Folding of Disulfide Proteins (Protein Reviews 14)

Protein Reviews Series Editor: M. Zouhair Atassi Baylor College of Medicine Houston, Texas

For further volumes: http://www.springer.com/series/6876

Rowen J.Y. Chang  •  Salvador Ventura Editors

Folding of Disulfide Proteins

Editors Rowen J.Y. Chang Research Center for Protein Chemistry Brown Foundation Inst. of Molecular Medicine, Dept. of Biochemistry and Molecular Biology The University of Texas Houston, TX 77030, USA [email protected]

Salvador Ventura Dept. de Bioquímica i Biologia Molecular Institut de Biotecnologia i de Biomed. Universitat Autònoma de Barcelona Bellaterra-08193, Spain [email protected]

ISBN 978-1-4419-7272-9 e-ISBN 978-1-4419-7273-6 DOI 10.1007/978-1-4419-7273-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011932496 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY  10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in ­connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The formation of selected intramolecular disulfide bonds is one of the most important postraductional modifications of proteins, influencing their folding, stability, and biological function. The folding of disulfide proteins is usually an intricate process in which, after their synthesis at the ribosome in a reduced and unfolded state, polypeptides gain coordinately their native disulfide bonds as well as their unique and stable conformation. In the cell, this process is exquisitely controlled and catalyzed by a complex protein machinery to avoid mispairing of cysteine residues, which might prevent the attaining of functional conformations leading to misfolding and, in some cases, triggering pathological processes. In vitro, the folding of disulfide proteins has constituted the bedrock for a long time on which to understand at the kinetic and structural levels the mechanisms by which a particular amino acid chain folds into a specific functional conformation. The aim of the present monograph is to provide the reader with a detailed view of our current structural and functional understanding of the complex process of protein oxidative folding and of their chemical, biotechnological, and biomedical implications; together with a historical perspective of a field that this year 2011 celebrates its 50th anniversary. The book presents a comprehensive description of the complexity and diversity of folding pathways of different disulfide protein models, including polypeptides with biomedical and biotechnological interest like insulin or plant cyclotides. It is discussed how the cellular machinery and more specifically protein disulfide isomerase promotes and proofreads the formation of native disulfides and how this process can be emulated in vitro exploiting the redox properties of small catalysts. Many therapeutically relevant proteins contain disulfides, which mispairing during recombinant production precludes their commercialization; top experts in the field describe why this occurs and how it can be avoided. The book focuses also on the emerging role played by certain disulfide bonds in the allosteric control of protein function and their implication in health and disease. In addition, the information

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accumulated in the present text constitutes a compendium of state of the art technical approaches in protein separation technology, in mass spectrometry and in highresolution structural characterization of folded and unfolded protein states of intrinsic high value for any protein chemist. Houston, TX, USA Bellaterra, Spain

Rowen J.Y. Chang Salvador Ventura

Contents

  1  Oxidative Folding: Coupling Conformational Folding and Disulfide Formation.......................................................................... Salvador Ventura and Rowen J.Y. Chang

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  2  The Case of Oxidative Folding of Ribonuclease A: Factors Impacting Fold Maturation of ER-Processed Proteins........... Mahesh Narayan

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  3  Cystine Knot Folding in Cyclotides........................................................ Norelle L. Daly, Christian W. Gruber, Ulf Göransson, and David J. Craik

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  4  In Vitro Folding of Single/Double Chain Insulins and Related Proteins................................................................................ You-Min Feng

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  5  Unfolding and Refolding of Disulfide Proteins Using the Method Disulfide Scrambling................................................ Rowen J.Y. Chang

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  6  Oxidative Protein Folding with Small Molecules . ............................... 109 Watson J. Lees   7  Protein Disulfide Isomerase and the Catalysis of Oxidative Protein Folding................................................................... 133 Hiram F. Gilbert   8  Allosteric Disulfide Bonds....................................................................... 151 Jason W.H. Wong and Philip J. Hogg   9  The Problem of Expression of Multidisulfide Bonded Recombinant Proteins in E. coli............................................................. 183 Silvia A. Arredondo and George Georgiou

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10  NMR-Spectroscopic Investigation of Disulfide Dynamics in Unfolded States of Proteins............................................... 217 Robert Silvers, Kai Schlepckow, Julia Wirmer-Bartoschek, and Harald Schwalbe 11  A Half-Century of Oxidative Folding and Protein Disulphide Formation......................................................... 257 Robert B. Freedman Index.................................................................................................................. 277

Contributors

Silvia A. Arredondo  Department of Chemical Engineering, University of Texas, Austin, TX 78712, USA [email protected] Rowen J.Y. Chang  Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine, 1825 Pressler Street, Houston, TX 77030, USA; Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, USA [email protected] David J. Craik  Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, QLD 4072, Brisbane, Australia [email protected] Norelle L. Daly  Institute for Molecular Bioscience, Division of Chemistry and Structural Biology, The University of Queensland, QLD 4072, Brisbane, Australia [email protected] You-Min Feng  Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China [email protected] Robert B. Freedman  School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK [email protected] Ulf Göransson  Division of Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre, Box 574, SE-751 23, Uppsala, Sweden [email protected]

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George Georgiou  Department of Chemical Engineering, University of Texas, Austin, TX 78712, USA; Department of Biomedical Engineering, University of Texas, Austin, TX 78712, USA; Department of Molecular Genetics and Microbiology, University of Texas, Austin, TX 78712, USA; Institute for Cell and Molecular Biology, University of Texas, Austin, TX 78712, USA [email protected] Hiram F. Gilbert  Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA [email protected] Christian W. Gruber  Center for Physiology and Pharmacology, University of Vienna, A-1090 Vienna, Austria [email protected] Philip J. Hogg  Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, 2052 Sydney, Australia [email protected] Watson J. Lees  Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL 33199, USA [email protected] Mahesh Narayan  Department of Chemistry, The University of Texas at El Paso, 500 W. Univ. Ave., El Paso, TX 79968, USA [email protected] Kai Schlepckow  Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438 Frankfurt/Main, Germany Harald Schwalbe  Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438 Frankfurt/Main, Germany [email protected] Robert Silvers  Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438 Frankfurt/Main, Germany [email protected] Salvador Ventura  Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra-08193, Barcelona, Spain [email protected] Julia Wirmer-Bartoschek  Institute of Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, Max-von-Laue-Straße 7, 60438 Frankfurt/Main, Germany Jason W.H. Wong  Lowy Cancer Research Centre, Prince of Wales Clinical School, University of New South Wales, 2052 Sydney, Australia [email protected]

Chapter 1

Oxidative Folding: Coupling Conformational Folding and Disulfide Formation Salvador Ventura and Rowen J.Y. Chang

Abstract  Determining how a string of amino acid residues folds into the ­biologically active protein conformation remains as one of the most important and challenging tasks in biology. Protein folding is usually a fast reaction in which transient intermediates in the folding pathway are short lived, highly dynamic, and very difficult to be trapped, isolated, and characterized. The technique of oxidative folding applied to study disulfide proteins overcomes some of these problems. During protein oxidative folding, the coupling between conformational folding and disulfide formation together with the possibility to selectively quench the progress of the oxidative reaction permits the isolation and further structural characterization of transient folding intermediates in atomic detail. With its unique chemistry and relatively slow kinetics of disulfide formation, the technique of oxidative folding has facilitated the detailed characterization of the folding pathways of an important number of disulfiderich proteins. The results reveal a high degree of diversity of folding mechanisms, which are mainly manifested by the extent of heterogeneity and native-like structures of their intermediate ensembles. Overall, as we will discuss in this chapter, the study of disulfide-containing polypeptides has contributed significantly to our current knowledge on the molecular basis of protein folding. Keywords  Disulfide bonds • Protein folding • Folding intermediates • Oxidative folding • Reductive unfolding • Folding pathways • Protein structure

S. Ventura (*) Institut de Biotecnologia i de Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra-08193, Barcelona, Spain e-mail: [email protected] R.J.Y. Chang and S. Ventura (eds.), Folding of Disulfide Proteins, Protein Reviews 14, DOI 10.1007/978-1-4419-7273-6_1, © Springer Science+Business Media, LLC 2011

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Abbreviations (des) species 1S 2S 3D structure 3S BPTI Cys–Cys DTTox DTTred EGF GSH GSSG H/D exchange LCI LDTI MCoTI-II MS PDI RNase A RP-HPLC S- SH TAP TCEP TCI

A folding intermediate lacking one disulfide bond One-disulfide intermediates Two-disulfide intermediates Three-dimensional structure Three-disulfide intermediates Bovine pancreatic trypsin inhibitor Cystine Oxidized dithiothreitol Reduced dithiothreitol Epidermal growth factor Reduced glutathione Oxidized glutathione Hydrogen to deuterium exchange Leech carboxypeptidase inhibitor Leech-derived trypsin inhibitor Momordica cochinchinensis trypsin inhibitor II Mass spectrometry Protein disulfide isomerase Bovine pancreatic ribonuclease A Reversed phase high-performance liquid chromatography Thiolate group Thiol group Tick anticoagulant peptide Tris(2-carboxyethyl)phosphine Tick carboxypeptidase inhibitor

1.1 Introduction: Protein Folding In the cell, proteins are synthesized as sequential strings of amino acids. However, these linear chains are in most cases inactive, since a protein becomes functional only when it folds into its specific and unique three-dimensional (3D) structure. Many proteins have been shown to attain spontaneously their functional conformation from an initially unfolded state in vitro, without needing the assistance of the cellular protein quality control (Daggett and Fersht 2003). This implies that most of the information required to form the extremely sophisticated 3D structures of proteins is imprinted somehow in their primary sequences (Anfinsen 1972). As any protein amino acid sequence can be univocally deduced from the corresponding gene, it can be inferred that the genome sequence of an organism contains all the information required to know its complete repertoire of protein structures and their associated functions (Eisenberg et al. 2000). However, although an overwhelming effort has been dedicated to understand the physicochemical determinants and the

