Molecular Bioelectronics

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Author: Claudio A. Nicolini | S. Carrara | P. Facci | V. Sivozhelezov | M. Adami | V. Erokhin | G. Picard | M. Sart

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MOLECULAR BIOELECTRONICS

Cover - logo of the EL.B.A. Foundation

MOLECULAR BIOELECTRONICS

Claudio Nicolini Institute of Biophysics, University of Genoa Italy

Scientific Singapore NewJersey*London Hong Kong

Published by World Scientific ~ b i i s ~ Co. n g Re. Ltd. P 0Box 128, Farrer Road, Singapore912805 USA oflee: Suite lB, 1060 Main Street, River Edge, NJ 07661 UK ofice: 57 Shclton Stnet,Covent Garden, London WC2H 9HE

British Library C a ~ ~ ~Data ~ ~ A catalogue record for this book is available from the British Library.

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~

b

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MOLECULAR BIOELECTRONICS Copyright 8 1996 by World Scientific Publishing Co. Re.Ltd. All rights reserved, This book or parts thereof; may not be reproduced in m y form or by m y mans, electronic or mechanical, including photocopying, recording or my information storage and retrieval system now known or to be invented, withut wriffenpermissionfrom the Publisher.

For phot~opyingof material in this voiume, please pay a copying fee through the Copyright ClearanceCenter, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher.

ISBN 981-02-2685-3

Printed in Singapore.

~

List of Contributors

Work written, coordinated and directed by: Claudio Nicolini

Full Professor and Director, Institute of Biophysics, School of Medicine, University of Genoa, Italy; President of Polo Nazionale Bioelettronica

With contributions and assistance for the indicated selected topics by: Manuela Adami

Technobiochip Scrl, Marciana (PAB)

Sandro Carrara

Institute of Biophysics, University of Genoa (conductive LB films)

Victor Erokhin

Technobiochip Scrl, Marciana (LB technology)

Paolo Facci

Institute of Biophysics, University of Genoa (STWAFM)

Gilles Picard

Technobiochip Scrl, Marciana (self-assembly)

Marco Sartore

Technobiochip Scrl, Marciana (transducers and biosensing elements)

Victor Sivozhelezov

Institute of Biophysics, University of Genoa (ab initio design)

Sergei Vakula

EL.B.A. Foundation, Portoferraio (editing and type-setting)

Acknowledgements We wish to thank Mrs. Cristina Berti, Marisa Chirninazzo, Mr. Fabrizio Nozza and Mr. Marco Zunino for their helpful cooperation in the preparation of this work. The work has been supported by Technobiochip (Marciana -LI, Italy), within the framework of the National Resexch Program 'Technologies for Bioelecuonics' sponsored by the Ministry of Universities and Technological and Scientific Research of Italy. We have to acknowledge also the support of the Polo Nazionale Bioelettronica- Scientific and Technological Park of the Elba Island (Portoferraio (LI), Italy), the EL.B.A. Foundation (Marciana Marina (LI), Italy) and the National Strategic Project 'Molecular Manufacturing' of the National Research Council of Italy.

CONTENTS List of Contributors

V

Acknowledgements

vii

. 2 . Active Bioelements 1 Introduction

1 5

Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Whole Cells . . . . . . . . . . . . . . . . . . . . . . . . . . .

.

5 5

9 13 14

15

3 Technologies 17 Langmuir-Blodgett and Its Modifications . . . . . . . . . . . . . . 18 Film Characterization . . . . . . . . . . . . . . . . . . . . . 24 Ellipsometry . . . . . . . . . . . . . . . . . . . . . . . 26 X-Ray Scattering . . . . . . . . . . . . . . . . . . . . . 26 Electron Diffraction and Microscopy . . . . . . . . . . . . . 31 Interferometry . . . . . . . . . . . . . . . . . . . . . . 31 Langrnuir-Blodgett Method . . . . . . . . . . . . . . . . . . 33 Langmuir-Schaefer Method . . . . . . . . . . . . . . . . . .33 Modified Langmuir-Schaefer Method . . . . . . . . . . . . 36 Is the Solution Structure and Function Preserved at the Air-Water Interface? . . . . . . . . . . . . . . . . . . . . . 38 Infrared Spectroscopy at the Air-Water Interface . . . . . . . 47 Self-Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . 50 High Density Subphase Method . . . . . . . . . . . . . . . . 50 Lipid Monolayer Method . . . . . . . . . . . . . . . . . . .51 Scanning Microscopy . . . . . . . . . . . . . . . . . . . . . . . 53 Basic Principles of STM . . . . . . . . . . . . . . . . . . . . 54 Basic Principles of AFM . . . . . . . . . . . . . . . . . . . . 55 Electron Tunnelling . . . . . . . . . . . . . . . . . . . . 56 Imaging Biopolymers and Biostructures . . . . . . . . . . . . . 58 DNA and Its Superstructures . . . . . . . . . . . . . . . . 61 Lipid and Protein Films . . . . . . . . . . . . . . . . . . 64 Biornolecular Information Read-Out and Interfacing . . . . . . . 65 71 Molecular Manipulation . . . . . . . . . . . . . . . . . . . . ix

Light-Directed Chemical Synthesis . . . . . . . . . . . . . . . . .75 A b Initio Molecular Design . . . . . . . . . . . . . . . . . . . . . 77 Multiple Electron Transfer Pathways . . . . . . . . . . . . . . 81 83 Cytochrome C . . . . . . . . . . . . . . . . . . . . . . Cytochrome B5 . . . . . . . . . . . . . . . . . . . . . . 86 Cytochrome C551 . . . . . . . . . . . . . . . . . . . . . 87 Azurin . . . . . . . . . . . . . . . . . . . . . . . . . . 87 89 Self-Exchange . . . . . . . . . . . . . . . . . . . . . . . . Cytochromes C. B5 and C551 . . . . . . . . . . . . . . . .89 Azurin . . . . . . . . . . . . . . . . . . . . . . . . . . 91 93 Cross-Exchange . . . . . . . . . . . . . . . . . . . . . . . Cytochrome C with Cytochrome B5 . . . . . . . . . . . . . 94 Cytochromes C551 and C with Azurin . . . . . . . . . . . 102 The P45Oscc Cytochrome: A Unique System for Bioelectronics . . . . . . . . . . . . . . . . . . . . . . 103

4 . Bioelectronic Materials 111 Heat-Proof and Long Range Storage Thin Protein Films . . . . . . 112 Structure and Function . . . . . . . . . . . . . . . . . . . 114 Electron Transfer and Redox . . . . . . . . . . . . . . . . . 118 Role of Water and 2D Order . . . . . . . . . . . . . . . . . 120 Thermostable Thin Lipid Films . . . . . . . . . . . . . . . . . 124 Alternating Bilayers of Valinomycin and Chemically Modified Bipolar Lipids from Archaea . . . . . . . . . . . . . . . . . 127 Fullerene and Other Organic Thin Films . . . . . . . . . . . . . 130 Deposition of Uniform Fullerene Films by LB Technique . . . . . 130 Heptadecylcarboxymethyl-BEDT-TTF . . . . . . . . . . . . 132 Conductive LB Films . . . . . . . . . . . . . . . . . . . . . . 134 2D Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Understanding 2D Crystallization . . . . . . . . . . . . . . 149 154 Nucleation . . . . . . . . . . . . . . . . . . . . . . . Flow Driven Assembly . . . . . . . . . . . . . . . . . . 157 Assisting 2D Crystallization . . . . . . . . . . . . . . . . . 158 LB Thin Films versus 2D Crystal Arrays . . . . . . . . . . . . . 161 Long Range Storage and Thermal Stability . . . . . . . . . . 164 Array Homogeneity and Size . . . . . . . . . . . . . . . . . 165 Recombinant versus Wild Type . . . . . . . . . . . . . . . 168 Electrical Properties . . . . . . . . . . . . . . . . . . . 171 5 . Bioelectronic Sensors Transducers and Biosensing Elements Electrochemical Transduction . X

175

. . . . . . . . . . . . . . 181 . . . . . . . . . . . . . . . 181

Amperometry . . . . . . . . . . . . . Potentiometry . . . . . . . . . . . . Optical Transduction . . . . . . . . . . . Absorbance . . . . . . . . . . . . . . Evanescent Wave . . . . . . . . . . . Surface Plasmon Resonance (SPR) . . Piezoelectric Transducers . . . . . . . . Calorimetric Transducers . . . . . . . . Integration of Transducers and Bio-Elements The PAB System . . . . . . . . . . . . . . Modelling of the Transducer Response . . Experimental Set-Up . . . . . . . . . . . Applications . . . . . . . . . . . . . . . Future Perspective . . . . . . . . . . . . . . 6. Bioelectronic Molecular Devices Monoelectronic Transistors . . . . . . . . Bioactuators . . . . . . . . . . . . . . . Photovoltaic Cells . . . . . . . . . . . . . DNA and Peptide Sequencing Microchips . . Drug Screening Chip . . . . . . . . . . Optical Filtering and Holography . . . . .

.

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . .

. . . .

. . . .

. . . .

. . . .

. . . . . 209 . . . . 220 . . . . 222 . . . . . 224

181 182 185 185 185 . . . . . . . . . . 186 . . . . . . . . . . 186 . . . . . . . . . . 187 . . . . . . . . . . 187 . . . . . . . . . 189 . . . . . . . . . . 189 . . . . . . . . . 196 . . . . . . . . . 199 . . . . . . . . . 206 209

. . . .

. . . . . . . . . . 227 . . . . . . . . . . . 228

7 Protein Automata: Year Zero

233

8. Conclusion

237

References

243

xi

1 INTRODUCTION In recent times new scientific frontiers at the crossing of life and physical sciences have been emerging. Among them bioelectronics, which operates at the frontiers of electronics and biotechnology, appears to have a significant potential impact on the industrial development of any country, previously unmatched. The term ‘molecular bioelectronics’ is utilized in a rather broad context merely to emphasize a new amalgam of electronics and biotechnology which is seen as the best way to achieve many objectives of industrial and scientific relevance, including biomolecular engineering, bioelectronic devices, materials and sensors capable of optimal hardware intelligence and molecular miniaturisation. The idea that a single molecule can work as a self-contained electronic device has been around for a long time. However, the long-range search towards the biochip and the biocomputer - so that production and assembly of molecular electronic components could be obtained with biotechnology in an absolutely integrated way is a concept which has been discussed among both the scientific community and the general public in the last two decades (Nicolini, 1988). There is no doubt that numerous open questions still need to be answered before the construction of ‘bona fide’ biochips and biological computers becomes a reality but many basic relevant discoveries and ‘proofs of principle’ have been recently made, as shown in this book. The potential revolutionary nature of applied bioelecu-onics appears thereby doubtless both in terms of fundamental scientific-technological knowledge and of potential industrial applications. This, especially, if certain superficial science fiction aspects are stripped off and if it is gradually faced through existing and technologically sound approaches. Legitimate concern has been frequently raised about many supporters of ‘molecularelectronics’,and any effort towards the biochip should avoid to adopt their approaches, frequently nothing more than a fishing expedition. Two major features of modern biology has attracted considerable attention, namely: 1

2

Molecular Bioelectronics

the coded self-assembly of biopolymers leading to complex structures in the human body and the brain architecture appearing capable to perform computation which is substantially a process of making a sequence of interacting decisions like those needed to reach for a glass of water or to recall a memory from an associated fact, to a level unmatchable by physical digital computers. Are these two facts to suggest that a biodevice could be assembled in the same way from individual biomolecular electronic components and constructed with a physical computation capable of emulating biological hardware, at least in terms of the essence of neurobiology, like the interaction of neuron with thousands of other neurons, their potentially analogue and non-linear response, and their dynamics and constantly changing behaviour. However, our current knowledge of the molecular basis of neurobiology is practically still lacking and the extensive exploitation of parallel processing architecture (to make compatible rapid information processing of the brain with slow propagation velocity of impulses along the axons and the dendrites) constitute nothing more than a good precedent, which recently led to the design and construction of new powerful brain-like architectures with existing silicon technology, namely to a kind of first generation bicelectronics, the neural VLSI chip (Kovacs et al., 1995). Similarly, it would appear highly questionable at present that biopolymers such as proteins or nucleic acids could be the primary building block of electronic circuits, at least as the active element, since they are rather unstable and possess large band gaps typical of electronic insulators. Only by means of suitable manipulation in vitro it might be possible to induce optimal semiconducting properties and stability, as indeed shown in this book. Indeed, contrary to traditional inorganic (likewise silicon) or organic (polypyrole or polyacetylene) materials which have unidirectional properties, several polypeptides (likewise metalloproteins) possess multiple pathways for the electron transfer which makes them uniquely attractive to mimic natural neurons and to yield by their proper assembly a real neural network (a sort of protein-based cell automata). The alternative approaches to use as molecular switching chemical and structural modifications in the biopolymers - like those relating to changes in

Introduction

3

geometry, bonding group or position of localized electrons and protons, or the chemical codification like the coding of genetic information within the DNA - appear presently still inadequate because these chemical processes are relatively slow. However, should it be possible to specify an amino acid sequence that will yield a protein with the three-dimensional structure needed for the described function (as redox or receptor) or properties (as electron transfer), be it for use as a passive matrix or as an active element, we would likely be able to obtain biomaterials with unique semiconductingproperties and self-bioassembly thereby making the first step toward the development of bioelectronics, namely by engineering from the extremely small to the big (upwards) rather than from the big to the small (downwards) as it is nowadays done by lithographic techniques. An important corollary of these important construction techniques is that the functional elements of such an architecture would be single molecules rather than macroscopic entities. These two aspects - the assembly of architectures from the single molecules and the functional units being individually accessible and controllable at molecular level - are the real revolutionary features of bioelectronics. As shown in this book, the necessary efforts to pursue both goals require not only outstanding scientific discoveries and new fundamental principles, but also significant imaginations and the solution of complex technological aspects. However these required efforts are of such a nature that they involve horizontally the most advanced and concrete aspects of industrial research not only that strictly electronic, but also that being at the frontier of biotechnology (namely protein engineering) and of material science. Despite the existing large semiconductor industries are still able to continue to capitalise on the next future successes of lithographic process (till the expected limit of 0.01 pm), a need for quite higher integration density, bypassing the physical limits of VLSI technology is already present. The advent of bioelectronics with all the numerous 'by-products' summarized in this book may be in fact facing us, as a necessary step toward the extremely small, extremely fast and towards the 'intelligent' electronic machine, which man and advanced society require to solve the numerous yet unsolved problems of biomedicine, of services, of security and of progressive pacific conquest of the space.

2 ACTIVE BIOELEMENTS

Several molecules of organic and biological origin being investigated in the last few years are here summarized as potentially useful active bioelements.

Organic compounds Among them we can list as examples fullerenes, fatty acids and C16H33-BEDT-

TTF. Fatty acids can be considered as the best representatives of amphiphilic molecules. Their foimula is usually the following: CH3(CH2),COOH where the usual range for n to obtain a good deposition in films ranges from 14 to 22. The structure of fullerenes instead consists of Cm molecules and has been investigated by X-ray diffraction (Kratschmer et al., 1990; Stephens et al., 1991; Meiney et al., 1991), electron diffraction (Krittschmer er al., 1990; Long et ul., 1992) and scanning tunnelling microscopy (STh4) (Wilson et al., 1991; Wragg et al., 1991) techniques. STM images of C a samples show close packing of spherical molecules with lattice spacing of about 1.1 nm. In accordance with X-ray diffraction studies at room temperature C,o molecules crystallize in face-centred cubic (FCC) lattice with lattice spacing of 1.404 - 1.411 nm.

Proteins Nearly all the chosen proteins have been well characterized in solution by X-ray crystallography and their three-dimensional structure is known at the atomic resolution (Table 2.1). Up to 13 different proteins, both water-soluble and 5

6


membrane-bound, have been utilized and extensively characterized by a wide variety of biophysical probes in our efforts to develop new protein-based nanotechnology (Nicolini, 1995).

A

Figure 2.1 PDB image of Glutathione-S-(ransferase,a) froiit view, b) top view, c) side view.

In the case of soluble proteins, the best way to examine the 3D structure is to make X-Ray analysis. However, some proteins are not suitable for this technique, as the size of the crystal is too small, or the protein is very difficult to produce and cost too much to make large (submillimeter) 3D crystals. Instead, for Transmission Electron Microscopy (TEM) only minute amount of protein is necessary to make 2D crystal. Thus, in spite of a low yield, or sub micrometric size 2D crystals, TEM is very suitable for this task.

Active Bioelements

7

TABLE 2.1 X-Ray Crystallographic Data of Proteins from B.N.L. Data Bank. PROTEIN Glutathione-Stransferase Alcohol dehydmgenase

SOURCE

rat liver

HEIGHT WIDTH

(4

(A)

DEPTH

(4

SHAPE

front view top view side view 54 32+ 32" 54 sphere

REFERENCE Jietal., 1992

70

54

48

ellipsoid

Escherichia coli Escherichia coli Azurin Pseudomoilas aeruginosa Cytochrome C551 Pseudomom aeruginosa Cytochrome c bonito heart

101

62

68

ellipsoid

Colonna-Cesari el al., 1986 Eklund er al., 1984 Kim et al., 1991

33

34

30

sphere

Katti er al., 1990

42

31

30

28

32

29

tetnhedron Admdi et al., 1981 sphere Matsuura et al., 1982

38

33

32

sphere

Cytochrome P450 bovine

73

71

66

125

40

90

temhedroe Vijayakumar er al., 1992 Y shape Brunger er al.,

horse liver

Alkaline phosphatase Thioredoxin

Tanakaetal.

1975

scc Antibodies

Fab fragment and 2 Fac fragment Photosynthetic Rhcdobacter Reaction Centers sphaeroides Photosynthetic Rhodopseudo Reaction Centers monas viridis Bacteriorhodopsin Halobactenum halobium

1991

90

28 + 28 *

68

125

55

60

48

43

45

ellipsoid

Yeateseral., 1988 ellipsoid Deisenhofer er ul., 1995 parallelepipedChou er al., 1992

* two subunits. As model systems for soluble proteins we have explored three enzymes, namely

glutathione-S-transferase (Fig. 2 . l), alcohol dehydrogenase and alkaline phosphatase, four metalloproteins, namely azurin, cytochromes c (Fig. 2.2), c55 1 and P45Oscc (Fig. 2.3), IgG antibodies (Fig. 2.4) and thioredoxin, an ubiquitous protein with many functions, such as in thiol-dependent redox reactions.


