molecules thermodynamics, 217-220 two-step process, 220-225 Distance dependence aromatic-aromatic interactions, 164 charge-charge interactions, 130 charge-dipole interactions, 137 polarizability effects, 134 potential energy of electrostatic interactions, hierarchy of, 150-151 quadrupole interactions, 140 sulfur-aromatic interactions, 161

258

SUBJECT INDEX

E Egg-white lywzyme intrinsic viscosity, temperature dependence, 230 thermodynamic parameters of denaturation, table, 197 EHS tumor BM-40 calcium-bindingprotein, 32-33 heparan sulfate proteoglycans, isolation, 28-29 laminin isolation, 21 nidogen isolation, 27 source of baacment-membrane proteins, 9 Elastase, collagen IV-cleaving, 42 Electron micrograph, collagen IV, network structure, 17 Electron micrographs collagen IV. laminin, and nidogen, 8 low- and high-demity heparan sulfate proteoglycans from EHS tumor,

so

Electronegativities biologically important atoms, 130-131 polarizability and, 133 Electronic charges partial atom of naturally occurring amino acids, table, 132-133 in proteins, 130-131 potential energy of interaction, 130 Electronic dipoles, see Dipoles, electronic Electrostatic interactions charge-charge interactions, 128-130 charge-dipole interactions, 134-137 classification, 185 electronic quadrupoles. 138-140 enthalpy estimates, 182 hierarchy, 150-151 hydrogen bonds, 142-150 London forces, 140-141 multipole repmentations of molecular charge distribution, 126-128 partial electronic charges, 130-131 polarizability, 131, 133-134 short-range electron shell repulsion, 140-141 structure and function of proteins, 184-185

Electrostatic potential energy, 126-128 fl-hdorphin amino acid sequence and structural dgnmenta, 97 helical net diagrams and proposed helical region, 98 model peptides, 97-101 Enkcphalin-bindingpeptide, design, 116-117 Enkephalins analogs, see specific analog cyclic, see Cyclic enkephalins Entactin, basement membranes, 26 Enthalpy of diaaolution, nonpolar molecules in water, 207-209 electrostatic interactions, estimates, 182 nonpolar gases in water, table, 208 protein denaturation, enthalpy of solution of liquid hydrocarbons and, 226 upper limit, 204-207 Entropy upon breakdown of native Structure, 204 upper limit, 204-207 Entropy difference, native and denatured protein states, 200-201 Entropy of solution, nonpolar gases in water, table, 215 Entropy of transfer equal to zero, nonpolar molecules, 216-217, 221 hydrocarbons from liquid phase to water, table, 215

F Fibronectin, basement membranes, 34 Folding, pressure effects, 181 Four-helix bundle pmtcins, design, 105-114 F m energies of tetramerization/dimerization,a1, au, a i ~and , ~ P B 112 ,

G Gene structure, collagen IV, 19-21 Geometry, energetically preferred hydrogen bonds, 146-148

SUBJEcr INDEX

259

Gibbs energy, hydrocarbons from liquid denaturation and, 225-228 phase to water, table, 215 cnthalpy of dissolution in water, 207-209 Gibbs energy difference thermodynamic parameters of maximum value, 202-203 solution into water, temperature dependence, 223 between native and denatured protein Hydrogen bonds states, 200 bifurcated, 143 Globular proteins bonding patterns in proteins, 147-150 compact thermodynamic parameters of with carbon as hydrogen donor atom, denaturation, table, 197 143-145 upper limit of specific enthalpy andclassification of electrostatic entropy of conformation interactions and, 185 ddinition, 145-146 transition, 204-207 folding and stability, 180-181 denaturation enthalpy and, 227-228 internal order, electrostatic dipole-dipole interactions, 138, interactions, 180 142-143 energetically preferred, geometry, mechanism of stabilization, 228-231 small, macroscopic states in 146-147 enthalpy of protein denaturation and, denaturation, 195 Glucapn, enhancement of helical 227 formation, 142-143 potential, 77-78 linearity, 148 Coodpasture antigen, basement membranes, 36 Met-enkephalin bonding patterns, 54 GPl glycoprotein, 21 nontraditional, 143-146 sulfur as donor and acceptor, 145 GP2 glycoprotein, 21 Hydrophobic bond, 178 Hydrophobic effect, 178 H Hydrophobic forces, effect on secondary structure formation, 71-75 Heat capacity Hydrophobic interactions, 177-181 change, role in regulating hydration globular proteins, 180-181 fm energy change, 224 internal order of globular proteins, 180 increment, nonpolar gases in water, micelle analog, 179 table, 208 between protein nonpolar groups, nonpolar molecules, change upon 230-231 aqueous dissolution, 211-212 Hydrophobic periodicity, role in partial, during transition from native secondary struct~reformation, 72-74 to denatured state, 196-206 Hydroxlysine, in basement membranes, 7 Helical domains, collagen IV (ul(1V) Hydroxyproline, in basement and a2(IV) chains, 12-13 membranes, 7 Heparan sulfate proteoglycan, basement membranes, 7, 28-32 low- and high-density, 29-31 1 size diveraity, 31 Host-guest method, 69 Isoforme, basement membrane laminin, Hydration 25-26 fm energy change, role of heat capacity change, 224 nonpolar molecules, 217-225 K Hydrocarbons, liquid dissolution in water, protein Knobs into holes packing, 103-104,106