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sequence of molecular events that allow a particular amino acid chain to fold into a specific functional conformation, we are still far from being able to assign gene sequences directly to protein structures (Sanchez et  al. 2000). The connection between protein sequence and function is lost, in many cases, in the black-box of protein folding (Nishimura et al. 2005). Therefore, it is clear that solving the socalled folding problem constitutes one of the most important and challenging tasks in biochemistry (Creighton 1988a; Gruebele 1999; Fetrow et al. 2002; Daggett and Fersht 2003; Dill et al. 2007). Protein folding obeys the laws of thermodynamics, which means that a protein always folds so that it achieves the conformation with the lowest possible free energy, which in most cases coincides with the functional state. Proteins are able to find the right functional structure, out of an astronomical number of potential 3D conformations in which they could randomly fold, in an extremely fast manner. Local structures can occur in 10–100 ns, loops can fold in 500 ns, and the minimum time required to complete the folding of a small protein is in the order of 1  ms (Gruebele 1999). Cyrus Levinthal in 1969 calculated that finding the right combination in the vast conformational universe by simple trial and error would be completely incompatible with proteins folding in a time scale that is biologically relevant (Honig et al. 1976). Therefore, protein folding must be somehow a directed process, which suggests the existence of defined pathways in which protein folding can occur rapidly. This idea was stimulating, because it implied that the characterization of a defined number of intermediates that populate along folding pathways would allow reconnection of a protein fold to its sequence (Kim and Baldwin 1982; Karplus and Weaver 1994; Ptitsyn 1991). Therefore, a large amount of experimental work has been pursued on the identification and characterization of the ensemble of folding intermediates that are generated during the folding reaction (Dobson and Evans 1988; Weissman 1995). However, due to the extremely fast time scales of protein folding, their transient nature and their highly flexible conformations, folding intermediates have been much more difficult to characterize that initially thought (Baldwin 1994). This is especially true for the initial folding intermediates, which are in fact the most informative species in terms of sequence/conformation relationship (Bryngelson et al. 1995). As it will be demonstrated in this and in subsequent chapters of this book, the study of the folding properties of disulfide-containing proteins has contributed much of our present knowledge about the way the intermediates shape the folding reactions of polypeptides (Creighton 1986).

1.2 Disulfide Bonds of Proteins The covalent link of cysteine residues by disulfide bonds constitutes an important and in many cases an essential structural feature of numerous proteins (Ventura 2008; Abkevich and Shakhnovich 2000; Darby and Creighton 1997). Disulfide bonds are thought to serve several functions. They thermodynamically stabilize the native conformation of proteins. By cross-linking sequentially distant regions of

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the polypeptide chain, they decrease the entropy of the unfolded ensemble, making it less favorable compared with the folded conformation. The increase in stability is directly related to the length of the polypeptide loop the disulfide bond closes, the longer the loop the larger the conformational stabilization (Poland and Scheraga 1965; Arolas et al. 2009; Camacho and Thirumalai 1995). In some cases, disulfide bonds also act enthalpically through the stabilization of favorable local interactions in the native conformation (Wedemeyer et al. 2000b). The extra stability provided by disulfides is especially important for protecting secreted proteins from oxidants and proteases present in the harsh extracellular environment, thus preventing their inactivation and increasing their half-life (Zavodszky et  al. 2001). In addition to their ability to form chemically inert structural disulfide bonds, cysteine residues can react with and be modified by their local environment. Accordingly, increasing evidence indicates that certain disulfide bonds are actually relevant from a functional point of view. Some are found in the active site of thiol/disulfide oxidoreductases and play a catalytic role in the formation, disruption, and exchange of protein disulfides, whereas others act as pH or redox sensors promoting structural changes that inhibit or activate the protein in which they reside (Wouters et al. 2010; Hogg 2003, 2009). The complement of disulfide bonds of a functional protein results from a complicated succession of covalent reactions, including oxidation or disulfide formation, reduction or disulfide disruption, and isomerization or disulfide reshuffling (Creighton 1986, 1997; Wedemeyer et al. 2000a). In vitro, the formation of a protein disulfide bond consists, in most of the cases, in two consecutive thiol/disulfide exchanges with a redox agent. In a first stage, the redox reagent forms a mixed disulfide with the free thiolate in the reduced protein. The mixed disulfide is subsequently attacked by another free cysteine in the protein, resulting in the formation of an intramolecular disulfide and the release of the reduced form of the redox agent (Fig. 1.1). In the absence of chemical reagents, molecular oxygen may act itself as a redox agent and promote the spontaneous formation of disulfide bonds in aqueous solutions. Also, free protein thiolate groups can attack intramolecularly preformed disulfide bonds, leading to an isomerization reaction that results in new disulfide connectivity in the protein. In proteins, the formation and disruption of disulfide bonds cannot be seen simply as chemical reactions, since the forward and backward reaction rates are sharply modulated by the effective concentration of thiolate anions as well as by the ­reactivity, proximity, and accessibility of both free cysteine residues and disulfide bonds. These specific features render disulfide bonds formation and disruption useful tools to approximate the conformational properties of folding intermediates and native states. Oxidation, reduction, and reshuffling reactions are impeded when the reactive groups are buried into stable and compact protein structure (Narayan et al. 2000; Wedemeyer et al. 2000a, 2002). Therefore, in polypeptides the equilibrium constant (Keq) for a thiol/disulfide exchange reaction is in fact a sensor of the accessibility of the involved groups (Wedemeyer et al. 2002). The Keq for such reactions can vary in more than eight orders of magnitude depending on whether they occur in an unfolded context or inside a compact globular structure (Darby and Creighton 1993). Therefore, they

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Fig. 1.1  Chemistry of a thiol–disulfide exchange reaction. A reactive thiolate group in the reduced form of a protein attacks the accessible disulfide bond of a (cyclic) redox compound. This results in the formation of a mixed disulfide precursor in which the protein and the redox reagent are covalently bound. The precursor is subsequently resolved by the attack of another free cysteine in the protein, which results in the formation of an intramolecular disulfide bond and the release of the reduced (linear) form of the redox reagent and the oxidized protein as final products of the chemical reaction

indirectly report on the conformational stability of the structural elements in their vicinity, in a similar manner to H/D exchange experiments (Darby and Creighton 1997). The rate of disulfide bond formation depends also on the spatial proximity of the two reactive groups. Proximity in this context refers to the probability of the two free thiolates coming within the minimal distance required for their covalent bonding (Wedemeyer et al. 2000b). Thus, disulfide formation kinetics provide us with a kind of molecular ruler to measure relative distances between residues in secondary, tertiary, or quaternary structures (Creighton 1997; Welker et al. 2001a). Finally, disulfide formation depends on the reactivity of the involved chemical groups. The thiolate form (S-) is the reactive form of cysteine whereas the thiol form (SH) cannot establish disulfide bonds. Therefore, both the specific cysteine’s pKa and the pH of the local environment control thiol/disulfide exchange reaction. The pKa of cysteine in an unfolded protein background is around 8.7 but this says little about the effective pKa of a given cysteine in a globular domain where the electrostatic environment is significantly different. As a general rule, it can be assumed that basic environments promote disulfide formation/disruption reactions whereas acidic conditions impede them. As we will see later this behavior allows the selective quenching of oxidative folding reactions.

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Overall, disulfide bonds display unique chemical and structural characteristics that allow us to use them as privileged sensors or reporters of the progress of protein folding pathways and of the structural properties of the intermediate or final species in the folding reaction (Wedemeyer et al. 2000a). (a) The reduction and oxidation of disulfide bonds follow a two-state mechanism and correspond to a specific and localized structural change. (b) The oxidation/reduction rates can be tuned experimentally by modifying the reaction conditions without affecting importantly secondary or tertiary protein contacts. (c) Disulfide bonds stabilize folding intermediates allowing their purification and subsequent structural characterization.

1.3 Experiments of Reductive Unfolding and Oxidative Folding Reactions of Disulfide Proteins 1.3.1 Trapping, Separation, and Characterization of Folding Intermediates The study of oxidative folding and the reductive unfolding pathways of disulfidecontaining protein requires fast and irreversible quenching methods to allow effective trapping of different conformational species that populate along the pathway in a time-course manner (Fig. 1.2). Creighton (1986) pioneered such approach by irreversibly alkylating the free cysteines present in these partially folded species using iodoacetate. Nevertheless, later on, it was observed that reshuffling of intermediates during the trapping step with iodoacetate can still occur, as observed for the oxidative folding of bovine pancreatic ribonuclease A (RNase A) and bovine pancreatic trypsin inhibitor (BPTI) (Weissman and Kim 1991). Although this undesired rearrangement could be reduced in the presence of high concentrations of iodoacetate, this might promote the modification of residues other than cysteine, like histidine, lysine, and methionine, and therefore the disruption or the establishment of anomalous intramolecular interactions. The use of faster and more selective molecules to block free protein thiols, such as 2-aminoethyl methanethiosulfonate, 1-cyano-4dimethylaminopyridinium tetrafluoroborate, and trans-dichloro(diethylenediamine) platinum (IV) ion, has come to solve this problem (Wu et al. 1998; Narayan et al. 2003a, b; Welker et  al. 2004). However, the irreversible binding of chemical ­modifiers to free cysteines usually distorts sterically the conformation of folding intermediates. Quenching of folding intermediates by acidification using aqueous trifluoroacetic acid or acetic acid at pH 2.0–3.5, has become almost the standard trapping method because it is a extremely rapid process that permits the thermodynamic, kinetic, and structural characterization of unmodified species and in addition allows to witness how a particular isolated intermediate explores the folding ­landscape toward the native structure after readjusting the pH in stop–go studies (Scheraga et al. 1987). It is important to note that because the reactivity of thiols decreases one order of magnitude for each pH unit below their effective pKa,