8

B

C

Figure 2.2 PDB image of Cytochrome c, a) front view, b) top view, c) side view.

As model system for niembrune-bound proteins we have chosen photosynthetic reaction centers from Rhodobucter sphaeroides (Fig. 2.5) and from Rhodopseudonzonas viridis, bovine rhodopsin and bacteriorhodopsin (BR). In the system of Hulobacteriimz halobiuni, bacteriorhodopsin is found as a 2D crystalline lattice in puiple membranes. The purple membrane is usually 1000 nm in diameter and 50 8, in thickness and makes part of the overall cell membrane of Halobacteria. Bacteiiorhodopsin is the main protein in the biological function of these bacteria. BR converts light energy into chemical energy by transferring protons from the inside to the outside of the cell membrane, thus functioning as a light-driven proton pump. As a result, ATP is regenerated from ADP by an enzyme called ATP-synthase.

A c t i v e Bioelements

9

A

B

C Figure 2.3 PDB image of Cytochrome P450scc. a) front view, b) top view, c) side view.

Recombinant Proteins While in most cases proteins are wild-rype, namely isolated from the corresponding cell tissues, in few cases they are of recombinurit origin, namely obtained from clones properly genetic engineered with quite high yield and extreme purity and homogeneity.


10

A

C Figure 2.1 PDB image of IgG antibody, a) front view, b) top view. c ) side view.

For example, the cDNA of the cytochrome P450scc obtained from genomic library of the bovine surrenal cortex (precursor gene) was cloned 'down-stream' the secretory signal of Kluvironiyces luctis in the ORF of GAL UAS promoter in the vector derived from pYeDP 1/10 (Figure 2.6). The yield of the product detected in the culture broth after induction of GAL UAS promoter was quite satisfactory. The signal-appended cytochrome P450scc was translocated across the cells compartments and processed to yield authentic, haem-assembled cytochrome P45Oscc and was exported from the yeast cells. The eventual processing of the P45Oscc precursor was somehow unstable but unusual with respect to the presence of mitochondria1 signal and the known reaction specificity of signal peptidase (Paschkevitch et al., 1996).

Active Bioelements

11

A

B

C Figure 2.5 PDB image of Photosynthetic reaction centers from Rhodobacter sphaeroides ,a) front view, b) top view, c) side view.

The secreted protein was tested spectrophotometrically, electrophoretically, immunologically and by visual transformation of the culture broth into red color. Thus, the mitochondria1 membrane protein (that carries the signal of posttranslational transfer) was efficiently processed to mature haemo-protein and secreted from S.cer-eviseue by way of co-translational transfer. On the basis of the HPLC data it was concluded that the purity level of this protein as secreted was extremely high (approximately 98%). The production rate of correctly matured exported cytochrome P45Oscc was 10.2 mg per litre of culture. The obtained mature haem-bound cytochrome P45Oscc was indistinguishable in many of its characteristics from the native counterpart or was even better, with the only exception that its production was quite unstable (Eldarov et al., in preparation). Because of this instability we have


12

then utilized homologous expression within the E . cofi bacteria and isolated the same cytochrome P45Oscc with similarly high degree of purity and yield, but with the needed reproducibility and stability.

Cy(P4SOacc

I

KI.Iadis 6.6.

CylP450su:

\

2mm-

Kl.ladls

S.S.

c--/

ORI

Figure -7.6. Subcloning of the gene of cytochrome P45Oscc, secretory signal of Kl.lacfis and GAL UAS CYC 1 block in expression-secretionvector.

The problem of obtaining metalloproteins from microorganisms is crucial for development of applied bioelectronics. In comparison with a method of extraction from natural source this approach is cheaper and more advantageous for obtaining high quality protein of interest in large amounts. Cytochrome P45Oscc in nature is localized in inner membrane of mitochondria of the bovine adrenal cortex and plays a crucial role in the steroid metabolism (Millet

Active Bioelements

13

and Geren, 1991). This protein is globular, contains the heme group and two active sites: 'oxygen pocket' and 'substrate pocket'. The NADPH, adrenodoxine and adrenodoxine reductase are necessary for the functioning of the protein as a 'mini chain of electron transfer' (Millet and Geren, 1991). The development of bioelectronics, creation of new biosensors and fundamental studies of this protein require an elevated amount of cytochrome P45Oscc. The haem-assembled P45Oscc in some of the characteristics is superior to its natural analogue.

Lipids

I

H~C-C-CH

I l l OHOHOH

-CH -CH-CH,

9I

I OH

I

OH

R2

R, = RZ = H, GONT (glycerol dulkylmnlol ieiraeii-ars) a-1

Figwe 2.7 Archaea lipids

Archaea, a philogenetically coherent separate group of microorganisms, which differs from Eubacteria and Eukaria, comprises a variety of extremophilic bacteria living under extreme conditions such as high temperature, acidic or alkaline pH, and saturated solutions (Kandler et (11.. 1992). The archaea membrane lipids have unusual structure (Gambacorta et al., 1994) based on isoprenoid chains of different lengths with ether linkage to glycerol or to more complex polyols. In particular, membrane lipids of thermophilic archaea, such as Sulfofobussolfaturicus (optimal growth conditions are 87 OC and pH 3), possess bipolar architecture (De Rosa et a!.,

1986; De Rosa et al., 1983; Luzzati et al., 1987) characterized by the presence of two polar heads and hydrophobic isoprenoid moiety of practically double average length with respect to that of classical ester lipids. The lipids play a key role in stabilization of the membrane under extreme conditions (Figure 2.7).

DNA

Em

a

F

A B C D

1’

-

16 compounds,

8 chemical steps

\I#------

/?\

I*)

Lel

w

IAl

Lel

M

n.uh

e

01

n each

27 ol

Figure 2.8 a) Orthogonal-stripe method of synthesis. A layer of monomers is formed by photolysing stripes for each block. using an approach similar to split-resin method (b). Dimers are formed by photolysing stripes orthogonal to the first set. b) Split-resin method. Peptide-synthesis beads are divided into specific sectors for chemical synthesis, combined, mixed to homogeneity and subdivided for subsequent reactions. (From Jacobs and Fodor, 1994).

Active Bioelements

15

Conventional solid-phase oligonucleotide synthesis involves the step- wise assembly of 5'-dimethoxyuityl (DMT)-protected nucleoside monomers in the 3' to 5' direction. In a typical coupling procedure, the 5'-hydroxyl of an immobilised nucleoside is deprotected with mild acid, the liberated hydroxyl group is phosphatylated with a DMT-protected deoxynucleoside 3'-phosphoramidite, and the resulting phosphate is oxidized to a phosphotriester (Caruthers, 1985). The process is repeated until the desired oligonucleotide has been prepared. This technique was adapted to include light-directed parallel chemical synthesis by replacing the 5'-protecting group DMT with a substituted nitroveratryl derivative, and incorporating a nitroveratryloxycarbonyl (Nvoc)-protected hydroxyl linker into the synthesis substrate (Fodor et ul., 1993). Hydroxyl groups are selectively photodeprotected as described previously, and arrays of oligonucleotides assembled using standard peptide chemistry. Using the orthogonal-stripe method illustrated in Figure 2.8, it is possible to assemble all 65 566 possible octanucleotides (4*) in only 32 chemical steps (4 X n; where n = 8) (Pease et ul., 1994).

Whole cells Several well-established cell culture systems, like mammalian CHO-K1 or HeLa cells or microorganisms, are utilized in different context for various biosensor applications (Adami ef al., 1992, 1995; Hafemann et al., 1988). Primary cultures of rat hepatocytes were also used for experiments with biosensing potentiometric systems because they are easy to obtain and, like all hepatocytes, they maintain, in the first hours in vitro, their metabolic skills practically unchanged with respect to the in vivo situation (Nicolini et al., 1995e). Hepatocytes were isolated from liver of Sprague-Dawley albino rats (200-250 g) by in situ collagenase perfusion according to Williams (1977). Isolated cells were suspended in Williams E Medium, supplemented with 10% foetal bovine serum and gentamicin (50 pg/ml) at the concentration of 5x105 hepatocytes/ml. Aliquots of this suspension were plated as follows: a) 1x105 cells were plated on the glass support (coated with collagen) for the measurements with PAB; b) 6x105 cells were plated on 35 mm dishes, coated with collagen, for conventional toxicity tests.

3 TECHNOLOGIES Recent work on protein monolayers (Nicolini et a / . , 1993, 1995; Shen et al., 1993; Facci et al., 1994b) appears to have for theirexceptional thermal stability 'the kind of implications that might capture the immediate interest of a wide audience', as testified by the enthusiastic News and Views published in Nature time ago (Hampp, 1993). Typically, LB films of most organic compounds are formed in a Langmuir trough by spreading the solutions at the liquid-gas interface and transferring by a vertical lift technique onto solid substrate. In our laboratories protein monolayers are instead usually prepared with horizontal lift (Langmuir-Schaefer)technique modified in a specific manner and deposited on solid substrates in the range of surface pressure between 25 and 45 mN m-*(Nicolini et al., 1993; Erokhin er al., 1991). In order to outline the unique structural and functional properties and the numerous potential applications of this nanotechnology as applied to proteins (the most difficult and promising undertaking), lipids, fatty acids and DNA complexes, we intend in this chapter to briefly summarize all the processes and procedures utilized for thin film formation (Langmuir-Blodgett and its modifications, selfassembly), manipulation (scanning microscopy at atomic resolution, chemical synthesis), optimal design (ab initio calculations) and characterization. Among existing technologies two seem to be the most suitable, since they provide the possibility of molecular manipulations at least in one direction. The techniques are Langmuir-Blodgett (LB) method and Self-Assembly (SA). In the LB method the monomolecular layer is formed at the aidwater interface and is then transferred onto the solid substrate, while in SA molecules are modified in such a way that one part of them has a group with affinity to the preceding layer and the other side contains groups for attaching the subsequent monolayer. Both methods were shown to be useful for fabrication of complex molecular structures with different sequences of layers. Both methods also allow to work with protein molecules which are rather 'delicate' and can be easily denatured. The LB method gives the possibility to conaol the layer structure during formation and deposition. It also yields layers of better 17

18

Molecular Baoelectronics

homogeneity, which is very important for device applications. From the other point of view, SA can provide the desirable connections of molecules in the film plane, realizing systems ordered with molecular resolution in all three dimensions. LB method cannot do it by itself and it will be necessary to make lithography on the film in order to reach the same goal. It seems very promising to try a combination of both techniques, namely, to deposit LB films where the molecules in the layer are arranged regularly in desirable way by means of SA.

Langmuir-Blodgett and its modifications The fact that oil forms thin layers over the water surface is known from the ancient time. First statement that such films must be monomolecular in thickness was done by Lord Rayleigh (Lord Rayleigh, 1879). Nevertheless systematic scientific study of such objects began from the works of Irving Langmuir and can be divided into several phases. During the first phase main attention was paid to the behaviour of monolayers of amphiphilic molecules at the aidwater interface. The phase is connected with early works of Langmuir (Langmuir, 1915, 1917, 1920). As a result, monolayer formation process was characterized and several phase transitions were determined in two-dimensional system at the adwater interface. During the second phase it was shown that the layers could be transferred onto surfaces of solid substrates. The works were performed by Langmuir in collaboration with Blodgett, whose names began to be used to term the method itself (Blodgett, 1935; Blodgett and Langmuir, 1937). Their technique consisted in deposition of monolayers when the substrate moved vertically through the monolayer. A little bit later another deposition technique was suggested by Langmuir and Schaefer, where the substrate touched the monolayer horizontally (Langmuir and Schaefer, 1939). The method now is called i n a literature as 'horizontal lift' or Langmuir-Schaefer technique. It is interesting to note that the method was developed for deposition of protein layers (first attempt to work with protein layers was done in 1938 by Langmuir and Schaefer on pepsin and urease (Langmuir and Schaefer, 1938)).

Technologies

19

After the initial interest in the subject at the beginning of the century resulting in a Nobel prize received by Langmuir in 1932, the activities in the field were not numerous involving only some academic interest for such two-dimensional systems. Third phase in the LB films investigations began with the works done in the group of Kuhn (Kuhn, 1965; Drexhage and Kuhn, 1966). The works have demonstrated that it was possible to form complex structures with desired mutual orientation of functional groups of molecules by the method (Bucher et al., 1967; hacker et ul., 1976). The works on energy transfer in the films (Kuhn, 1981) created big resonance in the scientific world attracting a huge number of researchers to be involved in investigation of films. It is possible to consider that the fourth stage of the LB investigations began when the first international LB conference was organized. The fourth phase of the investigation is still under way. It demonstrated that scientific forces of differing background began to be included into LB films investigations. During this stage the films became to be characterized by practically all experimental techniques available nowadays: circular dichroism, surface potential measurements, atomic force microscopy, scanning tunnelling microscopy, transmission electron microscopy, differential scanning calorimetry, NMR, infrared and EPR spectroscopy, X-ray diffractometry, absorption spectra, nanogravimetry, ellipsometry, CCD high resolution image analysis and electric (DC and AC) measurements. Application aspect of the films began also be taken into account. Even if direct competition with traditional electronics was not very successful, the research activity in LB films area was not stopped but even increased, because in case of success in the final goal, namely development of alternative technology for electronic device fabrication, a new generation of devices with decreased sizes and improved parameters would be achieved. As mentioned earlier in this book, biological objects, such as proteins, cannot be readily reproduced to behave as semiconductor devices. LB technique however allows to operate with them and to organise them in regular layers, which can be included into ordered molecular architectures. It is also worth mentioning that LB films are of fundamental interest as they can be really considered as 2D systems which can give origin to new types of phenomena not found in 3D objects.

20


Classic materials for the LB method are amphiphilic molecules, one side of which is a polar head-group and the other - a long hydrocarbon chain (Hann, 1990). In order to spread the molecules, they must be solved in a 'strong' solvent at concentration that does not peimit the formation of aggregates. Drops of the solution are placed on the water surface. The amount of the molecules in the drop must be rather small in order to have after spreading a monolayer where molecules are far from each other and do not interact. When such molecule is placed on the water surface, its polar head group interacts with water, while the hydrocarbon chain faces towards air, as it can not be surrounded by water for entropy reasons. Floating molecules can be compressed by the barrier until condensed state is achieved (Figure 3.1). Usually the parameter under control during the compression is the surface pressure, which characterises the decrease in the surface tension of the water surface due to the presence of the monolayer:

-

where OH,O is the surface tension of water without monolayer and oINl is the surface tension of the water surface covered by monolayer. Behaviour of insoluble monolayers on water surface was described in detail in the book of Gaines (Gaines, 1966). Here we present only some important features of the behaviow. One of important characteristics of the monolayers on the water surface is the dependence of surface pressure on the area occupied by single molecule in the monolayer. The dependence is usually called 'compression isotherm' or 'K - A isotherm' (the curve is measured at fixed temperature). This characteristic is rather important since it allows to calculate the area per molecule in the monolayer and to reveal phase transitions in the monolayer structure. Typical K -A isotherm of stearic acid (CH3(CH2)&00) is presented in Fig. 3.2. Initially molecules are rather far from each other and practically do not interact. So, initial part of the isotherm is characterised by zero surface pressure which does not increase upon compression. This region of the isotherm is called 'two dimensional gas'. Further Compression results in the beginning of molecular interactions.

21

Technologies

Hydrocarbon chains begin to rise from the water surface. This behaviour is accomplished by slight increase in the surface pressure. More significant increase in the surface pressure is possible to distinguish when molecules begin to interact. The monolayer in this region of the surface pressure is considered as 'two dimensional liquid. Further compression results in close packing of molecules in the monolayer. This condition is called 'two dimensional solid state'. There is a linear dependence of the surface pressure upon the area per molecule. For calculation of the area occupied by one molecule in the closely packed monolayer under conditions of zero compression the linear region of n -A isotherm is extrapolated to zero pressure, the intercept giving this value (Hann, 1990).

T (e)

6-fi:cdbark system

Figroe 3.1 Deposition process of films using Langmuir-Blodgett technique.

22

M o l e c d a r BioelectTonics

If the compression is further continued, i. e. area per molecule is decreased, collapse of the monolayer takes place. In collapse molecules do not form a monolayer anymore but aggregate at the water surface. Several types of collapses are described in the literature. They can be considered as two main types, namely, formation of small 3D crystals and bending of the monolayer in such a way that lamellar structure begins to be formed at the water surface (Gabrielli et al., 1976). Which type of collapse really takes place in a particular situation depends upon several parameters, such as presence of impurities in the environment, speed of the compression, temperature etc.

I

0 10

15

20

30

25

35

40

45

I

50

Area oer Molecule I A - A l

Figure 3.2 Typical isotherm of stearic acid.

For making measurements of n-A isotherms one needs to have a tool for recording the surface pressure value. There are two devices usually used for these reasons, namely the Langmuir balance (Langmuir, 1917) and the Wilhelmy balance (Wilhelmy, 1863). In the case of Langmuir balance (Figure 3.3a) one barrier is a sensitive element. The balance allows to monitor directly the surface pressure, as it measures the force acting on the barrier that arises from the difference in surface tensions at different sides of the barrier - from one side there is only pure water, while from the other - water with monolayer. Even if the described balance allows to measure directly the value of the surface pressure, it has several disadvantages. Such

23

Technologies

construction of the balance demands to provide the compression of the monolayer from only one side, which, as it was shown, can result in the appearance of concentration gradient in the monolayer in the compression direction (Petty and Barlow, 1990).

ECrnOI’

noatlng barrier

displaccmcnt sensor

Figure 3.3 Schematic preseiitatioii of a) Langmuir balance and b) Wilhelmy balance.