260

SUBJECT INDEX

L Lamina densa, in basement membranes, 3-4 Lamina fibroredcularb, in basement membranes, 3-4, 6 Lamina lucida, in basement membranes, 3 Laminin, basement membranes, 7, 21-22 A and B chains, 22 B1 chain domain model, 24 domains, 22-25 electron micrograph, 8 electrophoresis, 21-22 fragments, 22-25 isoforms, 25-26 isolation and preparation, 21 receptors, 41-42 self-assembly, 38 sequence data, 25 3-chain structure, model, 23 Large proteins, native structure, disruption stages, 195-196 Lennard-Jones 6-12 potential function, 140-141 Leu-enkcphalins,design, 61-68 Ligand binding, protein, see Proteinligand binding London forces, 140-141

M Macrocyclization. 61 enkephalins, 64 Mastorparan, amphiphilic a-helix formation, 84 Mellitin, analog, model, 82-83 Membrane-bindingpeptides, design, 81-85 Met-enkephalins crystalline forms, hydrogen-bonding patterns, 54 design, 61-68 Metalloproteinase, collagen IV-cleaving,

Morphology, basement membranes, 3-6 Multipole representations, molecular charge distribution, 126-128 Multipoles, atomic, estimation, 128 Myoglobin specific enthalpy of denaturation. temperature dependence, 201 thermodynamic parameters of denaturation, table, 197 Myosin light-chain kinate a-helix-forming peptides, 90 calmodulin-binding domains, helical net diagram, 91

N NCl domain, collagen IV, 11-12, 18 NC2 domain, collagen IV, 12 Network arrangement, collagen IV, 18-19

Nidogen basement membranes, 7. 27-28 electron micrograph, 8 Nonpolar gases enthalpy and heat capacity increment of solution, table, 208 entropy of solution in water, table, 215

Nonpolar molecules enthalpy of dissolution in water, 207-209

heat capacity change upon aqueous dissolution, 211-212 hydration, 217-225 thermodynamics of dissolution, 217-220

two-step dissolution process, 220-225

Nonpolar substances compact state, 224-225 entropy of solution in water, 212-217

42

C,-Methylamino acids, properties,

0

57-58

Nu-Methylamino acids, properties, 58-59 C,-Methylation, 58 Molecular charge distribution, multipole reprreentations, 126-128

Oligonucleotide synthesis, prebiotic template-directed, 172-173 Osteonectin, BM-40calcium-binding protein and, 32

SUBJECT INDEX Oxygen-aromatic interactions, 156-160 Oxytocin, aromatic-aromatic interactions, 169-170

P Papain, thermodynamic parameters of denaturation, table, 197 Parietal yolk sac cells GP1 and GP2 glycoproteins. 21 production of basement-membrane components, 10 Partial heat capacity, during transition from native to denatured state, 196-206 Parvalbumin aromatic-aromatic interactions, 164-165 thermodynamic parameters of denaturation, table, 197 Peanut proteins, chemistry, abstract, 235-238 Pepsin, collagen IV-cleaving, 42 Pepsinogen, thermodynamic parameters of denaturation, table, 197 Peptide design amino acids, conformational properties, 55-57 amino acids, conformationally constrained o-amino acids, 60 enkephalins bearing, 62-63 C,-methylamino acids, 57-58 Nu-methylamino acids, 58-59 a,B-unsaturated amino acids, 60-61 amphiphilic secondary structureforming peptides, 80-81 calmodulin- binding pept ides, 85-96 membrane-binding peptides, 81-85 peptide hormones, 96-101 cyclic peptides. 61 effects of conformational constraints, 52-53 enhancement of helical potential, 75-80 glucagon, 77-78 human analphylatoxin C3a. 78-80 ribonuclease S-peptide, 75-77 I r u - and Met-enlrephalin analogs, 61-68 medium-sized peptides, 68 small peptides, 55-55

261

stabilization of helix formation long-range interactions, 71-75 medium-range interactions, 69-71 short-range interactions, 68-69 Peptide hormones, design, 96-101 N-Phenylacetyl-L-phenylalanine molecular structure, stereodrawing, 134 partial electronic charges of phenyl rings, 131 Plasminogen, fragment K4, thermodynamic parameters of denaturation, table, 197 Polar coordinates, right-handed, 152-155 Polar interactions amino-aromatic interactions, 175-176 aromatic-aromatic interactions, 162-175 DNA structure and, 172 oxygen-aromatic interactions, 156-160 sulfur-aromatic interactions, 160-162 Polarizability, 131, 133-134 Potential energy charge-charge interactions, 150 charge-dipole interactions, 136 dipole-dipole interactions, 137-138 electrostatic, 126-127 polarizability, 134 Potential energy surfaces, minima, 182-185 Pressure effects, on protein folding, 181 Proline, conformational properties, 55, 57 Protein-ligand binding amino-aromatic interactions, 174-175 aromatic-aromatic interactions, 166 charged ligands, salt bridge mediation, 129-130 minima effects, 182 oxygen-aromatic interactions, 158, 160 Protein denaturation calorimetry, 194-195 discrete stages of protein structure disruption, 195-196 native and denatured states, 195 partial heat capacity changes, 196-204 specific enthalpy and entropy of conformation transition, upper limits, 204-207