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Fig. 1.2  Scheme of the succession of experimental techniques used to characterize the oxidative folding reaction of a disulfide-rich protein

a­ cidification strongly decreases the rate of thiol–disulfide exchange reactions but do not abolish them completely (Mamathambika and Bardwell 2008). The quenching method has been shown to have a strong influence on the apparent oxidative folding pathway of a disulfide-containing protein. This is best exemplified by the discrepancy of BPTI folding pathway elucidated by Creighton and Kim’s groups on the heterogeneity and native-like structure of BPTI folding intermediates. The difference is likely resulted from the dissimilar quenching method used in these studies, in which one group uses alkylation quenching (Creighton 1990) and the other uses acid quenching (Weissman and Kim 1991). Following sample quenching, the identity and/or conformational properties of the different intermediate species in the usually complex mixture of conformers need to be analyzed (Fig. 1.2). For a protein with only 3 disulfide bonds up to 74 different disulfide-bonded intermediates can populate along the folding pathway. As the number of disulfides in a protein increases, the complexity of the theoretical intermediate ensemble grows exponentially. Therefore, highly effective fractionation methods are required to analyze intermediates of oxidative folding reactions. The quenching method might condition the subsequent separation procedure. In this way, acid trapped intermediates should be kept at low pH, which precludes the use of many chromatographic and electrophoretic methods. For acid-trapped intermediates, reversed phase high-performance liquid chromatography (RP-HPLC) at low

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pH has become the most useful analysis technique. Although the ensemble of ­acid-trapped intermediates only differ in the number and the identity of their ­disulfide bonds, these covalent links endorse them with differential compactness and hydrophobicity, allowing their separation and isolation by RP-HPLC. When covalent chemical quenchers are used, the preferred separation method would depend on the nature of the cysteine-blocking reagent, ion-exchange chromatography­ can be used for molecules that alter the overall protein charge and SDS-PAGE can be used for large blocking reagents. Importantly, even when using relative small groups for derivatization, like vinyl-pyridine, the corresponding increase in molecular weight can be monitored by mass spectrometry (MS). Because the increase in molecular mass is proportional to the number of blocked cysteines, and accordingly to the number of remaining reactive thiols, MS analysis thus allows us to follow the kinetics of disulfide formation during oxidative folding. To determine the specific disulfide pairing of selected, purified, and derivatized folding intermediates, they are digested with endopeptidases in order to obtain peptide mass fingerprints under reducing and nonreducing conditions that would allow identification of the preformed covalent bonds in the isolated intermediate.

1.3.2 Reductive Unfolding Experiments In reductive unfolding experiments, native disulfide-containing proteins are treated with different concentrations of a reducing agent and the disruption of their disulfide bonds is monitored. In many cases, the disulfide bonds are totally or partially protected by the native protein structure and cannot be easily reduced, requiring first a total or local unfolding event that exposes the bond to the reducing agent. Therefore, the stability of a disulfide bond in front of reduction depends not only on its ­chemical environment and degree of exposition but also on the dynamic ­properties of the protein. The thermodynamic value of the structural transition leading to the exposition of a previously buried disulfide bond can be calculated from its rate of reduction at different reducing reagent concentrations (Welker et al. 2001a). In proteins with multiple disulfide bonds the reduction of the first bond renders a natively bonded des-species. Depending on its structure and stability, this species can be metastable and display a native conformation that is only lost after additional reduction of one or more disulfide bonds. Such des-species might also be highly unstable with the rest of the disulfides being exposed to solvent and rapidly reduced in a coordinated manner. Reductive unfolding pathways are sensitive to pH of the solution. Reshuffling reactions are favored when reductive unfolding is performed at basic or neutral pH and mostly avoided when the reaction takes places at acidic pH, since the new free cysteines are protonated and therefore not reactive. Reduced dithiothreitol (DTTred) is usually used for reductive unfolding experiments at basic to neutral pH, whereas tris(2-carboxyethyl)phosphine (TCEP) is the preferred reducing agent at acidic pH.

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1.3.3 Oxidative Folding Experiments In oxidative folding experiments, the protein is first denatured by using high ­concentrations of chaotropic agents, typically urea or guanidinium chloride, and placed under strong reducing conditions to completely reduce the disulfide bonds that still link covalently the polypeptide chain in the denatured state. This denatured and reduced state emulates the conformation of the protein molecules as they emerge from the ribosome after their synthesis. Denaturing and reducing agents are simultaneously removed and the proteins are allowed to refold. The sequential regeneration of their disulfide bonds is then monitored as described above. The kinetics of oxidative folding reactions are highly sensitive to the experimental conditions, specifically to the ionic strength, pH, and redox potential of the solution. As a general trend, the presence of salts accelerates oxidative folding, likely by competing with nonspecific ionic intramolecular interactions (Arolas et al. 2004). Because the oxidative reaction depends on the protonation state of the protein-free cysteines, the pH of the solution has a strong influence on the velocity and efficiency of the folding process. Most works employed pH 8.5 as the default choice to maximize the folding and recovery of native disulfide proteins. Oxidative folding experiments can be performed in the absence or presence of redox agents. When no external redox agents are added, the molecular oxygen acts as an oxidant. However, under these conditions, most oxidative folding reactions become extremely slow and inefficient. Accordingly, both oxidative and reducing agents are commonly used in oxidative folding experiments (Chatrenet and Chang 1993). Oxidative agents such as oxidized glutathione (GSSG), Cys–Cys, or oxidized protein disulfide isomerase (PDI) act as oxidases, accelerating the formation of disulfide-bonds, which often promotes the fast accumulation of fully oxidized isomers or scrambled species as major folding intermediates. Reducing agents such as reduced glutathione (GSH), Cys, 2-mercaptoethanol, or reduced PDI act as reductases. They catalyze and promote disulfide shuffling of already formed disulfide bonds by allowing the folding intermediates to fold toward a new energy minimum in the way to attain the native disulfide connectivity. Therefore, the optimal rates of in vitro oxidative folding are achieved at specific concentrations of oxidative and reducing agents and usually correspond to redox potentials in which the prevalence of thermodynamic and kinetic traps is minimized (Kibria and Lees 2008). Unfortunately, these concentrations should be empirically and individually adjusted for each particular disulfide-rich protein model under study. Despite different redox conditions might result in extremely different refolding rates and efficiencies for a given protein, it is important to note that, as a rule, the overall folding pathway of the protein remains essentially unaffected. This is best demonstrated in the case of oxidative folding of hirudin (Chang 1994). In other words, the conformational preferences, the identity, and the progression of intermediates that populate the folding reaction of a disulfide-rich protein are likely intrinsic­ properties depending on its specific primary sequence and mostly independent of the solution conditions, which affect mainly the folding kinetics.

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Although, in principle, during in vitro oxidative folding experiments one would like to approximate physiological conditions by reproducing the ionic strength, pH, and redox potential of biologically relevant setting, still little is known on the ­specific environment an unfolded and reduced disulfide-rich protein faces inside the cell or extracellularly. Importantly, natural redox agents such as ascorbate, tocopherol, and vitamin K are emerging as efficient catalyzers of oxidative folding ­reactions both in  vivo and in  vitro at physiological concentrations (Saaranen et  al. 2010; Margittai et al. 2009), which suggest links between oxidative protein folding and redox cellular metabolism.

1.4 Diversity of Oxidative Folding Pathways Beginning with the characterization of the oxidative folding pathway of bovine trypsin inhibitor (BPTI) by Creighton in the mid-1970s (Creighton 1974), the folding and unfolding reactions of a large number of disulfide-containing proteins have been studied (Arolas et al. 2006; Mamathambika and Bardwell 2008). Surprisingly, these studies illustrated a high degree of diversity of folding pathways, especially for small disulfide-rich proteins (Arolas et al. 2006). The diversity of folding mechanisms has precluded transforming this vast amount of detailed experimental information into a unified folding theory that might allow us to somehow predict the folding pathway from the primary sequence. More significantly, even with proteins which share sequence homology and similar 3D structure, it is impossible to forecast whether they would fold by similar pathway. This is best illustrated by the cases of tick anticoagulant peptide (TAP) and BPTI, two proteins that share very similar conformation and identical disulfide connectivity but exhibit strikingly different oxidative folding pathways (Weissman and Kim 1991; Chang 1996; Chang and Li 2005). An oxidative folding pathway is characterized by three main features: (a) the level of the heterogeneity of the folding intermediates that populate along the folding pathway, (b) the prevalence of intermediate species displaying native disulfide bonds and native-like structures, and (c) the accumulation along the folding reaction of fully oxidized scrambled isomers containing at least two non-native disulfides (Chang 2004, 2008). The two extreme mechanisms of oxidative folding are illustrated by (a) proteins that fold through a selected number of intermediates containing exclusively native disulfide bonds and (b) proteins fold via a highly heterogeneous population of intermediates containing mostly non-native disulfides, including fully oxidized scramble isomers. For many proteins, folding pathways share common characteristics of both extreme models.