Another disadvantage is associated to impossibility to measure the surface pressure near the point of deposition. It can be important, as the surface pressure can be different in different points, especially for rigid monolayers. Wilhelmy balance allows to overcome these disadvantages even if it does not measure directly the surface pressure. In the case of Wilhelmy balance (Figure 3.3b) a plate is used as sensitive element immersed in water. Several forces are acting at the plate, namely,

24


the weight, Archimedic force and suiface tension. If everything is equilibrated, after the monolayer formation one can register the difference in the total force acting at the plate and attribute it to the difference in the surface tension. In order to make the previous statement valid it is necessaxy to maintain the fixed position of the plate in the water, otherwise variations in the Archimedic force should necessarily be taken into account. Therefore. the construction of a Wilhelmy balance is usually such that feedback keeps the plate at fixed position.

Film Characterization 107

R

sjg'u1 *mt

LOCK-IN Amplifier

Ref.Lpd

output

1

1 -

Figure 3.4 Schematic presentation of a Kelvin probe.

An important parameter which can be measured in the monolayer at the adwater interface is surface potential (Holcroft et al., 1985; Tredgold and Smith, 1981; Tredgold and Smith, 1983; Jones et d., 1985; Christie et al., 1986). Orientation of molecules containing dipoles arising from the electron displacements in the bonds result in the appearance of surface potential. The other origin of the potential is the interaction of polar groups with water that provides specific arrangement of water

Technologies

25

molecules near the monolayer. It is clear that the potential can not be directly measured, as in the closed circuit it will not provide any current. There are two main methods for measuring the potential. In both cases one electrode is embedded into the water subphase while the second is placed above it. In one case it is necessary to have a source of ionising radiation between the upper electrode and water surface. The source generates carriers which can move due to applied potential. The other technique is more frequently used and is based on the Kelvin probe (Gaines, 1966). The upper electrode is a vibrating one (Figure 3.4). Oscillations of the electrode modulate the capacity of the gap between the electrode and water surface. As a results, alternative current appears in the circuit connecting the vibrating electrode and that in water subphase. The value of the cuirent depends upon the amplitude of the oscillations and upon the value of surface potential. By fixing the amplitude one can measure the surface potential.

BAM scanslone verticaie

Figure 3.5Brewster angle microscopy.

A useful tool for the visualisation of the monolayer domain structure is fluorescence microscopy (Losche and Mohwald, 1984; Fischer et ul., 1984; Losche et al., 1983; Losche et ul., 1984). Small amount of fluorescent dye which cannot penetrate the closely packed regions of the monolayer is introduced into it and, being excited, reveals the boundaries of the domains. Recently a new type of microscopy,

26


the Brewster angle microscopy appeared which provided the possibility to reveal the domain structure even without any additives in the monolayer, therefore without affecting its structure (Figure 3.5) The microscopy is based on the fact that light incident to the surface at Brewster angle is reflected being partially polarised. The value of the angle depends upon the material of the surface. Thus, it is possible to visualise the domain Structure of the monolayer acquiring the images through the analyser, as the presence of the monolayer vales the Brewster angle.

Elliusometrv Several other techniques were also successfully applied for the investigations of monolayers, among which ellipsometry should be mentioned (den Engelsen and de Konig. 1974). During ellipsontetq~two parameters are measured, namely - ratio of the vibration of the electric vector in the plane of incidence and perpendicular to it, and - difference of phases of these two vectors. The theory of the ellipsometry allows to connect these two parameters with the thickness and refractive index of the layer (Drude. 1902). Usually, the film is assumed to be not absorbing and isotropic. In principle, the assumption is not valid in the most of cases. Nevertheless, for the thickness estimation it seems to vary the value inside the experimental error. The technique allows to determine the thickness of the monolayer during compression, revealing the reorganisation of molecules at the water surface. It should be also mentioned that even X-ray reflectoinetry was provided for the study of monolayers at the water surface. Nevertheless, these techniques, especially X-ray reflectometry, are not so frequently used as they are rather delicate and demand special anti vibration conditions.

X-ray Scattering X-ray study is one of the most powerful tool for the study of LB film structure in the direction perpendicular to the film plane. As a result, numerous investigation of LB films by the method were reported (Holley, 1937; Bisset and Iball, 1954; Srivastava and Verma, 1966; Pomerantz er ul., 1975; Pomerantz and Segmiiller, 1980; Lvov et ul,, 1989; Von Frieling et al., 1988). As the periods of LB films are

Technologies

27

much higher with respect to the normal crystals (40 - 100 A with respect to 1.5 - 4 A) the X-ray equipment must have good resolution in the low angle region (Feigin et uf., 1989). Thus, the most of the experiments are carried out with small-angle diffractometers. It is possible to distinguish two main approaches in providing X-ray analysis of LB films, namely, diffractometry and reflectometry. In the case of diffractometry, Bragg reflections are taken into account. Their angular position immediately give the value of the spacing according to the Bragg equation:

where D is the spacing, 0 is a diffraction angle, h is the wavelength and N is the order of the reflection. Half of the reflection width allows to calculate the long rang order of the film. Typical X-ray pattern obtained from LB multilayer is presented in the Figure 3.6. If the film is well organised and numerous reflections were registered one can calculate the electron density profile across the period. Integral intensities of the reflections represent the modules of the structure amplitudes and as the structure of the most of LB films is symmetrical, the phases of the structure amplitudes can be only 0 or 1 (Feigin er al., 1989). Thus, the electron density will be the some of the harmonics where the only problem is to attribute plus or minus to each harmonic: F(o) +- - F F ( h / D ) c2 ahz p(z) = o s ~ D D h=l

(3.3)

where F(0)lD is the average electron density. The modules of the structure amplitudes are connected with the intensities according to the following equation:

where the Lorentz coefficient h takes into account the geometry of the experiment and k is a scale factor determined by Pwseval theorem (Belbeoch cf ul,, 1985). In order to draw the profile of the electron density, thus, is necessary to find the right signs for each harmonic in the equation for. The easiest way for doing it can be to make all possible 2’1 variants of the sign alterations ( n is a number of registered

28


reflections) and with some criteria to choose the best. The criteria can be the constancy of the electron density in the region of hydrocarbon chains packing, for example. The typical electron density profile is shown in the figure 3.7. It is possible to distinguish the region of the contact of adjacent hydrocarbon chains, plateau corresponding to the chain packing and 2 maxima of the electron density, corresponding to the position of metal atoms in the head groups of the molecule. The resolution of the electron density profile is determined by the half of the last harmonic D / h , where D is the period and ti the number of registered reflections.

0

10

2.0

3.0

4.0

5.0

6.0

Angle in dogmas

Figure 3.6 Typical X-Ray pattern from LB film (Nicklow er al, 1981).

X-ray diffractometry is very useful for study the structure of the superlattices and some unusual packing, as that presented in the Figure 3.8, where hydrocarbon chains of the adjacent layers do not simply touch each other but interpenetrate (Erokhin el uf., 1989). X-ray reflectometry is based on the analysis of the curves, applying to them the laws of X-ray optics. In this case one can even does not have Bragg reflections at all, working with small number of deposited layers or with polymer materials. Typical reflection curve is shown in the Figure 3.9. Oscillations in the initial region correspond to the total thickness of the film. In order to obtain the information about the electron density profile one must build a model, attributing

Technologies

29

some electron density to different regions of the film, calculate the X-ray curve and compare it with that obtained from the experiment. Summarising, both X-ray measurements give the infoiination about the structure of LB films. Diffractometry is more useful when there is a multilayer. as it does not demand any a priori information about the layer structure, while reflectometry is more useful for analysis of thin and weakly ordered films and demands some basic information about the possible organisation of the layer.

9

B

3 ' 2 '

Figure 3.7 Typical electron profile density of LB film (Feigin et ol. 1989).

30

Molecula+ Bioelectronics

X

Figure 3.8 An example of an unusual packing of lipids.

0

2

4

s. 10-2

ii-'

Figure 3 9 Typical X-Ray relleciion curve of LB film. From

Feigiii er 01 1989.

Technologies

31

Electron Diffraction and Microscopy Electron diffraction (Havinga and de Wael, 1937; de Wael and Havinga, 1940; Germer and Storks, 1938) and elecrruri microscopy (Fryer et al., 1985; Hann et al., 1985) can provide the information about the in-plane organisation of the films, what, being combined with the X-ray information, can give the complete picture of the film structure. Moreover, the amplitudes of electron scattering are about three times higher then that for X-ray scattering and the thickness of the films (100 - 1000 A) is just optimum for the electron diffraction study (Feigin et ul., 1989). Even if the determination of the film structure from experimental data is not easy problem, structure of several different films were solved, taking into account additional information obtained from the area per one molecule from the monolayer at the aidwater interface investigation, the thickness of the monolayer, obtained from interferometry, ellipsometry or X-ray measurements (Epstein, 1950; Fukui el ul., 1977; Russell et ul., 1984; Peterson and Russell, 1984; Bonnerot et ul., 1985; Earls er al., 1986; Robinson er ul., 1989). Electron microscopy of LB films allowed to conclude that in the most of LB films the degree of ordering is much lower with respect to the real crystals and the monolayer can be considered as the amorphous mauix with imbedded small crystal domains (Feigin, 1989).

Interferometry Interferometry is, probably, the first applied for the investigations of deposited LB films (Blodgett, 1935; Blodgett and Langmuir, 1937; Jenkins and Norris, 1939; Holley, 1937; Hartman, 1054; Mattuck, 1056; Courtney-Pratt, 1950; Srivastava and Verma, 1962; Drexhage, 1974). It allows (Figure 3.10) to estimate in a rather simple way the thickness of the deposited film according to the following formula:


32

where h is a wavelength, 11 is a refractive index of the film, and m is an integer. The difficulty here is some uncertainty in the deteimination of the refractive index. Nevertheless, several approaches for the overcoming the difficulty was suggested. Good accuracy in the thickness determination (approximately k 1 A) (Srivastava and Verma, 1966) was reached when the step in the film thickness was formed (the substrate was covered totally with some number of the layers and then some additional number of layers was deposited onto the part of the substrate). Then the structure was covered with metal layer deposited by thermal evaporation in vacuum. The structure was illuminated by white light and fringes of equal chromatic order

were obtained. The technique is very useful, but one limitation should be taken into account. One can apply the method only to films which are not affected by vacuum and are resisted from the evaporation process.

deposited fllm

,2040 am

evaporationo f A l through the mask

+ structure after evapom. tion of Al

treatment In air glow discharge

A1

+ structure after glow discharge treatment

etching of A1 (KOH : K J Fe(CNjgso1utiun) Al

,IUo-150 nm

/ formation of collodion flim over the structure

evaporationof A1 mirror

Figure 3.10 Estimation of the film thickness by means of interferometry.

Technologies

33

Langmuir-Blodgett method When the monolayer is formed at the water subphase it can be transferred onto the solid substrate. Two main techniques are used for the deposition: LB method (Blodgett and Langmuir, 1937) or vertical lift and Langmuir-Schaefer method (Langmuir and Schaefer, 1938) or horizontal lift. In the traditional LB method the formed monolayer is rather soft - the deposition pressure is in the beginning of 'two dimensional solid state' phase. The substrate moves vertically through the monolayer while feedback maintains the fixed surface tension (for fatty acid salts usually 27-29 mN m-l). The best deposition takes place when the surface of the solid substrate is hydrophobic. Hydrophobizaton of silicon can be easy done by etching the oxide layer with HF (Russell er al., 1984). More general procedure of hydrophobization is so called silanization, which allows to make hydrophobic not only silicon surface, but also surfaces of glass, quartz and some other materials (den Engelsen, 1971; Fariss et al., 1983; Peterson and Russell, 1984). During the procedure the washed substrates are placed into the solution of dimethyldichlorsilane, (CH3)2SiC12. In the solution C1 leaves the molecule and Si attaches itself to the oxide layer forming the structure, where CH3 groups are directed outside making the surface hydrophobic.

Langmuir-Schaefer method The technique is very useful for the deposition of proteins (Lvov et al., 1991) and rigid layers. The method implies horizontal touching with the substrate of the preformed monolayer. In some cases, when one makes the deposition of rigid layers, it is necessary to separate the monolayer at the surface with the special grid, otherwise it risks to have an absolutely non-homogeneous layer at the substrate, since after removal of some portion of the layer a 'hole' will form and will be maintained in the monolayer for several hours, before it will be compensated by the feedback system, For protein layers it is not necessary to use a grid as the layer is rather soft. For both above mentioned techniques it is necessary to compensate the charge of the molecules at the water surface before deposition (Hann, 1990). The forces of the

34


adhesion of the monolayer to the substrate surface have the hydrophobic origin and are rather week. If one does not compensate the charge of the molecule he risks to loose the possibility of the deposition, as the forces of the interaction with water dipoles can be higher than that with the substrate. As an example we can refer to the case of fatty acid films. Fatty acid molecules, being sprat at the water surface with pH 7.0, are practically dissociated. The COOH head group loses the proton and is converted into a COO- ion. It is impossible to transfer such monolayer to the solid substrate. In order to overcome the problem one must decrease pH value to 4.0, when the head groups will be again protonated and the deposition will be possible. The other way to provide the deposition is to add to the subphase some amount of salt of bivalent metals. Metal ions will compensate the negative charge of the head groups and will coordinate two fatty acid molecules (Newman, 1975; Vogel ei al., 1980; Pomeran tz ef ul., 1978). Dealing with the structure of deposited multilayers it is conventional to divide it into three groups, namely X-, Y- and Z-types (Figure 3.11a) (Fankuchen, 1938; Bernstein, 1938). Such division was done basing mainly on the analysis of the deposition ratios. If the film is deposited only during the upward or downward motion of the substrate it was drown a conclusion about X ore Z type of the deposition. In reality the situation is much more complicated. Even if the deposition takes place only in one direction of the substrate motion, mainly Y type of the deposition is realised (Fankuchen, 1938; Bernstein, 1938). It was reported experiments when the polar properties of LB multilayers were registered, and authors made conclusions about the polar structure of the film. In reality, the repetitive unit of the film remained still a bilayer. The polar phenomena, such as piro- and piezoelectricity, registered in such structures were due to the difference in the packing of add and even monolayers. Thus the question appear: how the bilayer structure can be foinied if the deposition takes place only in one particular direction? The answer is the following. When the substrate passes through the meniscus, molecules obtain the possibility to rearrange themselves in order to reach a packing with lower energy (Kato, 1988; Erokhin el a/., 1989). For the case of fatty acids it was shown that during the passing of the meniscus three last layers are involved into reorganisation, making flip-flop transition (Kato, 1988). It was also shown that in a

Technologies

35

case of protein films there is even the possibility of the molecular exchange between adjacent layer during passing through meniscus (Facci et af., 1995).

t

a

t X -DoposctKm

2

- oppositial

d

b

a

4-

-o--

44 0 4-

404-

4-

Subslrote Y - Deposition

PPP

b

1' 1 1 ?P I

.:ip

I l l

H-C-H

I 0

/

H

H-C-H

I

Figure 3.11 a) Classification of multilayers into different types. From Petty ai~dBarlow 1990 b) Fabrication of alternating layers of falty acid aiid aliphatic arniiE. From Christie ef a/ 1986

Thus, the most of the reported films have Y-type of the organisation. How, in this case, polar structures can be foimed? It was shown, that there is the possibility of

36

Molecular Bioeiectronics

fixing the molecular structures working with rather rigid monolayers (Kuhn and Mobius, 1971). More general solution of the problem is connected with deposition of superlattices, containing alternative layers of molecules, having different dipole moment (Christie et al., 1986; Smith et al., 1985; Jones et al., 1987). The approach is illustrated in Figure 3.1lb, where the structure with the alteration of fatty acid and aliphatic amine monolayers is fabricated (Christie et al., 1986).

Modified Langmuir-Schaefer method As proteins are not ordinary amphiphilic molecules suitable for LB technique, several points must be taken into account in the formation of thin protein films, hereby called Modified Langmuir-Schaefer method 'MLS' to underline the several chemical (reverse micelles, derivatization) and biotechnological (site-specific mutagenesis, subphase) modifications which needed to be introduced in the standard Langmuir-Blodgett technique to preserve the native protein structure and function at the air-water interface. All the proteins were transferred by Langmuir-Schaefer technique (horizontal lift) as it is the most suitable to provide homogeneous reproducible coverage of substrates with such type of monolayers. There is a big class of proteins, namely membrane proteins, for which their spreading over water surface does not affect the protein structure. Two possible cases of the spreading solutions can be distinguished. In the first case the spreading solution is a solution of separate proteins with hydrophobic area surrounded with surfactant molecules (as in a case of Reaction Centers). In this case, after spreading we have not only protein molecules at the aidwater interface but also the surfactant molecules. Taking into account that the surfactant molecules have rather short hydrocarbon chains (12 carbon atoms) not allowing to reach high values of the surface pressure. it is possible to remove them from the surface by applying compression-expansion cycles for several time. In the second case the spreading solution is a solution of membrane fragments. The deposition in this case is rather difficult as the fragments are mainly hydrophilic and only small hydrophobic parts are present. It results in the fact that the most of the protein molecules penetrate the volume of the subphase. instead of participating in the monolayer formation. To prevent this behaviour it is necessuy to use condensed salt solution as a subphase.

Technologies

37

But a new problem arises, namely, during the deposition salt is also transferred with the film onto the substrate. To remove it, it is necessary to wash the sample after deposition of each layer. To produce the film by classical LB technique (Fig. 3.1) the solution of amphiphilic molecules consisting of hydrophilic groups interacting strongly with water and long hydrocarbon chains is spread at the interface. After evaporation of the solvent the monolayer is compressed with barrier up to a definite surface pressure which is measured with the balance and is maintained at the constant level during the deposition.

5-rubslmte. 10-plate.

I I-pap between the

rvbstrsle and the plate

I

5

10

Figure 3.12 'Protecting plate' method for deposition of films.