262

SUBJECT INDEX

enthalpy enthalpy of mlution of liquid hydrocarbons and, 226 hydrogen bonds and, 227 hydrocarbon d h l u t i o n in water and, 225-228 Protein d&W, 101-102 a-helical proteins, 102-103 coiled coilr, 103-105 four-helix bundlm, 105-114 artificial DM"binding peptide, 115-116 6 proteins, 114-115 betabellin, 116-117 enkcphalin-bindingpeptide, 116-117 Protein structures, compact, stabilization mechanism, 228-231

Q Quadruple- quadrupok interactions, 139-140 strength of, 182 Quadrupolm, electronic, electrostatic interactions, 138-140

R Reaction microcalorimetry, 194 Ribonuclease A a-helix N terminal, 69-70 C-peptide, 70-71 intrinsic viscosity, temperature dependence, 230 pH dependence of helix formation, 70 salt bridge, 70-71 salt dependence of helix formation, 70

specific enthalpy of denaturation, temperature dependence, 201 thermodynamic parameters of denaturation, table, 197 Ribonuclease S-peptide, enhancement of helical potential, 75-77 Ridges into groova packing, 104, 106 Ring stacking, 162

S Salt bridges, 129 buried, 130 claaaification of electrostatic interactions and, 185 effect on protein thermal stability, 129 mediation of charged ligand-binding to proteh, 139-130 Scanning electron micrographs, human skin baaement membrane, 4 scanning m i ~ a l 0 r i m d C technique, 194 Secondary structum, amphiphilic, see Amphiphilic secondary structures 7 S domain, collagen IV, 13, 15-18 Single-crystalX-ray crystallographic data b m , 155-156 Solubility, hydrocarbons from liquid p h w to water, table, 215 Somatoetatin, aromatic- aromatic interactions, 168-169 SPARC, BM-40calcium-binding protein and, 32 Stabilization, compact protein structures, 228-231 Staphylococcus nuclease, thermodynamic parameters of denaturation, table, 197 Stenodrawings antigen-binding site of Fab fragment NEW, 165 N-benzoyl-5'O-tertbutyldimethylrilyl -2 'deoxyadenoeine monohydrate, molecular structure, 144 6-benzyl-S-chloro-2-pyrone bound to active site of y-chymotrypain, 167 conformations of carbon monoxymyoblobin arginine-45, 175 molecular structures of bisphenyl antigelling and antisickling compounds, 170 oxygm environment of 26 phenylalanine side chains, 157 N-phenylacetyl-L-phenylalanine, molecular structure, 134 succinyl-L-tryptophanyl-L-tryptophan bound to deoxyhemoglobin A, 171 van der Waals, interacting benzene rings, 163

263

SUBJECT INDEX Sulfur-aromatic interactions; 160-162 Sulfur, an hydrogen bond donor and acceptor, 145 Synthetic peptides q , (YIA, U ~ B ,and aPB9

112

amino acid sequences, 112 free energies of tetramerization or dimerization, 112

T Temperature dependence denaturational heat capacity increment, 198-200 intrinsic viscosity of egg-white lysozyme, apomyoglobin, and ribonuclease A, 230 specific mthalpy of denaturation of myoglobin and ribonuclease A,

Tertiary structure, stabilization by enthalpically favorable aminoaromatic pairs, 174 by enthalpically favorable aromatic pairs, 164 Thermal stability, effects of salt b r i d p , 129

Thermodynamics, dissolution of nonpolar molecules, 217-220 Troponin, aromatic- aromatic interactions, 164-165 &%sin, thermodynamic parameters of denaturation, table, 197 Tryptophan-containing peptides fluorescence properties, 95 orientation, schematic, 94 perturbational effect of tryptophan introduction, 93 Tryptophan introduction, perturbational effect, 93

201

thermodynamics parameters in dissolution of liquid hydrocarbons into water, 223 Temperature effects entropy of solution of nonpolar substances, 212-217 entropy of transfer equal to zero,

U Ultrastructure, basement membranes, 3-6

v

216-217, 221

heat capacity change upon dissolution of nonpolar molecules, 211-212

Teratocarcinoma cells, production of baerment-membrane components, 10

van van van van

der der der der

Waals Waals Waals Waals

distance, 141 interactions, 140-141 potential, 141 radius, 141

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