1.4.1 Folding Through Native Intermediates: The Case of Leech-Derived Trypsin Inhibitor BTPI represents the best characterized model of small disulfide-rich protein which folds through a limited number of intermediates that acquire predominantly native

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Fig.  1.3  Oxidative folding of LDTI. (a) Ribbon plot of the 3D structure of LDTI (3 disulfide bonds and 46 residues). The Protein Data Bank ID is 1LDT and the figure was prepared with PyMOL. The native disulfide bonds are indicated. (b) Scheme of the oxidative folding pathway of LDTI. R and N correspond to the fully reduced/unfolded and native states, respectively. The disulfide pairings of major folding intermediates are indicated. 1S is an ensemble of intermediates containing one disulfide bond. Species in which all the formed disulfide bonds are native are shown in gray and the rest in black

disulfide connectivity and native-like local structures. The folding pathway of BPTI has been widely discussed and reviewed (Creighton 1979, 1990; Creighton and Goldenberg 1984; Weissman and Kim 1991, 1992; Dadlez 1997). Therefore, in this chapter we have chosen to exemplify the folding behavior of this type of proteins, which we will denote as BPTI-like, by describing the folding pathway of leech-derived trypsin inhibitor (LDTI), characterized recently from the kinetic and structural point of view by one of our groups. LDTI is Kazal-type protease inhibitor isolated from the medicinal leech Hirudo medicinalis (Sommerhoff et al. 1994; Auerswald et al. 1994; Arolas et al. 2006). It binds tightly to human tryptase, a trypsin-like serine proteinase involved in allergic and inflammatory diseases. LDTI consists of 46 residues and contains three disulfide bonds (Cys4–Cys29, Cys6–Cys25, and Cys14–Cys40) (Fig. 1.3). LDTI folds into a defined conformation ­comprising a short central a-helix and a small triple-stranded antiparallel b-sheet (Di Marco and Priestle 1997; Stubbs et al. 1997; Muhlhahn et al. 1994). The first two disulfide bonds stabilize the a-helix by connecting

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this secondary structure ­element to the N terminus of the inhibitor forming a CSH motif that is also shared by other bioactive peptides. The third disulfide stabilizes the small b-sheet by linking­ the b1-strand and the end of the b3-strand. The folding of LDTI from the initially denatured and reduced state to the native conformation proceeds through a sequential oxidation of its cysteine residues (Fig. 1.3). Up to 74 different folding intermediates can theoretically populate along the oxidative folding pathway of LDTI. Nevertheless, similar to the case of BPTI, only five of these disulfide isomers accumulate significantly (Arolas et  al. 2008a). The initial oxidation of one of the LDTI disulfides renders an ensemble of 1S intermediates that rapidly reaches equilibrium. The distribution of disulfide bonds in this population does not obey to loop entropy contributions, but rather a preferential accumulation of the native bond linking b1- and b3-strand is observed. Structural studies of this 1S intermediate (PantojaUceda et al. 2009) indicate that, very likely, the triple-stranded antiparallel b-sheet of the inhibitor is already formed in this form. Secondary structure preferences might promote the establishment of transient interactions between the b-sheet elements and as a result Cys14 and Cys40 would come close to each other preferentially, relative to the other free Cys residues, allowing the formation of Cys14–Cys40 native disulfide bond. The new disulfide bond would significantly stabilize the preformed b-sheet and in turn this stable secondary structure would preferentially protect the disulfide from subsequent reduction reactions thus facilitating in kinetic terms the accumulation of this species as a major folding intermediate in the 1S ensemble. The formation of the native bond in the b-sheet of LDTI illustrates how, in BPTI-like proteins, conformational folding and native disulfide bond formation are tightly connected processes during oxidative folding. Only three 2S folding species accumulate in the oxidative folding of LDTI. Two of them, (Cys4–Cys29, Cys14–Cys40) and (Cys6–Cys25, Cys14–Cys40), originate directly from the further oxidation of the predominant 1S folding intermediate (Cys14–Cys40). The rest of 1S intermediates rearrange their disulfide bonds and oxidize their free thiols to form (Cys4–Cys29, Cys6–Cys25), a 2S intermediate which lacks the disulfide bond connecting the b-sheet. Importantly, only native ­disulfide bonds are present in the three LDTI 2S intermediates. This implies that, in principle, the three 2S have the potential to become productive species, since the direct oxidation of their remaining two free cysteines would form a natively bound, stable, and functional inhibitor in all cases. However, the experimental data clearly indicate that only (Cys6–Cys25, Cys14–Cys40) is the productive intermediate which is able to reach native LDTI. This suggests that the other two native-like 2S intermediates need to reshuffle their disulfide bonds to render the productive 2S species prior to their conversion to the native structure. In (Cys4–Cys29, Cys6–Cys25) the formation of the third disulfide bond is kinetically impeded whereas in (Cys4– Cys29, Cys14–Cys40) the free Cys25 is highly reactive, promoting the reshuffling of the preformed disulfide bonds instead of bonding to Cys6, which is in fact far away in this particular conformer. In summary, LDTI fold via BPTI-like mechanism in which the folding pathway comprises only four natively bonded intermediates without significant accumulation of scrambled forms. In BPTI-like proteins, the low complexity of the conformational space that the polypeptide chain has to explore to attain the native conformation makes the folding reaction fast and highly efficient.

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Fig. 1.4  Oxidative folding of hirudin. (a) Ribbon plot of the 3D structure of hirudin (3 disulfide bonds and 65 residues). The Protein Data Bank ID is 4HTC and the figure was prepared with PyMOL. The native disulfide bonds are indicated. (b) Scheme of the oxidative folding pathway of hirudin. R and N correspond to the fully reduced/unfolded and native states, respectively. 1S and 2S correspond to ensembles of intermediates containing one and two disulfide bonds, respectively. 3S indicates scrambled species in which all the cysteines are oxidized but they contain at least two non-native disulfide bonds. Species in which all the formed disulfide bonds are native are shown in gray and the rest in black

1.4.2 Folding Through Scrambled Isomers: The Case of Hirudin Similar to LDTI, hirudin is a protein isolated from the medicinal leech H. medicinalis (Chang 1983). Hirudin is a 65-residue long thrombin inhibitor that folds into two structurally and functionally distinguishable domains (Fig.  1.4). It consists of an N-terminal globular domain formed by a four-stranded b-sheet and stabilized by the presence of three disulfide bonds (Cys6–Cys14, Cys16–Cys28, and Cys22–Cys39). It also comprises an acidic C-terminal domain devoid of any regular secondary structure (Fig. 1.4) (Folkers et al. 1989; Rydel et al. 1990; Grutter et al. 1990). In contrast to LDTI, the oxidative folding reaction of hirudin is characterized by the absence of a predominant folding route, without any preferential accumulation of folding intermediates (Fig. 1.4) (Chatrenet and Chang 1992, 1993; Chang 1994). Apparently, the folding of hirudin from its initially denatured and reduced state is independent of any conformational constraint and responds to a “trial-and-error” process in which all the free cysteines become similarly involved in complex ­disulfide reshuffling reactions that finally result in the formation of the native state. Accordingly, the metastable intermediates that accumulate in the folding reaction of this protease inhibitor correspond mainly to 3S-scrambled isomers, that is, hirudin species with at least two non-native disulfide bonds. The folding of this kind of

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proteins, which we denote as hirudin-like proteins, comprises two different steps: an initial stage characterized by nonspecific packing into collapsed structures and a final stage in which these conformations consolidate to form the native functional protein. The first step consists of the sequential oxidation of hirudin-free cysteines to render equilibrated ensembles of 1S, 2S, and scrambled 3S intermediates. All these populations display a high degree of conformational heterogeneity without any apparent bias in the intermediate distribution resulting from protein structural propensities or noncovalent contacts. The theoretical number of 1S and 2S species for a three disulfide-containing protein is 60, from these, at least 30 have been shown to accumulate in the folding reaction, and 11 of the 14 theoretical 3S scrambled isomers have been identified (Chang et al. 1995). This sums ~40 folding intermediates. Therefore, the number of transient species that accumulate in the oxidative folding of hirudin is one order of magnitude higher than those populating the folding reaction of LDTI, strongly suggesting that the forces driving the packing-stage in hirudin have a nonspecific nature and probably respond to the collapse of previously exposed hydrophobic side chains. This lack of selectivity in the side chain and backbone interactions results in an almost unbiased formation of scrambled forms containing most of the possible disulfide bond combinations at this stage. In these species, all the six cysteine residues are involved in disulfide bonds and accordingly no free thiolates exist that can attack the preformed disulfides and promote their reshuffling toward new energy minima in the conformational space. This is the ­reason why scrambled isomers act as major kinetic traps, their conversion into the native structure being the rate-limiting step in the pathway. Accordingly, the consolidation step is strongly accelerated by the presence of thiol catalysts that allow newly formed scrambled isomers to initiate the required disulfide rearrangements in order to attain the native connectivity. In the past, it has long been debated if scrambled isomers constitute dead-end species or are instead on-pathway intermediates. It is now generally accepted, that they are essential and productive species in the folding pathways of hirudin-like proteins. In hirudin-like proteins, the formation of the native species from scrambled forms requires the establishment of nativelike noncovalent interactions. However, in contrast to BPTI or LDTI, there is apparently no sequential or even a preferential pathway for the formation of such contacts, in such a way that the consolidation stage follows again a “trial-and-error” process which renders the overall folding of hirudin-like proteins slow and inefficient.