The solid substrate is moved successively down and up through the air-water interface and successive monolayers are deposited onto the substrate. One important

38


detail is that the molecules of the last deposited monolayer are practically always arranged with inert hydrophobic tails in the direction of the air medium after pulling out the substrate from aqueous subphase. Immersion of the substrate with the film after completion of the process (shown in Fig. 3.ld) into some solution for adsorption of the dissolved compound, e.g. some soluble protein, onto hydrophilic groups of surfactant molecules will not give any result because active hydrophilic groups are protected with inert hydrophobic layer. Design of usual LB instruments with one compartment usually enables to deposit the films consisting of monolayers of one type. However, if two separate troughs or one trough with two separate compartments are used one can deposit the structure of alternating bilayers of different molecules similar to that shown in the figure 3. le. This latter design gives the possibility to deposit films of alternating monolayers, but always the last layer is aTanged with hydrophobic inert layer outside, so that dissolved molecules will not interact with hydrophilic groups of amphiphilic molecules if we put the deposited film in some solution. A new alternative approach is to close the substrate with the monolayer deposited by dipping down the latter with some plate situated very near to the substrate (Fig. 3.12). If then the substrate is removed together with the closing plate from the water subphase, water will be held between the plate and the substrate by capillary forces (Troitsky ef al., 1995). Thus the monolayer is protected by the layer of water and the hydrophilic surface becomes the external boundary of the film. Then the system consisting of the substxate and the plate can be transferred to any other compartment, allowing the successive deposition of new active monomolecular layers and the adsorption of any dissolved compound.

Is the Solution Structure and Function Preserved at the Air- Water Interface? The issue of the protein monolayer formation at the air/water inteiface is critical for the preservation of native protein structure and function. The structure of most proteins is such that the hydrophobic nucleus is inside the globule and the hydrophobic and partially polar interactions maintain the protein structure. The value of the forces are comparable with that of the surface tension. Thus, during spreading

39

Technologies

of protein monolayers over the water surface there is always a risk to denature protein structure. There are some examples in literature (see cytochrome c) where such behaviour really takes place. Nevertheless, several proteins being deposited onto solid substrates by LB method demonstrate preservation of structural and functional, properties, as it was confirmed by numerous techniques in different groups. There are two possible explanations of such non-denaturing behaviour, namely, either the proteins do not denature at the surface because of their inherent structure and binding energy, or the proteins are denatured at the interface, but by forming a layer, which decreases the value of the surface tension, leave the remaining proteins being subsequently adsorbed on this layer i n their native structure. What is happening in the reality appears still an open question.

"I =O

! ... P4W

...... i

. _ . - ..... GST

0

1

2

3

4

5

8

7

9

10

M b M l m .

Figure 3.13 Surface conceiitratioii of proteins in film measured by nanogravimeiiic technique.

From the applied point of view the answer to this question is irrelevant, because in both cases well organized 2D films with reproducible structure and properties can be obtained. On the other hand, it would be important in order to understand the real process of film formation to experimentally probe the existence or absence of a denatured layer. The surface concentration of proteins cannot be estimated from compression isotherm analysis, but can be measured on the solid substrate by


40

nanogravimetric technique (Figure 3.13 and Table 3.1).The sub-phase is typically a buffer solution of phosphate 10-3 M pH 7.2 (or other salt of varying concentration depending on the type of protein). The total volume of the subphase is -80 ml., with

M pH 7.2 deposited at a concentration of about a protein solution in phosphate 2 . 5 ~ 1 0 M. - ~ Several layers can be quickly and successively deposited with LS technique and their thickness determined by ellipsometry (Figure 3.14). loo

I

1

I

400

300

0 0

1

2

3

4

6

8

7

8

9

10

nmbrolbwr.

Figure 3.14 Thin film thickness determination by ellipsomeiry.

In order to assess the effect of film formation on the native protein structure CD spectra have been recorded in solution and in LS films at different surface pressure (see Fig. 3.15 for a corninon protein): by this and other structural (see above) and functional Table 3.2) probes optimal conditions can be deteimined for the LS film assembly of any protein, either water-soluble or membrane-bound. Monolayer thickness at each surface pressure was evaluated by the slope of the regression line through the ellipsometric data obtained as function of the number of depositions.

41

Technologies 30000

2sooo 20000

--2 I5000 3

$

10000

e" J

5000

.--

a

-

0

w

-m L

0

-5000

8.

-15000

-20000

\\'uvclcn~tli Innil

Figure 3.15 CD spectra of proteins in solution and thin films.

12

RAM monolayer thickness [nm]

15

20

25

30

35

40

45

Surface pressure [mN/rn]-

Figure 3.16 Dependence of film thickness 011 surface pressure.

The surface pressure strictly determines their orientation and subsequent monolayers thickness (Figure 3.16), in perfect agreement with the geometric features of the very same antibodies independently determined by X-ray crystallography (Table 3.2). STM analysis of large proteins (as IgG), while impossible in LS film

42


due to the large monolayer thickness (about 120 A in the case of IgG), strikingly confirms in solution the above conclusion (Figure 3.17).

Figure 3.1 7 STM image of IgG molecule.

Figure 3.18 CD spectra of Photosynthetic Reaction Centres (a) and cytochrome P450scc in solution aid in film (b).

43

Technologies

Table 3.1 LB film weight and thickness versus number of layers as determined by (1) nanogravimetry in 10- 10 d100nm2 and ( 2 ) ellipsometry in Angstrom a~ saturating surface pressure. PROTEIN

CST

AP

(1) 2.80 (2)21+3

(2) 7&4

one layer

(1) 1.88 (2) 25f5

(1) 3.2 (2) 35

(1) 4.98 two layers (2) 39k3

(2) 13&4

(1) 3.47 (2) 57f5

(1) 6.7 (2)77

( I ) 7.92 (2) 48k4

(2) 205f4

(1) 4.53 (2) 9&6

(1) 10 (2) 127

( I ) 1088

(2) 245f4

(1) 6.06

(1)

three layers

four layers six layers

eight layers

Thioredoxin P450scc

(2) 11S?r6

(2)56+4 (1) 16.63 (2)79+5

(2) 4oof4

(1) 21.26 (2) 107f6

(1) 9.88 (2) 17W10

(1) 13.06 (2) 231f12

(1) 26.13 ten layers (2) 135f7

13.2 (2) 177

IgC

Rhodopsin Reaction Centres (RC) (1) 1.3 (1) 7.54 (2) 48 (la) 3 (2)40 (2) 9W10 (2a) 35k10 (1) 2.7 (1) 11.18 (2)65 (1)6.2 (2)72 (2) 175T15 (2a) 8m10 (1) 4 (1) 15.23 (2) 97.5 (la) 8.9 (2) 90 (2) 29&20 (2a) 12W15 (1) 5.5

(1) 22.07

(la) 11.8 (2) 125 (2) 42W20 (2a) 15W15 (1) 19.6 (1) 8 (1) 33.87 (2) 271 (la) 17.5 (2) 179 (2) 63&30 (?a) 250+20 (1)26 (1) 46.04 (2) 367 (2) 264

(1) 16.54 (1) 32.4 (2) 285+12 (2) 457

(2) 164

(2) 32.5

(1) 58.84 (2) 385

(2) 30

(1) 5.7 (2) 42

(2) 29.2

(1) 64.4 (2) 907

twenty layers Average monolayer

(1) 2.62 (2) 14

(2) 66

Surface pressure (mN/m)

(1120 (2) 20

(2)45

(1) 1.6

(2) 27

(1) 3.2 (2) 44.8

(1) 1.6 (la) 4.2 (2) 40 (2a) 40 (1)25 (la)40 (2) 40

(2)45

44


There are stable proteins that are not destroyed at the interface. One of the examples of such proteins are antibodies. Numerous covalent S-S bonds prevent the denaturation of the protein molecules at the interface, as these interaction forces are much stronger with respect to surface tension. In this case the usual LangmuirBlodgett technique can be applied for the formation of such layers. Other examples are the Photosynthetic Reaction Centers and the P45Oscc cytochromes, the structure (Figure 3.18a,b) and function (Figure 3.19) of which remain invariant upon monolayer formation.

0.004

0 002 Ooo3

0 001

i

A

0

0

~0001 0 002

0 003 -0 004

(b)

*br

P460 WILD N P E

0 66

0 55 0 45

0 35 0 25 0 15

0 05

0

-0052 0 15

"rn

-025

Figure 3.19 CO reduced difference spectra of the P450scc wild-type in the LB film (a) and in solution (b).

There are proteins, which do undergo minor partial denaturation at the air/water interface. Glutathione-S-transferase (GST) is an example of such proteins, and its

Technologies

45

molecule is divided into two subunits at the water surface. One can apply usual LB technique also in this case, but it is necessary to take into account that both the structure and the function of the GST in the thin layer will be somehow decreased, even if largely preserved (Antolini et ul., 1995a). Similarly for most other protein, being metalloproteins, enzymes or antibodies, as it has been shown in recent overview (Nicolini, 1995 a, b). Finally, there are proteins that are absolutely unstable at the water surface. Cytochrome c is one example of such proteins, as conclusively shown by infrared spectroscopy (Figure 3.20). Indeed the bonds which stabilise cytochrome c secondary structures have energy per bond of about 4 kcal mol-l. Considering that the area occupied by one cytochrome c is about loo0 A2, with the area per one bond stabilising secondary structure being equal to 10 A2, the density of hydrogen bonds responsible for the tertiary structure is approximately 10 times less, i.e. area per one bond is equal to 100 A2. The maximum change of surface tension value due to 'optimal reorganisation' of the molecule (all hydrophilic groups are in water, all hydrophobic groups are in air) is 72 - 28 = 44 mN m-l = 44 mJ m-2 (really considerably less because such 'ideal reorganisation' is impossible). Assuming the above values for the area occupied by one bond responsible for secondary and tertiary structure the corresponding energy per bond becomes about 0.6 kcal mol-1 and 6 kcal mol-1respectively.

46

Molecular Bioelectronics lnrd

.-

dCwh-

c 1"

.d&

1.

0.05

0 04

.

...

0.06

0.05

,

0.0.

,

1 I 0 0 1

',

Figure 3.20 Infrared spectra of native Cytochrome C in solution (a) and in LB film (b). For reference the heat-denatured cytochrome c in given in pluiel (c). The panel C shows the spectra of denatured protein. It inust be noted that the frequency of the lunide I band shifted at 1645 cm-l.

Technologies

47

Infrared Spectroscopv at the air-water interface Three different techniques have been usefully integrated for the identification of the secondary structure of cytochrome C monolayer at the air-water interface: attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy, interferometry and FT-IR spectrometry. The spectra of native cytochrome c was obtained using KBr pellet preparation. Heat- denaturation in solution and LB film formation at room temperature appear to produce similar spectral alterations, which in turn can be associated to similar alteration in the native secondary structure of the protein. Transmission spectra were measured in a Perkin-Elmer 2000 spectrometer using a SPECAC grazing angle accessory under the condition of angle of incident of 82", 4 cm-1 resolution and 30 scans. Generally the bands at the 1650, 1630, 1670, cm-1 were respectively assigned to a-helix, P-sheet and random coil structures: that at 1309, 1240 cm-l to the a-helix and P-sheet. Following denaturation there is a clear shift to higher frequency at 1665 cm-1 and in the region of the Amide I11 band a striking difference is observed in that the band at 1309 cm-1 disappears and that at 1240 cm-1 increases significantly. Following film formation similar effects are appaent, pointing particularly in the peak region around 1665 cm-1 to the cytochrome c denaturation. The interference method confirms the above conclusion allowing to assess film thickness, even in this case where X-Ray diffraction patterns do not show any Bragg reflections and reproducible data cannot be obtained by ellipsometry because of significant inhomogeneity. Dipping the

cytochrome solution on the water suiface and waiting for some time, one can see the increase of the surface pressure even without compression, which means the enlargement and uncoiling of polypeptide chains. In order to make possible the deposition of such proteins it is necessary to diminish the surface tension. Two different approaches can be considered. First, to attach the protein to head groups of preformed monolayer of lipids (Figure 3.21a) and to analyse the corresponding films by X-ray scattering. As shown earlier, when we have Bragg reflections we know exactly the sizes of the repetitive unit, and when we know the composition of the unit we can estimate the sizes of the elements. For example, in the Figure 3,21a, the repetitive unit of 30 8, contains one layer of lipid and one layer of protein. By knowing that one CH2 group is 1.25 8, long, since we have 16

Molecular Bioelectronacs

48

groups the thickness of lipid layer is 20 A. Thus, for the cytochrome c we have less than 10 A. At the same time the X-ray pattern of Langmuir-Schaefer film of pure cytochrome c does not contain Bragg reflections, so there appears no lateral ordering or periodicity in the film. Nevertheless, there are Kissing oscillations, which indicate homogeneity of the film and allow to calculate the total thickness of the film. The thickness was found to be 526 A, which corresponds to the layer thickness a little bit more than 10 A. LS of Cytochronm C (a)

900

2

I

3

5

4

e

Wl

“I ?o

o

i 0

I

2

3

4

L d l l d r q 04..

.

5

6

7

Figure 3.21 X-Ray pattern of P cytochrome C film without (....) and with (-) adsorption on the head groups of performed layer of lipid (a). X-Ray pattern of the same cytochrome C deposited using reverse micelles as Ihe spreading solution (b).

Technologies

49

In a case of Kissing oscillations, we deal with the interference of X-ray beams, reflected from upper and lower boundaries of the layer, and we can estimate only the total thickness according to the following formula: L=

A 2(sin@, -sin@,)

(3.4)

where L is the total thickness of the film, h is the wavelength, sin 0 is sinus of angular position at two adjacent oscillations. The second attempted approach to overcome protein denaturation at the interface was to use reversed micellar approach. This seems to be more promising, as it does not use any adsorption of protein molecules to the monolayer and, thus, gives more possibilities to control the film formation process. Under these latter conditions the native structure of cytochrome c appears indeed preserved as indicated by the X-ray pattern (Figure 3.21b). Furthermore typically prior to deposition on the activated substrate, the films are not only dried, but washed in the water jet rather vigorously as well, and it is very important in order to exclude the eventual presence of denatured sublayer. As for the two-layer model utilized in computing thickness from ellipsometric data, the first, lower layer, cannot be the denatured sublayer since the determination of its parameters was undertaken before the deposition: this layer charactenses indeed the substrate only. Furthermore in the emblematic case of GST enzyme film earlier discussed (Antolini, et al., 1995a; Nicolini, 1995a), our procedure of deposition provides the presence of the GST molecules on the substrate only when is covalently bound with the surface. Everything that is not bound is washed from it by the water jet. Our experimentation conclusively shows that a single non-denatured protein monolayer is formed in most instances at the air-water interface, which remains deposi&edon the activated substrate after washing. Namely if the water surface should carry a bilayer of proteins, with the first (upper) layer being denatured and the second lower layer containing the native protein adsorbed onto the upper layer, we could not obtain the presence of the native protein in the deposited layer. In this case all covalent bonds would be covered by denatured protein and native protein would be washed out.


50

Self

-

Assembly

It is almost trivial to say that in order to favours self-assembly of proteins in two dimensions, we have to procure to the proteins a two dimensional space. This means practically an interface. Because the size of the proteins is typically I 5 nm, the

interface must be at least smoother that the nanometer' level, say a few Angstroms, which is one or two atoms at the most. Since this is practically impossible to have such perfect flatness for solids, this means that only liquids can be suitable for this task. The protein self-assembly leading to 2D crystallization is performed in a two dimensional interface that is entirely controlled. Consequently, the physicochemisuy is followed with a full range of instruments specially designed for surface study like ellipsometry, fluorescence, surface pressure and others, as described above. So, self-assembly of a single monolayer can be more understood, and then eventually improved.

High density subphase method The first expeiiment of this kind was presented recently (Yoshimura et al., 1994). Apoferritin was used, which is in fact holoferritin without the iron core. The set-up was relatively simple: a Teflon well of about 2 cm diameter and 3 mm' deep was filled with a 2% glucose, 0.15 M NaCI, and 10 mM CdSO4 solution. The protein solution, here only 1 pl of 1 mdml, is injected smoothly under the surface of the subphase with a syringe. After 20 minutes, a Transmission Electron Microscopy (TEM) grid is deposited onto the surface for the collection of the self-assembled monolayer, frequently as a 2D crystal. Non-distorted and up to 10 pm large protein 2D a m y s were observed. The explanation concerning this success is in fact simple. First, the glucose

increases the water density. So, when the protein solution, of low protein concentration, is injected, the Archirnede force lifts it to the air-water interface. Second, the glucose have lowered the surface tension of water. So, when the protein solution reaches the interface, the difference in surface tension makes the solution to spread spontaneously. Third, this spontaneous spreading thins instantaneously the

Technologies

51

protein solution to a thin film of some microns thick, so the bulk 3D protein solution becomes suddenly a protein 2D solution. Fourth, according to the authors, the protein close to the interface is suspected to denature and make a film I nm thick. No direct observations, however, have allowed these researchers to visualise or confirm this phenomena (which is instead excluded in LB-manufactured monolayer of most proteins). Fifth, the lateral free motion of the proteins will allow them to make contact each others by random motion. Then under close contact, the cadmium ion will make a bridge between proteins and start building a crystal. It was also demonstrated that the mild heating of the interface during the incubation increased the size of the crystal. Sixth, glucose itself may be necessary to keep the protein away from denaturation. Also, different glucose concentrations were tried: 2, 4, 10, and 20% (W/V), and NaCl (0.5 M). Other additives than glucose were tested: dimethyl sulphoxide. polyethylene glycol, glycerol. These additives could not make the process of spreading efficient. Moreover, other salts were tested: CdC12, MgS04, MgC12, ZnC12. They all gave self-assembled 2D arrays, but the best situation was with 2% glucose, 0.15 M NaCI, 10 mM CdS04, and 10 mM MOPS (pH 5.7). This experiment, very successful with apoferritin. did not work with other proteins, including holoferritin. In this last case, the reason is that holoferritin is loosing the iron core after denaturation, which becomes an obstacle to further close contact. All parameters must be very well controlled: salt concentration, protein concentration, temperature, glucose concentration, pH, and above all, the purity of all products that are entering in the preparation, including the glassware, the Teflon wells, and the air itself must be dust free for an optimum efficiency and reproducibility. Also, it was demonstrated that the procedure itself of the protein solution injection is a factor of success.

Lipid monolayer method It is clear that for two-dimensional self-assembly, the protein must be fixed in a

plane (Uzgiris and Kornberg, 1983). This condition alone is insufficient, because if the plane is rigid, it was calculated that a maximum coverage of 55% is expected for spherical molecules adsorbing on it. So, fluidity is necessary for the crystal growth for lateral and rotational random motion that allows the proteins to turn around the

52

Molecular ~ioe1ectTonics

vertical axis perpendicular to the lipid layer while moving laterally. This creates a favourable situation for trying many positions before a suitable one is found. Moreover, a high concentration is also necessary, to have the number of collisions high enough. However, in order to avoid a chaotic aggregation, there is a balance to keep between the binding energy, concentration, and temperature (Tiller, 1991). Practically, the two dimensions space for proteins is provided by a lipid monolayer at the air-water interface. The lipid monolayer is in the liquid phase, so lateral motion of the polar heads is occurring. Starting from this point, two ways are possible: either the protein is attached to the lipid electrostatically or chemically. Nevertheless, the protein is stabilised in a two dimension like solution, and crystallization happens in a classical way. The difficulty of this approach is on the side of biochemists that have to find or to design the molecular 'Lego' parts in order that they fit one in each other. Using a lipid monolayer, the indisputable champion is streptavidin (Antolini ct ul., in press). Typically, the equipment utilized is a Langmuir-Blodgett trough, with all the accessories usually available. In the case of electrostatic binding, the protein in the subphase moves by random motion and convective flow to the interface. There, a monolayer of lipid is unifoimly spread. By having the appropriate pH and salt concentration, an attractive force between the protein and the monolayer will keep the protein in a two dimension space. Thus, lateral motion, always by random motion, will bring the protein one beside the other, and will finally grow a two-dimensional protein crystal. This was successfully performed many years ago with holofemtin. Similar results were rather rare, as ferritin is very well known to be easily self-assembled and thereby crystallizable, due its high symmetry. Applied to other proteins this method was less successful. Consequently, an other swategy was used: chemical or specific binding. The method of specific binding is the most efficient for making relatively large protein 2D crystals. One of the major reason is that self-assembly can be well engineered by a suitable choice of molecules. They can interlocked one in each other, either laterally or vertically. This is more or less a molecular 'Lego game', that requires a small effort on physical chemistry, but a major effort on biochemistry. Recently, such a demonstration was proven to be efficient (Ku et ul., 1993).

Technologies

53

Scanning microscopy In the last decade, a fact that the scientific community have always thought of as a dream became reality: the possibility of imaging surfaces and adsorbates over them with the resolution level of single atoms. So far the atomic and molecular structure of both inorganic and organic matter have been studied only in an indirect way and even the experimental probes capable of achieving atomic or molecular resolution (e.g. Xray crystallography) provided only averaged information over rather extended molecular or atomic assemblies. With the development of the piezoelectric technology and the possibility of achieving angstrom accuracy in positioning macroscopic objects, Rhoren and Binnig in 1982 (Binnig et ul.) were able to exploit a well-known quantum-physical effect, the tunnel effect, to realise a first Scanning Tunnelling Microscope (STM). By means of this device they for the first time succeeded in visualising atomic structure of several conductors and semiconductors in the direct space. The origination of this device and the first fascinating results acted as a starting point for a large series of microscopes based on the principle of nanopositioning probes for the investigation of different physical surface properties. The second device which was developed by Binnig i n 1986 was named Atomic Force Microscope (AFM) (Binnig ef ul., 1986) and obtained atomic resolution images exploiting the interaction forces which appear between the sample and a sharp probe placed in the proximity of it (distances of the order of few angstrom). Since AFM did not require the sample to be conductive, it was immediately considered as the best candidate for imaging organic and biological molecules. Subsequently, a considerable number of other scanning probe microscopes that made use of different physical-chemical parameters were developed, namely photon scanning tunnelling microscope (PSTM), scanning ion microscope, thermal profile microscope, magnetic force microscope, and Maxwell stress microscopy. All these microscopes were characterized by a high resolution level and by raster scanning working principle for generating images. However, it is necessary to say immediately that the generic term 'microscopes' under which these devices are collected does not completely depict all their potentialities. More appropriately they should be considered very powerful instruments for investigation of surface

54


properties of various samples as some of them can provide also spectroscopic measurements and manipulation of the sample under study.

Basic principles of STM STM studies the surface of conductive samples by sensing the tunnelling current which occurs between a metallic stylus and the sample when they are placed at a distance of few Angstrom within each other. The stylus scans by a raster technique over the sample and the microscope can function according to different modes and register data that are displayed in the form of images. These modes are the 'constant current' (or topographic) mode, the 'constant height' mode and the barrier height (or STM spectroscopy) mode. In the first operational mode, a feedback system maintains the tunnelling current constant while the sample is scanned by varying stylus vertical position and the image is formed recording this position and mapping it onto a two-dimensional array which can be regarded as an image. In the constant height mode, instead, the vertical position of the scanning stylus is fixed (i.e. the height of the tip above the sample) and the current measured in each scanning point is registered and used to form the image. Finally, the barrier height mode allows to obtain information on the tunnelling barrier height over the sample by modulating the tip position during the scanning at a frequency outside the working band of the feedback system in a way that the demodulated signal is proportional to the value of the barrier height over the sample, giving information on the chemical-physical environment of the sample. Several different experimental approaches have been developed for STM imaging and at present STM investigations can be carried out in air, in conwolled atmosphere, under ultra high vacuum, in electrochemical cells, at liquid helium temperature and can match all the possible requirements of the experimentalists. Moreover, it turned out that STM, seemingly contrary to its working principles, is able to obtain images also on non-conductive samples such as biopolymers (DNA, proteins, nucleosomes, etc.), opening new prospective into the potentialities of such a device (Lindsay el al., 1992; Guckenberger et 01.. 1994 ; Nicolini et al., 1995, Amrein et al., 1989).

Technologies

55

Basic principles of AFM An atomic force microscope retrieves surface information on conductive and nonconductive samples exploiting the interaction between the tip of a microlever and the sample surface when they are placed in such a close proximity as to give rise to repulsive or attractive forces of molecular and atomic origin. Using the above mentioned potentialities of the piezoelectric technology AFM, as well as STM, can image in a raster way surfaces of different sizes up to atomic resolution. Different working modes are available also for the AFM, namely constant force, constant height and surface friction imaging. The first two modes correspond to the constant current and constant height of the STM and provide, therefore, similar information. The third, instead, exploits the shearing movement of the cantilever and can sometimes be very useful, providing contrast in the image even if that from vertical deflection is not sufficient. Due to its capabilities of imaging insulating samples, AFM was considered, since its origin, as the best candidate for imaging biopolymers and biostructures. DNA, nucleosomes, cells, microorganisms, proteins, Langmuir-Blodgett films, etc. were imaged by AFM (Yang et ul., 1993; Alliata et ui.,1996). However, the interaction of the cantilever with soft biological samples results often in the destruction of the sample since the distance at which the cantilever scans the sample is usually (repulsive mode, which can provide the highest resolution) closer than that at which the STM tip works. Moreover. for the aims of bioelectronics, it is essential to have a probe which can give information on the electronic behaviour of the sample under analysis. Therefore, it seems that despite the limits that STM displays in imaging non-conductive samples, it is a very powerful tool for investigating the electrical characteristics of the sample at molecular level. Most likely, the best results will come from a synergetic interaction of AFM and STM, taking into consideration for particular requirements also other scanning microscopes which can contribute to give a coherent picture of the sample under study.

56

Molecular B i o e l e c h n i c s

Electron Tunnelling As explained before, the working principle of an STM is based on the

phenomenon of electron tunnelling through a gap between two electrodes, the conductive substrate and the metallic tip, respectively. It can be useful, therefore, to recall the basic concepts underlying this quantum mechanical phenomenon.

vacuum l e v e l unoccupied states

ev

Figure 3.22 Shapes of poreiirial barriers: a) tunnelling between two metal electrodes: b) between two different materials arid c) for a sinall bias voltage.

Considering, for sake of simplicity, a one-dimensional problem of tunnelling between two metal electrodes a small distance, d , apart, the potential barrier can be approximated by a square one as shown in Figure 3.22a. If we think of conducting electrons in terms of plane waves, then the tunnelling probability contains the exponential factor exp(-2kd), where d is the barrier thickness and k is the wavevector of the electron within the barrier. At room temperature, few electrons are thermally excited and tunnelling occurs near the Ferini level. Hence, we can write:

Technologies

A

57

(3.5)

A

where U is the barrier height, E F is the Fermi energy and 0 is their work function. When two electrodes are of different materials (Figure 3.22b) the equilibrium conditions make the Fermi level constant across the junction barrier and a contact potential arises. At zero bias voltage, the tunnelling probability in both directions is the same and there is no net current. For a small bias voltage, (Figure 3.22c), the shape of the barrier does not change strongly and the tunnelling probability is still given by the early equation. However, in this case more electrons can tunnel in one direction, as indicated by the arrow, causing a net current to appear. Since the number of these electrons is proportional to Vat room temperature, the tunnelling junction is ohmic and one can write the tunnelling current as follows:

where A = 2 a / A = 1.025/&eV)1/2. For example, for a typical work function of 4eV, the current decreases by one order of magnitude when the gap is increased by 1 A. This fact accounts, of course, for the extremely high resolution attainable in STM microscopy.

experimental

theoretical

Figure 3.23 Comparison between a siinulnted and an experimental STM image of H O E .

58

M o l e c d a r Bioelectronics

'I'his simplified approach, however, although explaining the basic nature of electron tunnelling, cannot be used to interpret STM images because the real situation is much more complicated. For instance, the tunnelling tip cannot be considered as a plane and the electron wave functions at the Feimi level are, in general, much more complicated than plane waves. Therefore, the electronic structure of both sample and tip should be taken into account, yielding an expression for the tunnelling current in teims of first order perturbation theory (Bardeen, 1961). From this expression the tunnelling current can be calculated, in principle, for any given wave function, enabling, thus, the interpretation of STM images (Tersoff and Hamann, 1983). The potentialities of these ab initio calculations allow to produce images of the most common substrates used in STM imaging, such as highly oriented pyloric graphite ( H O E ) , Au (1 lo), etc. Moreover, improvements of this theory have led to the construction of images of adsorbates over such substrates as benzene molecules, quinones and nucleic acid bases. In Figure 3.23 the comparison between a simulated and an experimental STM image of HOPG is reported. However, despite its importance, this approach to the understanding of STM images seems to be not sufficiently powerful as it cannot account for all the various mechanisms of STM image formation, especially on organic and biological molecules.

Imaging biopolymers and biostrdctures Almost immediately after the birth of STM it began to be clear that such kind of device could be used to image successfully also samples for which it was impossible to forecast a priori suitable current levels. Convincing images of large molecules, assumed to be insulating, began to appear in literature. It was shown to be possible to image, by means of STM, double stranded DNA, and after that, images of other molecules and adsorbates were reported in literature. Therefore, it became clear that the principles undergoing STM image formation on large biological and organic adsorbates are not always and not simply (disregarding the numerous mistakes that appeared) pure tunnelling as described in the previous paragraph, but that also other phenomena, not necessary electronic tunnelling, contribute to image formation process in case of large adsorbates. For various existing cases, several different and complementary explanations have been

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proposed to explain such images, namely ionic current detection, resonant tunnelling, decrease of the tunnelling banier caused by the presence of adsorbate, and contact mode scanning. These phenomena in some cases can be used to explain the possibility of recording meaningful images on samples which should not provide enough current if considered within a simplified theoiy. It is, however, worth noting that STM imaging is always a very complicated and often tricky technique which exposes the experimentalist to serious risks of mistakes. In fact, periodical noises, both of electrical or mechanical origin, can very easily be confused for meaningful signals arising, for example, from periodical lattices if a careful analysis is not carried out. Towards this purpose, it is very useful to study STM images in the reciprocal space by means of two-dimensional fast Fourier transfoim algorithms, as this type of study allows to promptly reject, for example, images caused by peiiodical disturbances. Another approach which has to be considered in interpreting

STM images and uying to draw conclusions out of them is to integrate the incoming data with those from all other possible experimental techniques which are available for imaging and with the knowledge of the sample itself. This is the way STM is often used together with AFM or Transmission Electron Microscope (TEM). During its recent development, STM has been successfully used to image several kinds of biological systems achieving sometimes previously unmatched resolution levels and, in any case, very useful information on the structure and the spatial organisation of the samples. Biomembranes, bacteriophage particles, single stranded and double stranded DNA, proteins (metalloproteins, enzymes, membrane proteins), lipids organized in monolayer by the Langmuir-Blodgett technique are among the most investigated samples. Despite the popularity that these measurements achieved, it is important to stress that so far any STM experiment on biological objects is highly critical and requires the use of a set of techniques developed and used 'ad hoc' for each specific sample. Among the different experimental choices that the experimentalist has to face before starting STM studies on biological samples, the most important are surely the choice of the best material for the scanning tip and therefore the technique for its preparation. In fact, it is very important, especially imaging large isolated biomolecules, to use a very sharp scanning stylus (e.g. tungsten tip electrochemically etched) which is able to 'enter' the possible grooves without interacting with the sample by multiple tips which can be present on the tip

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conus. However tungsten tips are much easier to damage and to dirt, especially scanning on soft materials as biological ones, and from this point of view Pt-Lr tips mechanically cut could be better. Another very critical point is the sample preparation. It is essential to have a highly purified sample in order to minimise the presence of debris and of other undesired molecules or fragments. Moreover, it is of fundamental importance the choice of the proper buffer, if needed, and of its concentration in order to maintain the sample native structure and to avoid the formation of a thick salt film when the sample is dried for STM analysis in air or in vacuum. The substrate on which the sample will be deposited is another point which requires basic care and needs careful studies before starting the measurements. In fact, there are several basic requirements for the substrate to use in STM measurements. A very important parameter is the substrate flatness, which should display coirugation as smaller as possible in order not to affect the image and the molecular structure of the adsorbate itself. Another often critical point is the possibility of fixing the sample to the substrate in a fashion stable enough; it is essential to match this requirement in order not to move the sample under the scanning action of the tip, loosing, thereafter, the resolution. This problem, mainly connected with the hydrophobic or hydrophilic properties of the substrate and of the sample to image, is often faced by functionalizing the substrate before sample deposition in order to allow chemical binding between the substrate and the biomolecules. It is worth nothing, however, that this action requires a further detailed investigation of the surface structure of the functionalized substrate before imaging biopolymers. A further problem choosing the best substrate is the amazing fact that some of them can display by themselves features similar to those we are interested in! A typical example of this behaviour is the mimic of DNA and other biomolecules that HOPG display even when nothing is deposited on it (Clemmer and Beebe Jr., 1991). The functionalization of this surface, however, can solve this problem (Nevernov et al., 1994). Among the most important results, nucleic acids, molecular (Ciicenti et ul., 1989) and atomic (ultra-high-vacuum STM) resolution was achieved on single and double stranded DNA molecules, showing very fine details on the structure of the molecule, such as the sizes of minor and major grooves, the atoms of the phosphate backbone, etc. Towards the goal of enhancing the understanding of STM images of biopolymers, it turned out very useful to apply

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the gap modulation imaging mode together with the constant current mode. In fact, this method allows to outline the different physical-chemical parameters at the level of the single atom, helping in the identification, for example, of the different chemical groups of the DNA backbone which were deposited onto various samples. These results, showing atomic resolution images on nucleic acids have allowed even to speak about DNA sequencing by STM. In reality, however, this potentiality seems to be rather hard to be realised mainly because imaging such kind of biopolymers is not at all a routine work so far. Proteins have also been studied by STM, even if their size sometimes do not allow any imaging. Enzymes complexes such as phosphorylase b-phosphorylase kinase (Edstrom e f al., 1990) have been studied and differences in shape and size of the phosphorylase kinase molecule have been outlined when it bound phosphorylase b. Fragments of membranes, containing functioning protein membranes such as bacteriorhodopsin (Kononenko el al., 1990) have been imaged. Isolated Reaction Centres from Rhodobacrer sphaeroides (Alekperov er al., 1988) have been studied and their sizes have been found in agreement with the data from X-ray crystallography analysis. Oligomers of turtle a-macroglobulin on HOPG have shown features which were found also by AFM. confirming strongly the possibility of having STM images even from large proteins (Arakawa er al., 1992). STM on organic molecules has resulted to be a very powerful method for investigating their structures. For instance, in case of liquid crystals, atomic resolution is available on the single molecule and the film structure is exactly recovered (Spong ef al., 1989). Analysis on bilayers of fatty acid salts such as cadmium arachidate could elucidate the film structure showing molecular resolution and confiiing the data obtained by other techniques such as electron diffraction about the elementary cell parameters (Lang er al., 1988).

DNA and its suDerstructures Imaging DNA in ambient environment has been subject of great interest, due to its potential application to the study of DNA packing, protein-DNA interaction and, possibly, DNA sequencing. Several recently published sample preparation methods for DNA imaging by AFM allowed to achieve stable double and single-stranded

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DNA images as well as DNA-protein complexes. The obtained results matches well with the data from electron microscopy. Among the basic methods for improving the sample adhesion to the substrate there is the treatment of the substrate (mica, glass, etc.) with 3-aminopropyltriethoxy silane (Lyubchenko et al., 1992a, b). This treatment allows DNA and RNA to adsorb strongly to the substrate surface and peimits several scans over the sample. With this method a resolution of 6 nm was achieved with conventional cantilevers and 2-4 fold better using 'supertips' (Lyubchenko et al., 1993). Another method for fixing the sample to the substrate is to treat mica surface with magnesium acetate (Bustamante et 01.. 1992; Hansma et al., 1992; Vesenka et al., 1992). With this method and supertips under organic solvents such as butanol or propanol, a resolution of 3-5 nm was attained. Coating mica with a carbon film and depositing over it a solution of DNA, cytochrome C and formamide spread over a water surface gives also reliable images with 4-6 nm resolution if electrical grounding of the substrate is done with care (Yang et al., 1992; Yang and Shao, 1993). Imaging in organic solvent such as iso-propanol or n-butanol allowed to reach 3 nm in resolution. All the reviewed methods, however, did not allow so far to resolve the DNA double helix. Nevertheless, they suggest the possibility of imaging successfully DNA superstructures at nanometer resolution level. Study of higher order chromatin structures have been performed by means of STM (Nicolini et al., 1995g) and AFM (Alliata et al., 1996) and provided clear evidences for the superstructure organisation of DNA-protein complexes in fiber of about 25-30 nm and higher order 70-80 nm and 120-140 nm superfolding respectively. Atomic force microscope (AFM) has shown itself to be a powerful tool in high resolution imaging of biopolymers and biostructures up to molecular level. Large uncertainty still exists on the higher order chromatin superfolding in native nuclei. While earlier obsei-vations (Nicolini et al., 1989) and model (Nicolini, 1983) point out the drapery-like model of the regular folding of 300 8, fiber within the nucleus (as 'quinternary' chromatin-DNA superstructure), recently (Belmont et al., 1989) by scanning electron microscopy observed 130 nm wide fibers within both native interphase nuclei and intact metaphase chromosomes. To further prove these later findings, a native chromatin sample was isolated from GO calf thymocytes.