1.4.3 Mixed Oxidative Folding Pathways: The Case of Epidermal Growth Factor Folding pathways of many proteins exhibit both BPTI-like and hirudin-like mechanisms. One of these cases is the folding reaction of epidermal growth factor (EGF). EGF constitutes the founding member of a family of diverse proteins having very similar structure but different function. EGF is a 53-residue protein consisting of

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Fig. 1.5  Oxidative folding of EGF. (a) Ribbon plot of the 3D structure of EGF (3 disulfide bonds and 53 residues). The figure was prepared with PyMOL. The native disulfide bonds are indicated. (b)  Scheme of the oxidative folding pathway of EGF. R and N correspond to the fully reduced/ unfolded and native states, respectively. The disulfide pairings of the major folding intermediates is indicated. 1S is an ensemble of intermediates containing one disulfide bond. 3S indicate scrambled species in which all the cysteines are oxidize but they contain at least two non-native disulfide bonds. Species in which all the formed disulfide bonds are native are shown in gray and the rest in black

three different loops stabilized by three intramolecular disulfide bonds (Fig.  1.5) (Montelione et al. 1992; Ogiso et al. 2002). The N-terminal A loop is linked by the Cys6−Cys20 disulfide bond. The B loop forms a b-hairpin constrained by a disulfide bond between Cys14 and Cys31. Finally, the C-terminal C loop includes the third Cys33−Cys42 disulfide linkage. The folding of reduced and denatured EGF to the native conformation involves initially the formation of a heterogeneous and transient population of 1S intermediates (Wu et al. 1998; Chang et al. 2001). This ensemble transforms rapidly into a single, predominant 2S disulfide species, which represents up to 85% of the total protein along the folding pathway. This predominant 2S intermediate comprises two native disulfide bonds (Cys14−Cys31 and Cys33−Cys42), indicating that a single oxidative reaction between the remaining two free cysteines (Cys6 and Cys20) in this conformer would allow formation of native EGF. Surprisingly, the two free thiols of Cys6 and Cys20 cannot react directly. Instead, kinetic analysis indicates that substantial unfolding and disulfide rearrangement through the formation of scrambled isomers is an obligatory step to reach the

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native conformation. The reorganization of these totally oxidized species into the native form constitutes the major rate-limiting step in the folding of EGF, ­significantly slowing the overall folding reaction (Fig. 1.5). Therefore, the folding of EGF resembles that of hirudin in the initial accumulation of a complex ensemble of intermediates and at the final rate-limiting step involving the conversion of scrambled isomers into the functional structure. However, like in the case of LDTI, the major folding intermediate corresponds to a reduced native-like bonded species. The case of EGF illustrates how the search for a functional conformation might result in a complicated folding scenario in which already folded native-like structural elements are not preferentially retained, as will be expected, but lost during the progress of the reaction. This suggests that the folding pathway is shaped mainly on the basis of final thermodynamic requirements.

1.5 Structural Characterization of Folding Intermediates For small proteins without disulfide bonds, the topology of the native structure usually suffices to determine, in general terms, the folding rate and the location of the rate-limiting step in the pathway (transition state), independently of the proteinspecific sequence (Plaxco et al. 1998; Guerois and Serrano 2000). The divergent folding behavior of BPTI and TAP clearly illustrates that this is not the case for small disulfide-containing proteins, because conformational folding is necessarily linked to disulfide-bond formation. In these proteins, the formation of native disulfide bonds can promote and accelerate the arrangement of local or global secondary or tertiary structural elements and conversely strong structural propensities might force the preferential formation of native disulfide bonds. However, it also occurs that the rather unspecific hydrophobic collapse that initiates the folding process of many proteins lead to the formation of wrongly paired disulfide bonds which, by  restraining the polypeptide chain would prevent the subsequent formation of native-like structural elements. In turn, the conformational properties of both natively and non-natively connected folding intermediates affect the reactivity, accessibility, and proximity of their free cysteines and disulfide bonds, making very difficult to predict the flow of folding species along the pathway. Because in disulfide-rich proteins, neither the sequence nor the native structure appear to be informative by themselves about the conformational space these proteins have to explore to fold into the native conformation, we can only try to understand it by exploring at the atomic level the specific structural properties of the metastable folding intermediates that populate along the folding course. Chemically inert analogs for structural studies have been usually obtained by derivatization of the free thiols present in the intermediate (Kortemme et al. 1996). However, the packing of these disulfide intermediates is usually affected by the size and charge of the blocking reagent. Mutation of the intermediate free cysteines by protein engineering to alanines or serines generates inert analogs, which are expected to closely mimic the conformational properties of folding intermediates (Laity et  al. 1997;

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Fig. 1.6  Solution structures of genuine folding intermediates of oxidative folding. Ribbon plots of the 3D average structures of major folding intermediates of (a) LCI, (b) MCoTI-II, (c, d) TCI, and (e–g) LDTI, respectively. The Protein Data Bank ID codes are as follows: 1ZFL, 2PO8, 2K2Y, 2K2Z, 2KMP, 2KMQ, and 2KMR, respectively. The figure was prepared with PyMOL

Shimotakahara et al. 1997). However, the hydrophobic character of alanine might further stabilize the analog relative to the intermediate when the original ­thiols are buried in the structure and destabilize entropically the protein when they free cysteines that are originally exposed (Arolas et  al. 2009). The opposite effect is expected for cysteines to serines substitutions. To avoid these problems, efforts are now being focused on solving the structure of genuine folding intermediates isolated directly from the oxidative folding or reductive unfolding reactions under acid conditions, in which isolated and unmodified intermediates are stable yet thiol/disulfide exchange is almost unnoticeable. Because these intermediates display significant conformational flexibility in comparison to the native state, they are not amenable to crystallographic and X-ray studies and their structures should be addressed in solution by NMR spectrometry of the previously 15N and/or 13C labeled species. Seven of such structures have been solved in the last 5  years. Protease inhibitors are in many cases small-disulfide inhibitors and therefore have become important models to study oxidative folding reactions. Accordingly, all genuine oxidative folding intermediates structurally solved to date correspond to conformers of protease inhibitors: LDTI (Pantoja-Uceda et  al. 2009), Leech carboxypeptidase inhibitor (LCI) (Arolas et al. 2005), Momordica cochinchinensis trypsin inhibitor II (MCoTI-II) (Cemazar et al. 2008) and tick carboxypeptidase inhibitor (TCI) (Arolas et al. 2008b). It is important to note that, although the number of free cysteines in these species varies from two to six, in all cases, the formed disulfide bonds display native connectivity (Fig. 1.6). However, it remains a challenging task in the characterization of structural properties of non-natively bound intermediates and specifically scrambled species. This is crucial since it would allow us to decipher at the molecular level why they are such strong kinetic and, in some cases, thermodynamic

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traps in the folding pathway. This is also important because the accumulation of scrambled isomers has been associated with pathogenic phenotypes in genetic diseases like hypercolesterolemia (Arias-Moreno et al. 2008). The structural characterization of the above-mentioned set of folding intermediates (Fig. 1.6) has allowed us to decipher some molecular clues to understand why a particular productive intermediate is able to convert to the native state through direct oxidation of their free thiols and why other species are nonproductive, meaning that they have to rearrange the connectivity of preformed disulfide bonds to render a productive folding intermediate in order to convert to the native species. Nonproductive folding intermediates, usually posses a native-like conformation in which both the free thiols and the preformed disulfide bonds are buried in the structure and have a low accessibility to the solvent, in such a way that they become almost insensitive to external thiols (Arolas et al. 2004). The protection of the free cysteines in a stable structure impedes its direct oxidation and the progress of the folding reaction. Accordingly, structured nonproductive folding intermediates act as strong kinetic traps. As stated above, the lack of one or more covalent disulfide bonds render these conformers flexible, relative to the native state. On one hand, this flexibility stabilizes the intermediate from the entropic point of view, but on the other hand it promotes structural fluctuations that allow it to escape from this particular energy minimum (Arolas et  al. 2005). These intermediates usually correspond to what Scheraga and coworkers defined as “disulfide-insecure” species (Narayan et al. 2003b; Creighton 1988b), because, due to their similar burial, conformational changes that expose the free thiols also expose at the same time the disulfide bonds, which are attacked by the reactive cysteines promoting their reshuffling instead of the formation of the missing native disulfide bonds. In productive folding intermediates, the already formed native disulfide bonds are preferentially protected in regular secondary structural elements or located at the hydrophobic core, in both cases with reduced accessibility or exchange with the solvent. This is in contrast to their free thiols, which are either already accessible to solvent or, more frequently, located in flexible and exposed protein regions. They are “disulfide-secure” species (Welker et al. 2001b; Narayan et al. 2003b), in the sense that the rigidity of the native-like intermediate scaffold protects the native disulfide bonds from conformational fluctuations. This would allow the remaining and solvent accessible free cysteines to undergo direct oxidation to form the native protein. A long pursued goal in the field of oxidative folding has been the characterization of the structural properties of the complete ensemble of species that populate the folding pathway of a disulfide-rich protein. This objective has been recently attained by one of our groups for the LDTI protein model. The obtained results confirm that conformational folding and the assembly of native disulfide bonds need to occur coordinately in a productive folding reaction (Pantoja-Uceda et al. 2009). Importantly, although structural propensities define essentially the number and conformation of the different folding intermediates in the pathway, this effect is kinetically controlled by the reactivity and accessibility of thiols and disulfide bonds in these molecules. Therefore, the interplay of structural and kinetic constraints appears as a recurrent factor governing the folding of disulfide-rich proteins.

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1.6 Concluding Remarks The vast amounts of data obtained from the study of the oxidative folding reactions of an important number of evolutionarily unrelated disulfide-rich protein models constitute the ground work for our future efforts to understand how the linear information contained in the protein sequence is precisely translated into the 3D conformation. A first step in this direction would be the understanding of the sequential and structural determinants that determine the heterogeneity of a protein folding pathway. In this respect, the resolution of an increasing number of genuine folding intermediates at atomic level is shedding light on the structural, thermodynamic, and kinetic factors shaping the complex interplay between conformational folding and disulfide formation. This suggests that it is likely that in the near future we can arrive at the construction of a clear and uniform description of the mechanism of oxidative protein folding.