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mildly fixed in glutaraldehyde and deposited on a mica sheet in order to be investigated by atomic force microscopy. Chromatin was obtained by bursting the isolated nuclei in accordance to the cold water method (Nicolini er al., 1988) in order to yield native chromatin fibers longer than lo8 Da which display the topological constraints needed to maintain the higher order superstructures (Nicolini et al., 1988; Diaspro er al., 1991; Nicolini, 1983). Subsequently, the chromatin sample was mildly fixed with 1.6% glutaraldehyde, which does not alter native structural parameters of this sample and the degree of DNA supercoil, as monitored by ckcular intensity differential scattering (Nicolini et al., 1988; Diaspro er al., 1991; Nicolini, 1991) and differe.ntia1scanning calorimetry (Vergani er ul., 1992). Scanning force microscope was calibrated by means of mica lattice, by 1 pm grid and using polystyrene micro spheres of 91 nm of diameter with a standard deviation of 6 nrn (Sigma Chemicals Co.) i n Tris-HC1 10 mM at a concentration of 7.10* spheres/pl, to account for the effects of tip-sample convolution. For chromatin observation a freshly cleaved mica sheet was prepared in order to make its surface highly hydrophilic; indeed, this operation causes a temporary accumulation of electric charges at the surface, providing a good adhesion of the sample with a non invasive method. Immediately after this operation, a drop of sample solution of about 3 pl in volume was deposited and left drying for 15 minutes. The sample prepared in such a way was imaged by AFM in constant force mode (force = 10-8 N and scanning rate = 4 Hz)using microlevers of Si3N4 with a minimum tip radius smaller than 400 A and an elastic constant of 0.04 N/m. In Figure 3.24a, a fiber with a snake-like shape and an average diameter of 140+8 nm is visible across the picture. From this main fiber, a number of thinner 25-30 nm wide branches and loops depart, suggesting that they might concur to form the thicker one. Actually, it is impossible to say if they are single 25-30 nm fibers or the result of several of these fibers folded on themselves as earlier observed by electron microscopy. In Figure 3.24b. which is a physical zoom of Figure 3.24a, one can see that the same 'native' 140 nm fiber appears to result from the superfolding of a thinner fiber of about 25-30 nm. These data, which result from the first direct clear obseivation at the highest order chromatin structure by atomic force microscopy, are in good agreement with earlier experimental observations by other authors on large scale 3D chromatin domains using stereo pairs of human chromosomes and of

64


interphase nuclei froin 0.5 pin thick sections. These observations would appear to suggest that, contrary to earlier inference from freeze-etching (Nicolini et al., 1989) and viscoelastometry data (Nicolini et ul., 1982) on rat liver chromatin, in native chromatin from calf thymocytes the 25-30 nm 'tube like' fibers superfold into successive wider 'tube like' 130-200 nm fiber as suggested time ago by polarized light scattering microscopy and by three-dimensional stereo electron microscopy of nuclei stripped of their envelope (Nicolini, 1983; Belmont et uf.,1989).

Figure 3.24 AFM image ot' a chromatin sample a) and its zoomed part b).

LiDid and protein films Exploiting the potential of the Langmuir-Blodgett technique, it is possible to foim and deposit onto solid substrates many kind of films both from lipids, phospholipids and proteins (membrane proteins, protein containing membranes, enzymes, antibodies, etc.) which are very interesting subjects for AFM investigation and for bioelectronic applications. The possibility of studying these molecules in form of films is veiy important both for the thin film science and because it represents a good

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way to organize the samples in a stable enough way in order to be successfully scanned and manipulated by AFM. Considering lipid films at first, LangmuirBlodgett films of cadmium arachidate, stearic acid, and several phospholipid monolayers and bilayers have been imaged by AFM. Langmuir-Blodgett films of cadmium (barium) arachidate are among the most robust for transfer onto solid substrate. Several experiments have shown molecular resolution on these samples both under air and under water (Schwartz el al., 1992). Surprisingly, even with probe forces larger than 200 nN, the lattice structure of either head groups or tails, according to the orientation of the top layer of the multilayer structure, was resolved in air with about 5 resolution (Buldt el (11.. 1978; Dorset, 1978). Even if other techniques such as X-ray, electron or neutron diffraction give better resolution, AFM

a

images the membrane surface (rather then the bulk or the mass density) and offers the additional possibility of visualisinp the domain structure of the membranes (Gamaes et ul., 1992). Phospholipid molecules in biological membranes have larger head groups with larger hydration shells than fatty acids. This fact causes more difficulties in the film formation process (less order) and in the transfer to solid substrates. AFM can image them under water and allow the study of the films which mimic natural membranes under native conditions. For instance, on bilayers of dimyristoylphosphatidylethanolamine(DMPE) under water, resolution level of 7-9 A have been achieved (Zasadzinski et ul., 1991).

Biomolecular Information Read-out and Interfacing Among the technique for sample preparation, it seems that a very suitable approach is that of making Langmuir-Blodgett films of the molecules to be investigated. As it was shown in an earlier paragraph of this chapter, in recent years it has been developing several techniques for Langmuir-Blodgett film formation of proteins and also nucleic acids. These samples can solve many of the problems connected with STM imaging of biopolymers if molecular resolution is sought for. In fact, LB film structure is such that can anchor the sample to the substrate surface in a rather effective way. avoiding problems connected with the sample motion under the tip effect. Besides, LB samples solve rather simply the problem, non trivial in such kind of microscopies, to find the sample on the substrate surface. An important

66


example of the potentialities of the STM used together with LB technique is the study of proteins monolayers. STM has been applied to study the structure and the functional properties of monolayers of Reaction Centres (RC) from Rhodobacter Sphaeroides organised in LB film (Facci et ul., 1993). Such a study is a meaningful example of the potentialities and the limits of STM as an investigating tool for Bioelectronics. RC, a large (100 kDa) protein formed by 3 subunits (Allen and Feher, 1984) and carrying out light-induced electron transport across bacterial membranes (Moser and Dutton, 1988), are subject of detailed investigations because they enter the photosynthesis process and are among the most promising molecules for bioelectronics. The existence of electron transfer chains inside this protein facilitates the flow of current through it and points out RC as one of the best object for STM study among proteins. In order to exploit the properties of RC as active elements of bioelectronic devices, it is useful to organize them in a regular twodimensional lattice, study their structure and function and in which way these properties depend upon temperature. Langmuir-Blodgett (LB) technique providing these regular structures, is considered as one of the most useful technologies for bioelectronic purposes (Lvov et al., 1991). The film prepared according to the procedure described above was deposited over a plate of freshly cleaved highly oriented pyrolytic graphite by the horizontal lift technique, which has been shown to be the best for protein film deposition. RC samples were dried by means of a nitrogen flow in order to remove the excess proteins from the surface of the sample. PtIr (80:20) mechanically cut tips were used throughout all the experiments because of the softness of the sample (less risk to dirt them). Samples were first studied without modifications after keeping them in the dark for a couple of hours before scanning them with STM. All the images were acquired in constant current mode in the tunnelling voltage range -1.5 t 1.5 V and in the tunnelling current range 0.1 t

0.5 nA. Outside the specified voltage range, it was impossible to get meaningful images, but a marked spike-like noise began to take place. STM Voltage-current (VI ) characteristics were also registered by placing the scanning tip over the desired region with the feedback system switched on. Then, the feedback was switched off, the bias voltage was swept in the range -4 + 4 V and the corresponding output current was recorded. RC were imaged in the dark (Figure 3.25) and in the light. Irradiation was provided by a 100 W tungsten lamp. using 3 cm water filter in order

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to avoid sample heating. In the dark, the images revealed two different periodic structures: a low frequency one, giving average spacing value of about 64 8, and a higher one corresponding to a periodicity of 30+1 A. In the light, no periodic features but graphite steps were obtained. The higher frequency corrugation can account for the subunits constituting each molecule, while the 64 A periodicity correlates well with dimensions of the protein molecule itself. Besides, these values are in agreement with the protein size obtained from X-ray crystallography data of RC structure (Yeates e t a ] . , 1988) from Protein Data Bank (Bernstein er al., 1977; Abola et ul., 1987). V-I characteristics, in single points, were measured both in the dark and in the light to try to show the functional activity of the single RC protein, Figure 3.26.

Figure 3.25 STM images of RC in the dark.

The results outline the basic difference in the RC film under two different experimental conditions. In fact, the V -l characteristics, measured in the dark on several bumps, show a resonance at a tip-substrate voltage of 3.16 V. This peak disappears completely in the light. This resonance is due to the functional properties of RC, namely the electron transfer process between bacteriochlorophyll donor and quinone acceptor. This hypothesis is corroborated strongly by the different trend of positive and negative branches of V/I curve confirming the asymmetry of the electron transfer chain inside the protein. Thus, it is possible to induce the electron transfer

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even without light irradiation by applying a proper voltage between tip and substrate. A simple estimation of the energy associated with a photon of 867 nm (maximum of BChl2 optical absorption) gives a value which is about half the value we obtained for the resonance. Therefore. this result agrees with the previous hypothesis, as energy is required not only to induce the electron separation between the groups constituting the electron transfer chain, but also to overcome the sample-tip tunnelling barrier.

?

1 0.8

E!

t

0.6 c

f

I .

light

0.4

-0.2 -0.4

-0.6 , 4 -4

-+--I

-3

-2

-1

1

0 voltage

2

3

4

M

Figure 3.26 Functional activily ol' RC measured in the dark and in the light.

In the light the process of charge separation inside the protein is caused directly by the incoming photons and therefore, V-I measurements cannot elicit any detectable resonance. Under these conditions RC can be considered as a permanent dipole. Surface potential measurements were applied to quantify the effect of this dipole-like behaviour, namely the potential generated by the monolayer when irradiated by light, Table 3.3. Thus, to explain the loss of resolution in the light, it was settled the hypothesis that the tip could rearrange the molecules under itself due to their dipolelike behaviour induced by light. It is worth noting that this phenomenon can take place even in closely packed systems as that at issue. Therefore, RC monolayer were studied after a chemical treatment for fixing the protein matrix. In particular, it was chosen glutaraldehyde (GA), a fixative which binds amino and hydroxyl groups and

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that is known to foim a network of covalent bonds. First, the deposited sample was immersed into 1% GA solution for 2 minutes, then washed and dried with nitrogen. This action was expected to 'freeze' the film into a rather rigid network, avoiding any rearrangement of the molecules under the tip action. Figure 3.27 reports a STM image obtained on the fixed sample during irradiation. The same features were imaged in the dark. The periodicity measured in this case is again 30fl 8,. as in the just deposited film. However, likely due to the fixative effect, the 64 8, modulation disappears. The similarity of the images obtained during darkness and light confirms that the fixation of the molecules inside the LB film prevents the molecule reorientation during the scanning process. Surface potential measurements on this cross-linked filin give results similar to the one in Table 3.3, confilming the protein activity (data not shown). Afterwardb, i t was faced the problem of understanding the effect of heating upon the filin stiucture, knowing that protein secondary structure is

preserved even at 200°C for protein arranged in LB films. Therefore, an RC sample was heated at 150 "C for 10 minutes and then it was imaged, Figure 3.28a. The images obtained on such a sample were absolutely the same both in the dark and during irradiation. In these images, a highly increased order was evident and a spacing of 27fl 8, appeared (see also zoomed image) together with a new hexagonal packing (according to the symmetry of Fourier transform). The increased order in the film structure, in comparison with that in Figure 3.28a,b, seems to be due to the closer packing of the previously identified subunits which have undergone a recrystallization process induced by temperature. Moreover, both in the dark and light, the V-I characteristics were absolutely straight lines, pointing out the fact that the functional activity of the proteins was lost. Table 3.3 Surface potential measurements of RC films.

One RC monolayer Ddk

Light

surl';lce Poieiitial (mV) 25f 1

344f1

70


Figure 3 2 7 STM image of glutxaldehyde-fixed RC film.

Figure 3.28 STM image of RC sample after heating.

Surface potential measurements on the film in dark and light (Table 3.3) show clearly that heating induces the loss of protein activity. Further, the measured values

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of the surface potential, being almost constant in both cases and nevertheless intermediate between the values obtained on the noii heated film, are in agreement with the hypothesis of recrystallization after heating. The previous is, therefore, an example of the application of STM within the framework of Bioelectronics. In fact, it has been shown that this investigation tool can be fruitfully used for studying the characteristics of sample of interest for biomolecular electronics, achieving a resolution level both in the imaging and i n the functional investigations which is uiiinatched by any other instrument. STM investigation, in fact, provided molecular and submolecular resolution on these protein samples; moreover, allowed to analyze the effect of the scanning tip action on the protein assembly finding evidence for the effect of the interaction between the proteins in the film and the tip. This interaction resulted to be due to the interaction between the dipole field generated by RC during illumination and that present between the tip and the substrate due to the bias voltage. Furthermore, the reported study shows a clear example of interfacing single biomolecules with an investigating probe. The STM tip can therefore be used to read-out information at the molecular level, representing the first step towards the experimental realization of systems based on biomolecular basic elements (proteins, quantum-sized structures, etc.) constituting the single functioning units.

Molecular manipulation Nanoengineering - the creation of structures with resolution better than 100 nanometers, holds particular allure for the semiconductor industry, where smaller is always better. Until now, however, the promise has remained largely theoretical, because conventional chip-making techniques have trouble with anything smaller than 0.2 pm. and newer methods have been confined to the laboratory. First indications of the feasibility of suiface modifications on the atomic scale by means of scanning probe microscopy instrumentation were found by Becker et 01. (1987) and this was finally demonstrated by Eigler and Schweizer in 1990. To move an atom, Eigler and Schweizer lowered the tip to the atom. The tip was then moved, dragging the Xe atom with it. Afterwards the tip was withdrawn, leaving the xenon at the desired position. To perform this experiment STM was operated in ultra high vacuum (UHV) at the temperature as low as 4 Kelvin to avoid

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the sample drift and suiface contamination. This success, showing the possibility of fabrication of atomic scale su-uctures, have not brought until now to the realisation of really working electronic devices due to the very high requirements of the technological conditions (ultra high vacuum and low temperature).

FiguriJ 3 2 Y The lirst electronic device made by STM lithography. This thin-film resistor was made by exposing PMMA and subsequently lifting off a 13.5 nm film of gold-palladium. The device showed a room-temperature resistance of 2.5U.From McCord et a l 1988.

Attempts to use the scanning probe lithography on bulk material had greater success in fabrication of really working devices. The basic ideas of this type of surface modification on nanometer scale are summarized in the review of Shedd and Russell (1990). The basic idea of this type of surface modification is to use the STM as a low-energy electron-beam lithograph (Figure 3.29). The surface of bulk material over which the lithography is to be performed is covered by a thin layer of e-beam resist, which can be locally passivated by the STM tip working in the field-emission mode. Short voltage pulses of tens of nanoseconds are used to perform the e-beam etching of the resist without heating the tip and the sample. Further chemical treatment allows to produce nanostructures in bulk inateiial with resolution up to 10 20 nm. Recently, the group of Quate (Gardner, 1994) reported the creation of a working transistor using the atomic force microscope with metal coated tip.

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The main advantage of this technology is that it is compatible in some limits with the traditional chip-making technologies. Changing the parameters of voltage pulses it is possible also to perform the deposition of metallic features onto the sample surface. Foster et at. (1988) pinned organic molecules with the STM onto graphite. They were also able to subsequently cleave or remove the deposit. Tens of nanoseconds pulse of 3.7 V was applied to the tip, leading to the pinning of the organic molecule. Later Yuqiu er ul. (1992) showed the possibility of DNA manipulation with 100 n s pulses. In this work a DNA molecule was picked up and attached to the STM tip when a short voltage pulse was applied and was redeposited in another place by reversing the pulse polarity. This opens new perspectives for the nanometer scale manipulation (Figure 3.30), thus the technology can work in ambient conditions and even the biological material can be patterned without its destruction. Moreover, the recent success of Eigen and Rigler in single biological molecule individualisation (Eigen and Rigler, 1994) shows that the selective assembly procedures can be developed. The proposed way to build a submicron device is to deposit the construction elements, onto a solid substrate in such way that further lateral arrangement of those elements is possible by means of a probe of the scanning probe microscope. Preliminary studies of the deposition methods with polystyrene particles show that it is possible to obtain a random homogeneous lateral distribution of particles with a controlled density. In order to arrange the construction elements on the substrate suiface several technologies may be used. Nanometer-size particles can be moved over the substrate surface when a mechanical force is applied by moving probe of SXM. When the force of mechanical interaction between the probe and the particle becomes greater than the friction force caused by particle adhesion to the surface, F,, > Ff , the particle will start to move with the SXM probe. The most promising method of biological molecule manipulation is, however, electric field trapping. Utilisation of this technology in volume have already brought to a great success in sorting single molecules. Several attempts of transferring single molecules by electric field pulse were successfully made. The main idea of this method is to apply a voltage pulse between the metallic probe and the substrate in

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Molecdar

Baoelectronics

order to pick up the charged object. The probe can be placed in any desired point and then, by reversing the bias voltage, it is possible to release the molecule. The iterative use of this approach can give rise to supramolecular architectures such as nanowires and nanostructures made of the desired molecules and, therefore, pei-forining the desired functions.

Figitw 3.30 Spatial manipulation of T4 DNA molecules with an STM tip. (a) STM tip (large arrow) i n the presence of a few tluoresccntly stained DNA molecules (small arrow). The DNA solution is very dilute so there are just a few DNA molecules in the filed of view. (b) A potential of 1.5 V has now been applied to the STM tip and the DNA molecule has been 'picked up' and attached. (c) The tip is now moved. carrying the DNA molecule with it. (d) By reversing the voltage and rapidly shaking the tip thc DNA molecule can be detached. (e) DNA molecule pulled into an extended configuratioii by viscous d u g as thc tip inoves rapidly diagonally upwards (~UTOW). (from Yuqui e/ ul.)