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Plaxco KW, Simons KT, Baker D (1998) Contact order, transition state placement and the refolding rates of single domain proteins. J Mol Biol 277:985–994 Poland DC, Scheraga HA (1965) Comparison of theories of the helix-coil transition in polypeptides. J Chem Phys 43:2071–2074 Ptitsyn OB (1991) How does protein synthesis give rise to the 3D-structure? FEBS Lett 285:176–181 Rydel TJ, Ravichandran KG, Tulinsky A, Bode W, Huber R, Roitsch C, Fenton JW II (1990) The structure of a complex of recombinant hirudin and human alpha-thrombin. Science 249:277–280 Saaranen MJ, Karala AR, Lappi AK, Ruddock LW (2010) The role of dehydroascorbate in disulfide bond formation. Antioxid Redox Signal 12:15–25 Sanchez R, Pieper U, Melo F, Eswar N, Marti-Renom MA, Madhusudhan MS, Mirkovic N, Sali A (2000) Protein structure modeling for structural genomics. Nat Struct Biol 7(Suppl):986–990 Scheraga HA, Konishi Y, Rothwarf DM, Mui PW (1987) Toward an understanding of the folding of ribonuclease A. Proc Natl Acad Sci USA 84:5740–5744 Shimotakahara S, Rios CB, Laity JH, Zimmerman DE, Scheraga HA, Montelione GT (1997) NMR structural analysis of an analog of an intermediate formed in the rate-determining step of one pathway in the oxidative folding of bovine pancreatic ribonuclease A: automated analysis of 1H, 13C, and 15N resonance assignments for wild-type and [C65S, C72S] mutant forms. Biochemistry 36:6915–6929 Sommerhoff CP, Sollner C, Mentele R, Piechottka GP, Auerswald EA, Fritz H (1994) A Kazaltype inhibitor of human mast cell tryptase: isolation from the medical leech Hirudo medicinalis, characterization, and sequence analysis. Biol Chem Hoppe Seyler 375:685–694 Stubbs MT, Morenweiser R, Sturzebecher J, Bauer M, Bode W, Huber R, Piechottka GP, Matschiner G, Sommerhoff CP, Fritz H, Auerswald EA (1997) The three-dimensional structure of recombinant leech-derived tryptase inhibitor in complex with trypsin. Implications for the structure of human mast cell tryptase and its inhibition. J Biol Chem 272:19931–19937 Ventura S (2008) Oxidative protein folding: from the test tube to in vivo insights. Antioxid Redox Signal 10:51–53 Wedemeyer WJ, Welker E, Narayan M, Scheraga HA (2000a) Disulfide bonds and protein folding. Biochemistry 39:7032 Wedemeyer WJ, Welker E, Narayan M, Scheraga HA (2000b) Disulfide bonds and protein folding. Biochemistry 39:4207–4216 Wedemeyer WJ, Xu X, Welker E, Scheraga HA (2002) Conformational propensities of protein folding intermediates: distribution of species in the 1S, 2S, and 3S ensembles of the [C40A, C95A] mutant of bovine pancreatic ribonuclease A. Biochemistry 41:1483–1491 Weissman JS (1995) All roads lead to Rome? The multiple pathways of protein folding. Chem Biol 2:255–260 Weissman JS, Kim PS (1991) Reexamination of the folding of BPTI: predominance of native intermediates. Science 253:1386–1393 Weissman JS, Kim PS (1992) Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor. Proc Natl Acad Sci USA 89:9900–9904 Welker E, Narayan M, Wedemeyer WJ, Scheraga HA (2001a) Structural determinants of oxidative folding in proteins. Proc Natl Acad Sci 98:2312–2316 Welker E, Wedemeyer WJ, Narayan M, Scheraga HA (2001b) Coupling of conformational folding and disulfide-bond reactions in oxidative folding of proteins. Biochemistry 40:9059–9064 Welker E, Hathaway L, Scheraga HA (2004) A new method for rapid characterization of the folding pathways of multidisulfide-containing proteins. J Am Chem Soc 126:3720–3721 Wouters MA, Fan SW, Haworth NL (2010) Disulfides as redox switches: from molecular mechanisms to functional significance. Antioxid Redox Signal 12:53–91 Wu J, Yang Y, Watson JT (1998) Trapping of intermediates during the refolding of recombinant human epidermal growth factor (hEGF) by cyanylation, and subsequent structural elucidation by mass spectrometry. Protein Sci 7:1017–1028 Zavodszky M, Chen CW, Huang JK, Zolkiewski M, Wen L, Krishnamoorthi R (2001) Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V. Protein Sci 10:149–160

Chapter 2

The Case of Oxidative Folding of Ribonuclease A: Factors Impacting Fold Maturation of ER-Processed Proteins Mahesh Narayan

Abstract  Proteins that are membrane-bound or secreted outside the cell often possess disulfide bonds. The maturation of such proteins is termed oxidative protein folding and takes place within the endoplasmic reticulum (ER) of eukaryotic cells prior to their export. The events that comprise oxidative folding are complex. A variety of intrinsic and extrinsic factors finely orchestrate the formation of a biologically viable molecule almost from the time of its genesis in the ribosome. This chapter recapitulates our current understanding of factors impacting fold maturation of ER-processed proteins which emerges from a large number of in  vitro regeneration experiments performed in a number of laboratories on several small single-domain disulfide bondcontaining proteins. The focus here is on advances made in our understanding of oxidative folding through studies on the folding pathways of bovine pancreatic ribonuclease A which were primarily undertaken by the Scheraga laboratory over 30 years. In addition, laboratory techniques and biophysical and biochemical manipulations which facilitate the study of oxidative protein folding are also discussed. Keywords  Fold maturation • Thiol–disulfide exchange • Endoplasmic reticulum • Misfolding • Disulfide bond • Oxidative folding • Conformational folding • Trafficking

Abbreviations 1S 2S 3S

One-disulfide intermediates Two-disulfide intermediates Three-disulfide intermediates

M. Narayan (*) Department of Chemistry, The University of Texas at El Paso, 500 W. Univ. Ave., El Paso, TX 79968, USA e-mail: [email protected] R.J.Y. Chang and S. Ventura (eds.), Folding of Disulfide Proteins, Protein Reviews 14, DOI 10.1007/978-1-4419-7273-6_2, © Springer Science+Business Media, LLC 2011

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AEMTS 2-Aminoethyl methanethiosulfonate [(NH2) C2H5SSO2CH3] BPTI Bovine pancreatic trypsin inhibitor des [40, 95] RNase A Ribonuclease A lacking the disulfide bond between the cysteine residues denoted in the brackets des [65–72] RNase A Ribonuclease A lacking the disulfide bond between the cysteine residues denoted in the brackets DTT ox Oxidized dithiothreitol DTT red Reduced dithiothreitol EDTA Ethylenediaminetetraacetic acid GSH Reduced glutathione GSSG Oxidized glutathione HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High-performance liquid chromatography MS Mass spectrometry RNase A Bovine pancreatic ribonuclease A Tris Tris(hydroxymethyl)aminomethane

2.1 Introduction The eukaryotic cell contains a specialized compartment, the endoplasmic reticulum, which is charged with the task of ensuring the maturation of those proteins that reside within membranes of cells, are secreted into specialized compartments, or secreted outside the cell (Tu and Weissman 2004). Such proteins normally contain disulfide bonds formed by oxidation of cysteine residues (Narayan et al. 2000). The acquisition of the biologically active, tertiary structure of such proteins is a complex event termed as oxidative folding (or oxidative regeneration) (Narayan et  al. 2000; Arolas et  al. 2006; Mamathambika and Bardwell 2008; Wedemeyer et  al. 2000; Welker et  al. 2001a, b; Chang 2004; Chang 2008; Woycechowsky and Raines 2000). It involves the acquisition of the native set of disulfide bonds from the fully reduced nascent polypeptide chain and a conformational folding reaction which preserves the native disulfide bonds within stable tertiary structure (Welker et al. 2001b; Wedemeyer et al. 2002). The endoplasmic reticulum in contrast to the cytosol has an environment that is oxidizing in nature and facilitates the formation of disulfide bonds. The chief redox couple in the ER is glutathione with the ratio of GSSG to GSH varying between 1:1 and 1:3, in contrast to 1:30 for the cytosol (Hwang et al. 1992). In addition to glutathione, there are a variety of chaperones and cofactors that facilitate the maturation of ER-processed substrate proteins prior to their secretion. Principal among these is the ER-resident oxidoreductase chaperone, protein disulfide isomerase (PDI) (Gilbert 1998; Wilkinson and Gilbert 2004; Hawkins and Freedman 1991; Fewell et al. 2001; Shin and Scheraga 2004; Tian et al. 2006; Xiao et al. 2004), which is similar in function to the prokaryotic DSB oxidoreductases (Hiniker and Bardwell 2003; Gleiter and Bardwell 2008). Lastly, molecular oxygen also plays a pivotal role in the ER by serving as the terminal electron acceptor via Ero1 (Romisch 2004, 2005).

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The following sections describe the process by which the nascent polypeptide oxidatively regenerates in the ER. A variety of biochemical and biophysical studies and methodological inroads, referenced herein, have facilitated our current understanding of this process. These studies have focused on what can now be termed as “model” or “classical” disulfide bond-containing proteins. The list of proteins include, but is not limited to, bovine pancreatic ribonuclease A (RNase A), onconase, hirudin, hen-egg white lysozyme, tick anticoagulant peptide, leech carboxypeptidase, bovine pancreatic trypsin inhibitor (BPTI), etc. (Weissman and Kim 1995; Arolas et al. 2004, 2006; Narayan et al. 2000; Chang and Li 2005; Salamanca and Chang 2005; Chang et al. 2006; Lin and Chang 2007; Ardelt et al. 2008; Gahl et al. 2008; Mamathambika and Bardwell 2008). In this chapter, we have chosen to describe the folding pathway of RNase A as the paradigm for oxidative folding of ER-processed proteins (Narayan et al. 2000). This is especially appropriate considering that the protein adopts several different pathways by which it regenerates, possessing features that are representative of facets prevalent in the oxidative folding landscape of other disulfide bond-containing proteins. The chapter is divided according to the stages observed in the oxidative folding pathway of RNase A which generally applies to other proteins as well. In each step of the folding process factors that impact the regeneration rate and regeneration pathway are discussed. Extensive work performed in the Scheraga laboratory in unraveling the regeneration pathway of RNase A is widely referenced. It is also becoming increasingly clear that despite the presence of a finely tuned mechanism complete with an orchestra of folding adjuvants and highly evolved folding pathways, the traffic that enters the ER does not always exit via the secretory pathway (Romisch 2004, 2005; Pal et  al. 2010a); the hazards are many and misfolding is common. Such misfolding, if terminal, becomes victim to the subcellular housekeeping machinery and exits the ER via the ERAD (ER associated degradation) pathway (Romisch 2004). Recent work in our lab is aimed at understanding how (mis)folding events within the ER can impact accumulation of cellular debris which is the bedrock of several neurodegenerative disorders (Wang and Narayan 2008; Pal et al. 2010a).

2.2 General Terms and Conditions Pertaining to the Oxidative Folding of Bovine Pancreatic Ribonuclease A Bovine pancreatic RNase A is a 124-amino acid endonuclease that cleaves single-stranded RNA (Crook et al. 1960) (Fig. 2.1). This activity was leveraged to determine refolding rate and yield during the early years when folding studies on this protein were initiated (Konishi et al. 1982). It is a member of the ribonuclease superfamily which includes onconase, RNase H, and RNase T1 among others (Bientema 1998). RNase A has four disulfide bonds at positions: Cys26– Cys84, Cys58–Cys110, Cys40–Cys95, and Cys65–Cys72. The tertiary structure is a two-layer a + b protein folded in half, with a deep cleft for binding RNA substrate.

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Fig. 2.1  Bovine pancreatic ribonuclease A (ribbon diagram)

2.2.1 Terminology Before proceeding toward description of its folding pathway, we describe terminology that the reader should become familiarized with: R, refers to the fully reduced RNase A. Thus, R would constitute unfolded RNase A possessing eight cysteines; des [40, 95], ribonuclease A lacking the disulfide bond between the cysteine residues denoted in the brackets; des [65, 72], ribonuclease A lacking the disulfide bond between the cysteine residues denoted in the brackets; 1S, one-disulfide-containing intermediates; 2S, two-disulfide-containing intermediates; 3S, three-disulfide-containing intermediates; 4S, four-disulfide-containing intermediates (also known as scrambled or dead-end species); nS, an isomeric ensemble of species containing n number of disulfide bonds; N, native protein.

2.2.2 Folding Conditions Folding conditions at which RNase A regeneration was studied in the Scheraga laboratory generally involved pH values at which chemical reactions required for successful disulfide bond formation could be easily captured. A folding buffer at

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pH 8 and composed of 100 mM Tris–HCl and 1 mM EDTA was mostly employed. The choice of redox reagent, though initially GSH/GSSG, was dithiothreitol (DTT red)/trans-4,5-dihydroxy-1,2-dithiane (DTT ox). DTT ox/red was selected over glutathione for two reasons. (1) DTT ox is a weak oxidizing agent. Therefore, kinetic control over the folding is possible over a wide range of DTT ox concentrations. Furthermore, (2) DTT red is an unusually strong reducing agent, owing to its high conformational propensity to form a six-membered ring with an internal disulfide bond (Rothwarf and Scheraga 1992). Thus, intermediate mixed-disulfide states between protein thiols and DTT is unstable and poorly populated because the second thiol of DTT has a high propensity to close the ring, forming oxidized DTT and leaving behind a reduced disulfide bond (Rothwarf and Scheraga 1992, 1993a). This facilitates analytical techniques such as HPLC fractionation and mass analysis of intermediates with differing numbers of disulfide bonds (Rothwarf and Scheraga 1993a, b, c, d). A noteworthy disadvantage of DTT red is that it being a strong reducing agent, the regeneration rate of the reaction at higher protein concentrations becomes sensitive to the reducing equivalents (i.e., DTT red) generated during the course of the reaction. This can be overcome by maintaining low concentrations of the protein under study (Rothwarf and Scheraga 1993a).

2.2.3 Analytical Techniques and Methods Used to Study Regeneration Rates and Pathways Characterization of fold regeneration requires a good analytical handle on kinetics of formation and disappearance of all intermediates and the formation of the native species. This requirement in turn necessitates the separation of intermediates from one another, ability to isolate them, and requires their structural characterization. The Scheraga laboratory has pioneered the use of 2-aminoethyl methanethiosulfonate (AEMTS; Bruice and Kenyon 1982), a potent thiol-blocking agent, to facilitate analytical study of the regeneration process (Rothwarf and Scheraga 1993b). AEMTS attaches to free thiols and imparts a charge of +1 per blocked thiol. Blocking the thiol is necessary to determine the reaction progress. However, use of AEMTS facilitates fractionation of intermediates by charge. On a cation-exchange column, the AEMTS-blocked R with a positive charge of +8 relative to N is most strongly retained relative to other intermediates (Fig. 2.2). The order of elution of the intermediates is N, 3S, 2S, 1S, and R which are well separated on a strong cationexchange column (Xu et  al. 1996). Furthermore, their separation into isomer ensembles upon AEMTS-blocking and fractionation (see Sect.  2.3.1) facilitates characterization of their disulfide bond connectivity, mass , and kinetic study (order and rate of appearance/disappearance) (Rothwarf and Scheraga 1993a; Xu et  al. 1996; Volles et al. 1999; Xu et al. 2005) (Fig. 2.2). A reduction-pulse is a convenient tool to convert unstructured intermediates to the fully reduced protein (R) by the application of a small amount of reducing

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M. Narayan equilibrium constants rate-determining step pathway rate-constants

N

intermediates DTTred GdnSCN

reduction

data analysis

N R

DTTox at 25 OC, pH 8.0

regeneration

fractionation R

3S

N

ion-exchange R HPLC N

R AEMTS-block quenching

1S

2S

4S

3S

1S

2S

4S

Fig. 2.2  Typical regeneration schematic of a multidisulfide protein (e.g., RNase A). RNase A is first fully reduced and unfolded by application of strongly denaturing and reducing conditions. The fully reduced protein is then introduced into refolding conditions and aliquots are periodically withdrawn, blocked with AEMTS, and fractionated using cation-exchange chromatography. Kinetic analysis of the folding rate is performed by integrating the peak areas under the chromatogram as previously described (Rothwarf and Scheraga 1993b)

equivalents (1–5 mM DTT red) for a short period of time (1–2 min) (Rothwarf et al. 1998a; Narayan et al. 2000; Narayan et al. 2008). This simplifies the regeneration chromatogram and facilitates the collection of structured intermediates for further characterization (Welker et al. 1999). It has also been successfully applied to characterize and contrast the regeneration pathways of proteins within and across families and to expedite analysis of the regeneration rates of proteins (Welker et al. 2004; Wang and Narayan 2008).

2.3 The Folding Pathways of RNase A RNase A folds via multiple (parallel) pathways as shown in Fig. 2.3 (Rothwarf et al. 1998a, b). In order to understand the mechanism(s) by which the native fold is generated, the folding of this protein from its fully reduced state I can be divided into three stages: initial stage of oxidative folding; the rate-determining step in regeneration; and post-folding steps (Narayan et  al. 2000). The folding pathway of most disulfide-proteins can also be divided into the above stages with minor differences

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R↔1S ↔ 2S ↔ 3S ↔ 4S 3S*

Des [40-95] Des [65-72]

N Fig. 2.3  Regeneration scheme of RNase A resulting in the formation of the native protein (N) from its unstructured precursors (R, 1S, 2S, 3S, and 4S). The rate-determining step involves the formation of two structured 3S* species (des [40–95] and des [65–72]) from its unstructured 3S ensemble isomers. The two 3S* species then form N by the oxidation of the remaining cysteines

observed among proteins (Narayan et al. 2000). Various factors impact the folding of this protein (and by extension others) during each step of the folding process.

2.3.1 Initial Stages of Oxidative Folding The initial stages of the oxidative regeneration involve sequential oxidation of the cysteines of the fully reduced polypeptide (R) by an extrinsic oxidizing agent (Rothwarf and Scheraga 1993a, b, c, d). In this stage, the rates of formation of disulfide bonds from cysteines are governed by pH of the folding medium and choice of the oxidizing agent (redox reagent). Typical oxidative folding conditions include an appropriate choice of pH (between 7 and 9; this is important because the thiolate is the active form of the sulfhydryl capable of being involved in thiol–­ disulfide exchange reactions) (Rothwarf and Scheraga 1993a). The pKas of free thiols generally vary around 8.3. At a pH of 8.3, 50% of the cysteines would be in the active, deprotonated form and the formation of disulfide bonds would have a certain rate. However, if the pH is lowered by one unit, there would be a tenfold increase in protonation of thiolates to form thiols resulting in a decreased oxidative folding rate. Nevertheless, pH serves as a valuable handle to study intermediates in oxidative folding because control over the reaction rate can be achieved by tuning the pH of the folding buffer. Of the large number of available redox reagents (Singh et al. 1995; Gough et al. 2006), the Scheraga laboratory has resorted to the use of DTT ox after initially working with glutathione because it does not form mixed disulfides with protein thiols (Rothwarf and Scheraga 1992). DTT ox is also a relatively weak oxidizing agent and thus incapable of rapidly oxidizing R to a fully oxidized state. Again, the use of a relatively weak oxidizing agent, compared to GSSG, provides greater control over the folding rate. Under folding conditions (pH 8, 100 mM DTT ox, 1 mM EDTA), as regeneration progresses, a percentage of R becomes oxidized to 1S; a fraction of the latter species then becomes oxidized to 2S and so on, until R, 1S, 2S, 3S, and 4S are populated