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Light-Directed Chemical Synthesis In the laboratory of Fodor, a technology for constructing large compound arrays has been developed which combines techniques of solid-phase chemistry, photolabile protecting-groups and photolithography (Fodor el ul., 1991). The synthesis of compact library matrices is made possible by the specific characteristics of photolithography (precision and high resolution). Moreover, a binding agent of interest, such as an antibody, enzyme or receptor assays in a single incubation all immobilised compounds. Thus, for every compound synthesised activity data is obtained noting both positive and negative binding.

Figure 3.3I Lightdirected chemical synthesis (according to Jacobs and Fodor, 1994)

Figure 3.31 depicts the synthesis procedure. In the first step, linker molecules are attached to the substrate. The amines of linker molecules are blocked by photochemically cleavable protecting group like Nvoc (nitroveratryloxycarbonyl). By using a photolithographic mask, specific surface sites are deprotected by illumination, creating a specific pattern. The exposed regions are linked to Nvocmodified amino acid by standard procedures. By using as many new masks as required, arrays of desired composition and length are built up, where the product set-up is defined solely by the mask pattern and synthesis sequence (order of amino

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acid additionj. The whole process is rendered more precise by computer control feedbacked by a specific database . The obtained arrays serve not only as sources of moleculx diversity, but also as hybridization probes for sequencing fragments of DNA. The application of combinatorial chemistry to the synthesis of peptide and oligonucleotide libraries is a powerful new tool for studying biomolecular interactions. Peptide and oligonucleotide synthesis was a logical starting point for developing combinatorial synthesis, as both classes of compounds include biologically active representatives, and can be prepared using either chemically or biochemically driven techniques (Wells and Lowman, 1992; Dower, 1992; Birnbaum and Mosbach, 1992; Pavia et a / . , 1993). In the light-directed chemical synthesis the location of ever compound synthesised is defined and detection is independent of the composition of the compound being investigated. Light-directed chemical synthesis involves the construction of large ai-rays of compounds i n a spatially directed fashion. Simultaneous detection of positive and negative binding events is carried out utilizing high-sensitivity fluorescence microscopy of the bound agent. I n principle, any solid-phase synthesis strategy could make use of this procedure BS long as the protecting groups of interest can be removed photochemically. With the identification of solid-phase techniques used for synthesis of novel compounds, these methods are apt to become adopted in lightdirected chemical synthesis procedures.

Tec hnulugies

T7

Ab Initio Molecular Design The purpose of this subchapter is to provide, basing on the available literature data, a structural and mechanistic background for the choice of metalloproteins currently under study at the Institute of Biophysics, University of Genoa, for the implementation of materials for electronics, and analyse their properties in view of their association into structures that could be used in bioelectronic devices. It should be noted that molecular manipulation techniques have already been developed and involve: self-assembly (Morgan er al., 1992 ; Hoffmann et al., 1992); Langmuir-Blodgett/Langmuir-Shaeffertechniques (Facci et al., 1994a). including utilisation of reverse lipid micelles to form protein films (Erokhin er ul., 1994); site-directed chemical modifications complementing the above two techniques (Eldarov eta/.,in preparation). The choice of metalloproteins as the most plausible materials to be used in bioelectronics is determined by several of their inherent well-documented properties, of which the most important is that the metal sites present in those proteins are redox centers across which electron transfer occurs along highly selective pathways; this is not the case for low-molecular-weight electron can-iers that are more uniformly (i.e., less selectively) reactive with respect to electron transfer. These pathways are considered later in the subchapter. Other considerations are as follows: those metal sites are well characterized by a number of spectral methods, so useful structural and mechanistic information can be obtained using relatively inexpensive techniques; from X-ray or NMR experiments, three-dimensional structures are known for many of these proteins which are good starting points for designing or improving their stabilities and electron-transfer characteristics; as a result of genetic engineering effort, the genes for most of these systems are available and can be expressed in large quantities in yeast or E. coli ( Eldarov el al., in preparation). the proteins i n question have well-studied biophysical, biochemical, and enzymatic properties since they have served as routine objects for many types of

78

Molecular Bioe~ectronics

investigations, particularly, cytochrome c which is commonly referred to as the 'textbook protein'; those proteins have highly asymmetric distributions of electrostatic fields around them. Some also have asymmetric distribution of hydrophilichydrophobic amino acid residues over their surfaces. Both of the two factors can be used for controlled association of those proteins into supramolecular structures like thin films suitable for macroscopic characterization and/or direct industrial applications. It should be noted that, while there are several fundamentally different approaches to biocomputer architecture (reviewed i n Nicolini, 1995), the choice of metalloproteins for the basic material for bioelectronics does not, in principle, impose any limitations on the actual architecture of the biocomputing device to be designed except that the electron is adopted as the information carrier, and hence, approaches like solution propagation are ruled out. However, there is one type of computer architecture for which the applicability of metalloproteins and their complexes has a very strong background. Surprisingly, it is the conventional computer architecture, and the background is the discovery of correlated singleelectron tunneling by (Fulton and Dolan, 1987) and subsequent effort (Avenn er d., 1991) i n which the conventional logic circuits were shown to function on the singleelectron level. In other words, the 'one electron for one bit of information' principle was implemented, with the problem of the inteiface between molecular-scale and macroscopic-scale devices also solved. Although those results were obtained on metal granules, the physical principle behind them is the same as that in the electron transfer acrossbetween metalloproteins i.e. electron tunneling. The universally accepted key factor justifying the use of a particular material in bioelectronics is the time required for triggering an electronic device based on it compared to the one based on the conventional material. Table 3.4 below shows that: the biomaterials have advantage in the spatial scale - provided the circuit design is planar, the factor is (100 nd(0.l-1 nm)*=I@ - 106; in the theoretical limit, the advantage in speed is lo3 which makes the overall advantage in performance per unit of volume of as much as 107-109 which provides a considerable margin for material designers.

79

Technologies

It should be noted, however, that the data quoted refer to the fastest known electron transfer reaction in protein systems. For the more practical cases, including the metalloprotein systems discussed below, the time is a few magnitudes more, s. But this is where artificial improvement of electron transfer typically, to properties of metalloprotein systems comes in. The most important classes of metalloproteins involved in electron transfer are listed in Table 3.5, along with examples currently under study at the Institute of Biophysics and at the EL.B.A. Foundation: l a b l e 3.4. Orders of magnitude of triggering time and for the conventional and the single-

electron devices. Triggering time Conventional (theoretical l i m i t ) Single-electron (Metalloprotein) S i ng 1e e lec t r o n (Metal granules)

-

10-9 s

Device size 100 nm

10-12 s

0.1-1 nm

10-13 s

10 nm

Table 3.5 Lisi of metallopro~cins Metalloprotein type Heme proteins Blue copper proteins Iron-sulphur proteins

Example(s)

cytochromes C. C551, B5, P45oscc hutin

Thioredoxin

The most advanced area of basic research relating to metalloprotein electron transfer is studies of electron winsfer between them in aqueous solutions. In such an experiment, the following reaction is typically studied:

where A and B can be any of the metalloproteins (typically, physiological partners are chosen), or one of them is a small electron carrier like ascorbate ion or superoxide ion radical. The varying conditions include temperature, solution pH and

80


ionic strength. Also, changes are introduced into A or B by means of chemical modifications or (a more recent and advanced technique) protein engineering. A special case is when A = B which is termed electron self exchange. The aims of such studies are: identification of the contact sites on A and B through which electron transfer proceeds - this allows to design mutations in order to optimise the interprotein contacts in the new structures including thin films; elucidating the electron transfer mechanisms and paths within A and/or B to design proteins with increased electron transfer rates in the desired direction while hampering the undesired (parasite) electron transfer; monitoring the effect of various factors on the system integrity to design proteins able to perfoiin their electron transfer function under more drastic conditions like increased temperature. The overall electron transfer process in such systems proceeding as shown by the equation (3.7) and is best described as a complex chemical reaction comprising the following stages (Marcus and Sutin, 1985):

1) collision of two protein molecules with or without subsequent formation of a complex stabilised by electrostatic or hydrophobic interactions. To distinguish between the latter two factors, dependencies of the rate of the overall electron transfer process on ionic strength are usually studied, and attempts are made to theoretically simulate such dependencies (Meyer at al., 1993; Mauk and Ma&, 1989; Cheddar et ul., 1989). In this review, this stage will be discussed later under 'Selfexchange' and 'Cross-exchange', accordingly. because the key question for solution studies is whether this stage determines the overall electron transfer rate. Besides, understanding this stage is a key to modelling protein association into structures with pre-defined properties such as thin films. 2) electron transfer itself in the fonned complex or during the collision time (in the latter case, the electron transfer time should be smaller than the average lifetime of the species called 'encounter complex' formed in the first stage). This stage is discussed mostly in 'Multiple electron transfer pathways' because it is determined mostly by those pathways.

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Multiple electron transfer pathways The classic theories describing electron transfer within proteins is found in (De Vault, 1984; Marcus and Sutin, 1985) and they relate electron transfer rates to the distance between the redox centers (in our case, metal sites), and two thermodynamic parameters - AGO, the driving force of the reaction i.e. the difference in redox midpoint potentials of the two reacting species (zero in the case of self-exchange) and As, the reorganisation energy characterizing the overall difference in coordination of nuclei between the initial and final state of the system in equation 3.8:

Apart from cytochrome P450,all the proteins under study are compact globular proteins that do not feature large reorganisation energies which is, of course, related to electron transfer of their main biological transfer. For instance, cytochrome c (Takano and Dickerson, 1981) has very small redox state-related conformation changes. A more recent study (Moser et al., 1992) largely confirms this view. In that work, analysis of intraprotein electron transfer was developed from electron-transfer measurements both in biological and in chemical systems. A variation of 20 A in the distance between donors and acceptors in protein changes the electron-transfer rate by 1012-fold i.e., basically, electron transfer rate decays exponentially with distance. Protein was shown to present a unifoim electronic barrier to electron tunnelling and a uniform nuclear characteristic frequency, properties similar to an organic glass. Selection of distance, free energy and reorganisation energy are sufficient to define rate and directional specificity of biological electron transfer. This meets the main bioelectronic requirement (see above) in protein systems. So, basically, the most important for electron transfer is the distance between the redox sites. However, the body of data on long range electron transfer in proteins (where the site-to-site distance is prohibitively large) raises the question of whether a protein structure can influence the rate or path of such transfers, and if yes, what is the mechanism. Answering these questions requires infoiination on which of the various

a2


structural elements composing proteins support long range electron transfer. In (Lee et ul., 1992), evidence is presented for long range electron transfer along the alphahelix of a synthetic leucine diiner. In more detailed theories (Beratan er ul., 1991; Beratan ei ul., 1992; Koga er ul., 1993; Liang and Newton, 1992; Liang and Newton, 1993; Onuchic et ul., 1992; Tang, 1994; Wheeler, 1990) quantum mechanical calculations are performed to generate electron transfer trajectories for electron transfer. These are, however, quite expensive computationally, but the main conclusion that can be derived from their results confirms that the rate of long-distance electron transfer in proteins rapidly decreases with distance, which is indicative of an electron tunneling process. It was also found that the distance dependence of electron transfer in native proteins is controlled by the protein's structural motif. The helix and sheet content of a protein and the tertiary amangement of these secondary structural units define the distance dependence of electronic coupling in that protein. The tunneling pathway model applied i n (Beratan er ul., 1991) was also successfully used to model intraprotein-protein electron transfer i n ruthenium-modified proteins i.e. the transfer from the newly designed ruthenium sites on the suiface of the protein to the native metal site inside the protein (Nocera er ul., 1984; Bechtold et ul., 1986; Willie el uf., 1992). The analysis ranks the average distance decay cotisrant for electronic coupling in electron transfer proteins and identifies the amino acids that are coupled to the charge localization site more strongly or weakly than average for their distance. The conclusion is that pi-electronic system play a major role is assisting electron transfer

so the aromatic side chains are more preferable for electron transfer than nonaromatic. Also, sulphur a t o m can play a major role. Below, we shall present the probable electron transfer pathways in metalloproteins under study according to the above considerations. In subsequent sections, we shall identify which of these pathways dominate the particular electron-transfer reactions of metalloproteins including self-exchange and cross-reactions.

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Cvtochrome C We are starting from this protein because it is the most extensively studied metalloprotein, suffice to say the three-dimensional structure for it was first obtained over 20 years ago (Dickerson eral., 1971). From that structure, three major pathways can be identified (Figure 3.32): via the sulphur atom of Cys 17 by which heme is covalently attached to the protein; via Phe82 residue in the immediate vicinity of the heme which is highly conserved in cytochromes C of practically all species; via Tyr74 residue which is also present in most cytochromes.

Figure 3.32 Major electron trmsfeer pathways for cytochrome c.

At this point, it is worth noting that the dominating pathway depends on the actual electron-transfer system used. For instance, to determine whether the philogenetically conserved Phe-87 of yeast iso- I-cytochrome c helps to mediate electron transfer between cytoclu-ome c and cytochrome c peroxidase, authors (Liang

84


e t a / . , 1987) constructed mutants of cytochrome c that are altered at this position and studied the kinetics of long-range electron transfer within their complexes with zincsubstituted cytochrome c peroxidase. The rate of electron transfer from reduced cytochrome c to the zinc cytochrome c peroxidase action radical is four orders of magnitude greater when phenylalanine or tyrosine is present at position 87 than when serine or glycine is present. So for the particular case of transfer to zincsubstituted peroxidase, PIie82 is the key factor. Note that, in the yeast protein, five additional amino acids are present at the N-end of the protein. Although there is no way they can contribute to the electron transfer, the numbering of amino acids is affected by the fact. While some authors prefer a horse cytochrome c - aligned numbering, others (the above-quoted paper) use the original numbering so Phe 82 will be Phe87 i n the yeast protein. Similar numbering problem occurs in cytochromes P450 (see later). In contrast to that, the electron transfer in the complex of cytochrome c with native peroxidase (not zinc-substituted) probably proceeds via Cys 17 sulphur, as follows from (Hazzard rr 01.. 1988) where the kinetics of reduction of wild type and several site-specific mutants of yeast iso- 1 cytochrome c (Arg- 13----Ile, Gln- 16----Ser, Gln16----Lys, Lys-27----GIn, Lys-72----Asp), both free and in 1: 1 complexes with yeast cytochrome c peroxidase, by free flavine semiquinones have been studied. Intramolecular one-electron transfer from the ferrous cytochromes c to the H202oxidized peroxidase at both low (8 mM) and high (275 mM) ionic strengths was also studied. The accessibility of the cytochrome c heme which determines the overall electron transfer rate for the Cys 17 pathway within the electrostatically stabilised complex at both low and high ionic strength are highly dependent on the specific amino acids present at the protein-protein interface. Replacement by uncharged amino acids of Arg or Lys residues that are important i n orientation and/or stabilization of the electron-transfer complex resulted, in increased rates of electron transfer. Increase of ionic strengths from 8 to 275 mM also produced increased intramolecular electron-transfer rate constants. The results suggest that the electrostatically stabilised 1: 1 complex has its electron-transfer pathways misaligned, and that by neutralizing, by mutation, the key positively charged residues, or by an increase in the ionic strength thereby masking the ionic interactions, the two proteins can re-orient themselves within a complex for more efficient electron

Technologies

a5

transfer. This shows that electrostatic effect in cytochrome C interactions with negatively charged proteins only favours formation of the precursor complex while may in fact be adversary for the electron transfer act itself. One other factor influencing the preferred pathway is inclusion of the protein(s) into ordered structures like charged lipid bilayers (Cheddar and Tollin, 1991). Binding strongly stimulated (up to 100-fold) the rate of reaction with the positively charged cobalt phenantliroline ion, whereas the rate of reaction with the negatively charged femicyanide ion was greatly inhibited (up to 60-fold), as compared with the same systems either at high ionic strength or at low ionic strength either in the presence of electrically neutral vesicles or in the absence of vesicles. Reactions of tuna cytochrome c with uncharged or electrically neutral oxidants (such as benzoquinone) were unaffected by binding to vesicles, suggesting no effect of membrane association on the preference of the electron transfer pathway. The same Cys 17 site, as follows from the results of mutating the nearby Argl3 in the yeast cytochrome C, is used in the reaction with its oxidase (Huang er ul., 1994). The other mutation, that of Asp90-->Cys. resulted in a marked loss of stability. The above considerations bring about a conclusion that there is experimental evidence for two of three electron transfer paths i n cytochrome C mentioned here. This is supported by a very recent theoretical study (Nakagawa el af., 1994) in which seventy-four kinds of cytochrome c sequences have been compared in order to determine the conserved residues and residues of which the types (e.g. aromatic) are conserved. Their coordinates, along with those of the redox site (heme) in the structure of cytochrome c were used for pelforming an extended Huckel molecular orbital calculations on the molecule (based on tuna cytochrome, but representative of the Cytochrome c). The examination of the shapes and the energy levels of the resulting MOs has suggested that three nearly degenerate HOMOs might play an important role in the electron transfer. These HOMOs are exposed to the protein surface around the heme, Cys-17, and Phe-82. A delocalized electron system developing in these regions is proposed to be the electron transfer pathway of cytochrome c.

86


Cvtochrome B5 In this protein, the three-dimensional structure (Mathews ef ul., 1972) the heme is more exposed than in cytochrome C which makes intraprotein electron transfer pathways less relevant (Figure 3.33). However, i n a recent work (Vergeres et al., 1993), the role of Tyr74 was investigated which is part of a hydrophobic patch on the surface of cytochrome b5, also foiming van der Waals contacts with the heme prosthetic group of the protein. In addition it is a member of an aromatic network of amino acids which includes Phe-35 and the axial ligand, His-39. The Tyr-74 residue was mutated to a lysine to investigate how it affected the interaction of heme with the protein and whether it might be an alternative binding site and an electron transfer path which cytochrome b5 utilizes for electron transfer to cytochroine P450.The results were: the elecnon transfer rate remained the same as in &hewild-type protein, but the stability of the protein reduced 10 times, causing heme to be less tightly bound. Similar results were obtained when mutating the aromatic residue of Trp in cytochrome P450cam near the heme (see below).