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(Rothwarf and Scheraga 1993a, b, c, d). The ratio of these populations depends upon pH, choice of oxidizing agent, and sampling time (after initiation of regeneration). As discussed, a strong oxidizing agent would favor the populations of species with larger number of disulfide bonds (i.e., 4S and 3S over 2S and 1S). The reduction of DTT ox by the protein thiolates produces DTT red, a strong reducing agent. This can result in 1S → 4S becoming reduced. The tendency to become reduced is further promoted by the fact that in RNase A and many other proteins, R → nS is not structured and thus susceptible to reduction (Narayan et al. 2000). The potency of reducing equivalents generated during the course of the regeneration process therefore depends upon the nature of the redox system and also on the protein concentration and numbers of protein cysteines. High protein concentration (i.e., high concentrations of R) coupled with a large number of free cysteines can arrest regeneration from progressing to completion because of the high concentration of reducing equivalents generated. 2.3.1.1 Nature of the nS Species of RNase A The 1S → 4S species are ensemble mixtures of such species (Xu et al. 1996; Volles et al. 1999). In case of a four-disulfide-bond-containing protein such as RNase A, the 1S ensemble consists of 28 isomers that are in equilibrium with one another. Each isomer interconverts to another via intramolecular thiol–disulfide exchange reactions between existing disulfide bonds and free cysteines (Xu et  al. 1996). Similarly, 210 species in the 2S ensemble, 420 in the 3S ensemble, and 104 isomers in the 4S ensemble are predicted to exist (Rothwarf and Scheraga 1993a). Nevertheless, the isomers are not present in equal concentrations and the factors affecting their distribution are discussed next. 2.3.1.2 “Structure” and Disulfide-Connectivity During the Initial Stages of Regeneration A variety of structural probes and application of enzyme activity assays have revealed that little or no structure exists in the R → 4S ensembles of RNase A (Saito et al. 2001; Narayan et al. 2003). Nevertheless, these intermediates are not “random coils” but instead can be described as “statistical coils” (Matheson and Scheraga 1978). Fully reduced RNase A was examined thoroughly by a variety of techniques and found to be “locally ordered.” Fluorescence labeling of select residues and distance measurements revealed a native-like trace of the backbone with select side-chain distances not very different from the native protein (Navon et  al. 2001a, b). Nevertheless, when “native tendency” of R was measured, the value was 0.6% suggesting that cysteines destined to become paired in the native structure were not spatially aligned in R (Narayan et al. 2003). (Note: In the oxidative folding pathways of certain proteins, predominant occurrence of native disulfide bonds has been observed.) A valuable tool has been developed to assess the “native tendency” of the fully reduced forms of ER-processed proteins (Narayan et al. 2003). The tool permits

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estimation of the impact of chain conformation, sans disulfides, on the tendency to form native disulfides without resorting to corrective thiol–disulfide reactions. The “native tendency factor” therefore is a report on the “native-like” orientation of the chain in its fully reduced form which suggests that the amino acid residues are involved in promoting native interactions in the initial stages of the folding process. Native tendency values can range from 0 to 100%. The acquisition of the first disulfide bond impacts the chain significantly. Chain entropy is lost upon loop (disulfide bond) formation (Flory 1953; Xu et al. 1996). An examination of the population distribution of one disulfide connectivities in the 1S ensemble of RNase A indicates that the distribution of disulfide bonds in the 1S ensemble are mostly entropically ordained (Xu et al. 1996), i.e., loops that result in the least loss of chain entropy are those that are favored to the greatest extent. Therefore, proximal cysteines tend to form disulfide bonds to a greater extent than cysteines that are further apart in primary sequence. A similar trend was observed upon mapping the 2S ensemble of RNase A (Volles et al. 1999). Nevertheless, in both populations, deviations were found from loop entropy correlations. These deviations were attributed to enthalpic interactions that might bring sequentially distant residues in close special proximity and reduce the loss in entropy that might be encountered by the chain in the absence of such interactions (Xu et al. 1996; Volles et al. 1999). Such enthalpic interactions can include hydrophobic interactions, electrostatic interactions, etc. which would likely manifest themselves in a polar environment prior to the formation of a disulfide bond. The loss in chain entropy upon formation of a disulfide bound would thus be reduced compared to conditions in which such enthalpic interactions were absent. Notably, the 65–72 disulfide bond of RNase A is favored well-above the entropically ordained value and fourfold over the entropically equi-probable 58–65 disulfide bond (Xu et  al. 1996). Disulfide bond distribution of a synthetic 58–72 peptide fragment of RNase A had revealed a similar fourfold bias of the 65–72 disulfide over the 58–65 disulfide bond (Altmann and Scheraga 1990). These results suggested that the enthalpic interactions favoring the formation of the 65–72 disulfide bond over other reside within the 58–72 peptide segment of RNase A. Structural, computational, and mutational studies of this peptide and native RNase A suggested the presence of a type III b-turn encompassing Cys65Lys66-Asn67-Gly68 which reduced the entropy loss associated with the formation of 65–72 disulfide bond, thus favoring it over 58–65.

2.3.2 The Rate-Determining Steps in the Oxidative Folding of RNase A The unstructured ensembles of RNase A initially populate the folding landscape of the protein. Gradually, there is the appearance of enzymatic activity. Chromatographic separations, mass spectrometric studies, disulfide mapping, and structural studies reveal that the appearance of the native protein (N) is preceded by the appearance of two structured (native-like) three disulfide bond-containing isomers from their unstructured 3S isomers (Rothwarf et  al. 1998a, b). These intermediates were

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Fig. 2.4  Schematic of the rate-determining step in the regeneration of RNase A. The 3S ensemble consists of 416 non-native disulfide bond-containing isomers and four native disulfide bond-containing isomers in equilibrium with each other. The chemical exchange between these species is shown where a 3Snonnatbnd species reshuffles to form an intermediate with three native disulfide bonds (3Snatbnd). The physical conformational folding of 3Snatbnd to 3S* results in the protection of native disulfide bonds within tertiary structure and prevention of further isomerization reactions; conformational folding is pivotal to regeneration because it separates a structured intermediate from the pool of unstructured intermediates. The overall 3S → 3S* step is rate determining because the formation of 3Snatbnd is stochastic and because the formation of 3S* in RNase A is proline-isomerization limited and hence slow

determined to be des [40–95] and des [65–72] (see Sect. 2.2.1) and referred to as 3S* species (Laity et al. 1997; Shimotakahara et al. 1997). Fitting of the regeneration data as well as isolated 3S → 3S* experiments demonstrated that the reshuffling and conformational folding of the 3S ensemble to form the two 3S* species was the ratedetermining step in RNase A regeneration (at pH 8, 100 mM DTT ox, 100 mM Tris– HCl, 1 mM EDTA) (Rothwarf et al. 1998b). Note that the rate-determining step is dependent upon the folding conditions. It is possible to conduct regeneration experiments under a highly oxidizing environment, which would rapidly oxidize R to the scrambled (dead-end) 4S ensemble. Under such conditions, the rate-determining step is likely to be the reduction of 4S back to 3S which is necessary before the 3S → 3S* reaction can take place. Similarly, for the purpose of argument, under a reducing environment, the rate-determining step would be the oxidation of R to 1S and so on. Under physiological conditions, the rate-determining step might also be influenced by the presence of cofactors and chaperones. Nevertheless, in vitro folding studies have permitted us to infer important facets of the regeneration process and the rate-determining step of the regeneration pathway. In RNase A and several other proteins, the rate-determining step involves a competition between chemical and physical reactions (Welker et al. 2001a, b; Wang and Narayan 2008; Pal et al. 2010b) (Fig. 2.4). The chemical reactions are thiol–disulfide

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exchange reactions that are necessary to populate an intermediate with the native set, or native subset, of disulfide bonds. Such an intermediate can fold in a physical conformational folding reaction and protect its newly formed native disulfide bonds from attack by solvent thiolates (redox agents) or intramolecular thiolates. The formation of the native protein or native-like structured intermediate first requires the formation of a critical number of native disulfide bonds. In RNase A, this number is three (of the four possible disulfide bonds). However, of the 420 possible three disulfide bond-containing intermediates in an eight-cysteine protein such as RNase A, only four species possessing three native disulfide bonds exist. Thus, statistically, less than 1% (4/420) of all species in the 3S ensemble possesses native disulfide bonds. The process of forming the native-like 3S* intermediates in RNase A from its unstructured 3S ensemble isomers is depicted in Fig. 2.4 (Wang and Narayan 2008). The process of forming 3S* from its unstructured 3S precursors constitutes the ratedetermining step in RNase A folding (Rothwarf et al. 1998b). The four native-disulfide-bond-containing intermediates (3Snative) must first form from the pool of non-native-disulfide-bond-containing isomers through intramolecular thiol–disulfide exchange reactions (Fig. 2.4). The 3Snative intermediate is at the cross-roads of the rate-determining step. It has two possible fates. It can physically (conformationally) fold to form the structured (native-like) 3S* species. Or it can be attacked by the thiolates and become back-reshuffled and revert to the pool of 3Snonnative species (Saito et  al. 2001). The forward reaction is fruitful to the regeneration process because it removes the molecule from the “unstructured pool” of intermediates that constitute the folding landscape. This is because the newly formed native disulfide (as those in the 3Snative intermediate) disulfides become buried within the stable tertiary structure of the native-like intermediate and are protected from back-reshuffling reactions or attack by extrinsic redox reagents. Furthermore, the forward reaction results in a structure that has the remainder of the cysteines aligned to form disulfide bonds (Gonzalez et al. 2010). 2.3.2.1 Factors Influencing the Rate-Determining Step in the Regeneration of RNase A There are several factors impacting the rates of the chemical and physical halves of the rate-determining step. At pH 8, in RNase A ~30% of the 3Snative species back reshuffles to form 3Snonnative (Welker et al. 2001a; Saito et al. 2001). This is because conformational folding in RNase A is slowed by proline isomerization (Houry et al. 1994; Iwaoka et al. 1999). In RNase A, isomerization of three of the four prolines contribute significantly to the conformational folding rate (Schmid et  al. 1993; Schmid 2001). Therefore, RNase A conformational folding is multiphasic with discrete populations and lifetimes attributable to the dispositions of the prolines in the unfolded states of the molecule (Houry et al. 1994). If conformational folding is not initiated from the equilibrium unfolded state, then the conformational folding phases will have a different set of lifetimes. For example, if native RNase A (or 3S*) is

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introduced into unfolding conditions for a very brief period (