Heme propionates

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87

Cvtochrome C55 1 This is the smallest of the proteins considered here which has, according to the stiucture revealed by NMR (Timkovich and Cai, 1993) the most exposed heme edge which makes it certain that the electron transfer occurs exclusively via the exposed heme edge. Although attempts have been made to identify an alternative electron transfer pathway (Osvath et ul., 1988) using the ruthenium-modified proteins, the rate turned out to be slow. This suggests that there is no necessity to search for intraprotein electron transfer paths in this protein while attempts should be made to mutate its external residues to enhance its binding into various assemblies such as to favour its orientation with respect to the heme group.

Azurin Unlike cytochromes, azurin is a blue copper protein which makes i t a particularly attractive model system for bioelectronics since the copper site present in it can be readily grafted to other electron-transfer proteins (Canters and Gilardi, 1993) (compare the necessity to perform a de novo synthesis for heme proteins, above). The weak point of azurin is that its redox state change is accompanied by large conformational changes (Rogers and Moore, 1988) which makes it a rather inefficient electt.on-transfer protein. As well as for cytochrome C, its structure suggests there are three possible elecaon transfer pathways (Figure 3.34): via solvent-accessible His1 17 which is a copper ligand; via His46, another ligand, and surface His35; via axial ligand Met121 and the neighbouring PhelS. There is also experimental evidence for electron transfer via the Cys3-Cys26 disulphide bridge which is f i r from the copper site (labelled '4' on the figure) but utilizing it requires C02- radicals obtained by pulse radiolysis, and the electron transfer rate is very small. There is some reliable data supporting the first pathway as to be the most important (van de Kamp er ul., 1990, Pascher er ul., 1989). where electron-transfer reactions of site-specific mutants of the blue copper protein azurin from

88


Pseudomonas aeruginosa with its presumed physiological redox partners cytochrome c55 1 and nitrite reductase were investigated by temperature-jump and stopped-flow experiments. In the hydrophobic patch (site 1) of azurin. Met44 was replaced by Lys, and in the His35 (site 2) patch His35 was replaced by Phe, Leu and Gln. The observed changes in midpoint potentials (driving force) could be attributed to electrostatic effects. Analysis of the kinetic data demonstrated the involvement of the hydrophobic but not the His35 patch of azurin in the electron transfer reactions.

Figure 3.34 Possible elcctron iransl'er pathways in azurin.

Site-directed mutagenesis has been also used to prepare azurins i n which amino acid residues in two separate electron-transfer sites have been changed: His-35-Lys and Glu-91-Gln at site 2, and Phe-114-Ala at site 1. The results suggest that Glu-91 is not important for the interaction with cytochrorne c551 and that His-35 plays no critical role in the electron transfer to the copper site.

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Self-exchange Cvtochromes C. B5 and C551 For cytochrome C, the self exchange rate value measured at ionic strength 0.1M (Gupta et al., 1972) was only 1x103 M-Is-1 at 0.1 M ionic strength, so one should expect still lower (less than lo2 M-ls-') values without salt. Since, in this type of reactions, electrostatic effects usually hamper the electron transfer, ionic strength dependencies are measured and the rate constants for the overall process (equation (3.7)) are measured and then extrapolated to infinite ionic strength. In (Dixon et ul., 1989; Timkovich et ul., 1988), this was done for self-exchange rate constants of cytochromes C, C551, and B5. The theoretical dependence used was based on the monopole-dipole formalism (van Leeuwen, 1983). The dipole moment projection - a the key model parameter determining by which pathway the electron is proposed to follow - was set by the heme edges of all three proteins (near Cysl7 in cytochrome c in which the heme is less exposed of all). The rate constants obtained this way for cytochromes C, C551, and B5 are 5.1~105M-Is-', 2x107 M-ls-', and 3.7~105MI S - ~ , respectively. Of these. the value for C551 stands out (2 orders of magnitude greater) and suggest diffusion control. In this case, the characteristic time for electron transfer should be less than the lifetime of the short-lived protein assembly called 'encounter complex' i.e. less than 10-6 sec. For comparison, the selfexchange rate for synthetic heme-containing peptides (Dixon et ul., 1984) is 107 to 108 M-1s-1, depending on the ligation type, i.e. close to those for cytochrome C551, and, most likely, diffusion controlled. Note that the theoretical formulae of (van Leeuwen, 1983) used for fitting the ionic-strength data, were derived under the assumption that the electron transfer, not diffusion, is rate limiting. The question of whether electron transfer or diffusion is the limiting stage stays unanswered for cytochromes C and B5; but, in any case, the electron transfer act for them should be slower. Note that the theoretical formulae of (van Leeuwen, 1983) used for fitting the ionic-strength data, were derived under the assumption that the electron transfer, not diffusion, is rate limiting, so extrapolation used may not be valid for cytochrome (2551. However, the dependence is so weak (only one order over the whole ionic

90


strength range) that almost any function chosen for data fitting may produce the correct results. As to reasons for differences i n those values, the three-dimensional structures of those proteins (Figure 3.35) suggests a much larger heme exposure for C551. However, while the heme exposure is greater in cytochrome B5 than in cytochrome C. this effect may be cancelled out by presence of the two heme propionate groups at the electron transfer site, and absence of positive charges in the vicinity of heme to neutxalise thein (positive charges of one B5 molecule neutralizing propionates of the other. and vice versa). The ionic strength effects are also absent when the proteins are in immediate contact as water is, at least in patLLt, expelled from the contact area.

Figure 3.35Three-dimensional sttucture of cytochrome cS5 1.

Other evidence i n favour of diffusion control during cytochrome C551 self exchange was found i n the work at this Institute when Pro58 nearby a heme cleft was changed into Ala (Mariani et al., unpiblished data). If the electron transfer and/or accompanying confortnational changes were rate-limiting, this mutation could substantially improve the dynamic characteristics of the heme edge environment thereby enhancing the electron transfer. In the case of diffusion control, however, the rate is a function of steric factor which, i n this case. is static accessibility of the

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91

heme only, and does not depend on dynamic properties of its environment. It was shown that the mutation does not lead to any change in the electron self-exchange rate, within the experimental error. Theoretical simulation of cytochrome C55 1 self-exchange by Brownian dynamics (Nonhrup et al., 1993) gave perfect agreement with experimental data. The method focuses on the diffusion stage and is at its best for diffusion-controlledreactions, so, again, the results favour diffusion control for this case. The overall good agreement of theory with experiment in self-exchange rate ionic strength dependencies suggests that the heme edge is the electron transfer pathway for self-exchange in all globular cytochromes. However, no other sites were explored by the authors (Dixon et ul., 1989) to make this conclusion more reliable.

Azurin For azurin (Van Pouderoyen et ul., 1994), by changing Met64 into a glutamate by means of site-directed mutagenesis a negative charge was introduced into the hydrophobic electron transfer site (His1 17, Site 1, Figure 3.34). While the threedimensional structure of the protein (including the structure of the metal site) was unaffected by the mutation, the electron-self-exchangerate constant was two orders of magnitude less (105 vs. lo7 M-ls-l) at high pH although equal to that of the wildtype protein at pH 4.5. Electron transfer is inhibited only when both of the reacting azunn molecules have an ionised glutamate at position 64 in their hydrophobic patch. These results confirm that once again that the Site 1 in Fig. 3.34 is the likely entry and exit point for electrons. More evidence on Site 1 is in the study of the effects of the replacement of Met44 (also within that site) by Lys as a function of the ionization state of the introduced lysine (Van de Kamp et a/., 1993). The strong pH dependence (the effect is again around two orders of magnitude) of the electron selfexchange rate of the mutant azurin demonstrated the importance of Site 1 for selfexchange. Theoretical calculation of electronic coupling between the copper atoms in an azurin dimer put together with Sites 1 of the two proteins facing each other by using many-electronic wave functions (Mikkelsen et ul., 1993). When one of the two water molecules forming intennolecular hydrogen bonds between Sites 1 of the two

Molecular Baoelectronics

92

azurins is removed, the calculated coupling element is reduced from 2 . 5 ~ 1 0 to -~ l . l ~ l O eV - ~ which translates, according to equation (3.9), into over two orders of magnitude decrease in the electron transfer rate: (3.9) where h is the Planck constant, AG# is the free energy of activation. The meaning of As is related to the fact that. due to charge transfer, the polarization patterns i n the a t o m surrounding the electron-transfer sites (whether protein or solvent atoms) are different before transfer and after transfer. The difference of energies between the "before transfer" and "after transfer" states is called the "solvent reorganization energy" (As). If (Marcus and Sutin, 1985) (a) fluctuation is assumed to be able to bring the polarization pattern of the "before trmsfer" state to that close to the "after transfer'' state. and (b) the potential energy functions near the electron-transfer point are parabolic (harmonic approximation), the following equation is valid:

AC* = (AG" +A,)' 1 4AA

(3.10)

where AGO is the driving force, negative when electron transfer is favorable. A simple test for this theory is based on its evident prediction that, when AGO exceeds h,, it should have adverse effect on electron transfer (the so called "inverted region"). For several types of moiecular systems, such effect was observed experimentally (Closs and Miller, 1988; h-vine ef ul., 1986). All these parameters depend on the distance between the redox sites. The one with the steepest dependence, however, is the coupling matrix element present in equation (3.9):

in,

I' =IH,,, I' xexp(-PR>

(3.11)

where HABO corresponds to the van der Waals conract between redox sites. So, if the distance dependence of other variables of equation (3.9) are ignored (considering

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93

only the steepest-dependent factor is a common practice in chemical kinetics), it becomes surpiisingly simple: (3.12) where kg includes all other factors i.e. protein and solvent structure, pH and ionic strength dependencies. The results show that water molecules may play an important role as switches for biological electron transfer. Note that these water molecules are bound to the protein surface by hydrogen-bond interactions and are completely different in their physical properties from those of the bulk solvent. Notably, wild-type azurin shows no ionic strength dependence of the self-exchange rate. It also has an unusually high selfexchange activation entropy (the entropy part in AC# in equation (3.9). Both effects (Groeneveld et ul., 1988; Broo and Larsson, 1991) mean that hydrophobic interactions prevail and, accordingly, confirm Site 1 to be the site dominating azurin self-exchange.

Cross-exchange The cross-exchange bimolecular electron transfer rate constants are summarized in Table 3.6. Different values for yeast cytochrome redox potential for the same pH value are caused by different methods of measuring redox potential in (Cutler ef ul., 1987) and (Rafferty et ul,, 1990). Instead of the yeast cytochrome, the protein from horse was used in (Eltis ef ul., 1991). Although the structures of these two species of the cytochrome seem highly conserved, the charge distribution around the heme edges are similar, and the electron transfer rates from cytochrome B5 are close, the two proteins happen to be different in their electrostatic properties, namely, the yeast protein interacts with other redox partners like cytochrome B5 to foiin complexes of greater thermodynamic stability (including higher stability at higher ionic strength) than in the case of coinplexes formed by the horse cytochrome. This difference shows in the study of the electrostatic properties of the cytochrome c - cytochrome B5 complex (Mauk er ul., 1991b) where proton uptake upon complex formation is measured as a function of pH. Observations such as this have led to the conclusion

94


that hydrophobic effects contribute more to the stability of protein-protein complexes foiined by the yeast cytochrome than is the case for complexes formed by the horse cytochroine.

Cvtochrome C with cvtochrome B5 Although the physiological significance of this reaction is not known, it proceeds, at low ionic strength, with rates, at low ionic strength, up to 5x108 M-1s-I (Dickerson and Timkovich, 1975) typical of the physiological electron transfer reactions. These rates are only an order of magnitude less than the diffusion limit for identical spherical particles (7x 109 M- *s-lj and, unless the diffusiotVassociation stage is proven to be equilibrium, suggest diffusion control. For this system, however, the data quoted below clearly prove equilibrium association, i.e. existence of a stable and detectable complex. Since the structure of cytochrome C was revealed (Dickerson et a/., 1971j, attempts have been made to elucidate the role of electrostatic forces in the interaction, particularly, the role of the ring of positively charged lysine residues surrounding the heme crevice. In (Salemme, 1977). as a result of manual docking of 3D structures of the two proteins, a theoretical complex structure was proposed in which lysines 27, 13,72, and 79, of cytochrome C were participating in complementuy electrostatic interactions with acidic residues 44,46, 60, and one of the exposed hetile propionates of cytochrome B5, respectively. In (Wendoloski er u / . , 1987). this static modelling studies were extended by molecular dynamics simulations of a cytochrome c-cytochrome B5 intermolecular complex. The simulations indicate that electrostatic interactions at the molecular interface results in a flexible association complex that samples alternative relative heme positions and molecular confortnations. Many of these transient geometries appear to be more favourable for electron transfer than those formed in the initial model complex including a conformational change that occuned in phenylalanine 82 of cytochrome C that allowed the phenyl side chain to bridge the two cytochrome heme groups suggesting this pathway to be dominant in the complex analysed. In the subsequent theoretical work based, instead of docking, on more sophisticated Brownian Dynamics techniques (Guillemette et ul., 1994), while this result was confirmed, there appeared. to be one other structure of the complex (see below).

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Technologies

Table 3.6. Bimolecular electrori lrnnsfer rate constants (M-lsec-I) at 25°C.

Protein

~

-

Redox potential

4 1

C - c v w o m c BS (redox potenlial quoted for cytochrome C ) :rate constant values should be multiplied by lo7 Mutations of iiilernal iesidues Wild typea 272 18.3 14.8 Arg38His 245 Arg38Gln 242 10.8 Aq438Ala 22s 8.9 Ty67Phe 222 19.0 Ans5211e 221 19.5 Mutations of extemnl residues Wildtype 279 18.3 Lys79Ala 282 6.8 Lys72Ala 27.5 3.7 Lys72Ala/Lys79Ala 280 3 .O Witdtypeb 290 8.9 Phe82TWLeu85Ala 283 9.7 Phe82Tyr 280 9.9 18.5 Phe82 Ile 273 Phe82AIa 260 22.7 Phe82Ser 255 13.9 Leu85Cys 288 7.5 Leu85Ala 28.5 8.8 Leu8SPhe 285 9.2 Leu85Met 284 8.4 ' (native or chemically modified cytochrome C), Cvtoch-r rate constant values should be multiplied by lo3 Native (pH4.I) 279 6.6 CDNP-72 (pHs6.1) nla 41 Native (pH=8.3) n/a 4.00 CDNP-72 (pH4.3) nh 27 SV I - z ii (wildtype or mutant azurin) Wildtypea 306 I .8x 106 Phell4Ala Met44Lys His35Phe His35Leu His3SGln His3SLys Glu91Gln Phel14Ala

Ratio mutant/wild type

1.oo 0.81 0.59 0.49 1.04 I .07

I .oo 0.37 0.20 0.16

I .oo 1.09 1.11

2.08 2.55 1.56 0.84 0.99 1.03 0.94 I .oo 6.21 I .oo 6.75

"Here and furthcr below, pH=7.0: bHere and further below (for cytochrome C - cytochrome 85). pH=6.0.

96


I n (Stonehurner et ul., l979), the reduction of cytochrome C by cytochrome B5 was studied over a wide range of ionic strengths in four different buffer systems. The reaction rate was found to decreased linearly as the 11/2 was increased, suggesting that electrostatic interactions are important in the interaction. The ionic strength dependence of the reaction rate was in quantitative agreement with the theory (Wherland and Gray, 1976) only if the effective radius of the interaction was 2 8, which, in terms of that theory, means that the interaction between the two proteins is best described as the sum of n complementary charge interactions, each involving a specific lysine on cytochrome C and a specific carboxyl group on cytochrome B5. The number of complementruy charge interactions was calculated to be five to seven. The same result was obtained when the electron transfer rates were measured for several chemically modified cytochrome C species in each of which the charge of a specific lysine was neutralised 01'reversed (Koppenol and Margoliash, 1982, and refs. therein). I n (Stonehuerner et ul., 1979), existence of a stable complex of the two proteins at low ionic strength was proven by ultra centrifugation and gel pelmeation techniques. The interaction between cytochrome C and cytochrome B5 was also studied by 1H-NMR spectroscopy (Eley and Moore, 1985). At pH 6.3 (27OC, I = 0.04), the two fet-ricytochroines were found to form a 1:1 complex with an association constant of approx. 103 M-1. Note that the intra-complex electron transfer characteristic time will be around 10-5 sec (compare with direct measurements results, below). The protein-protein-interaction region was found to encompass the region of the surface of horse cytochrome C that includes Ile-81, Phe-82, Ala-83 and Ile-85, and Lys-13 and Lys-72 of horse cytochrome C were suggested to be involved in two important intermolecular interactions. These results suggested involvement of Phe82 in the electron transfer pathway. However, when the complex formed in solution by chemically modified (as well as native) cytochrome C with cytochrome B5 was later studied by I3C (as well as IH) NMR (Burch ef ul., 1990). lH-NMR spectroscopy showed that there is no major movement of cytochrome C residue Phe82 on binding to cytochrome B5. The greater resolution provided by 13C-NMR spectroscopy allowed detection of small perturbations in the environments of cytochrome C residues Ile75 and lleX5 on binding with cytochrome B5. All residues mentioned are in the vicinity of the heme edge. As individual cytochrome C lysyl residues are

Technologies

97

resolved in the H-NMR spectrum of N-acetimidylated cytochrome C , the interaction of thus modified protein with cytochrome B5 has been studied to evaluate the number of cytochrome C lysyl residues involved in binding to cytochrome B5, and that was found to be at least six coinpared to four expected from the theoretical model of the complex. It was concluded that cytochrome C and cytochrome B5 foim two or more structurally similar 1 : 1 complexes in solution. Stability of the cytochrome c-cytochrome B5 complex as a function of solvent stress was analyzed in (Kornblatt er ul., 1993). High concentrations of glycerol were used to displace the two equilibrium. Glycerol was found to promote complex formation between cytochrome C and cytochrome B5. These results mean that the association of this complex is largely entropy driven, most likely, because the complex expels water in the process of foimation. One of the important features of the cytochrome B5 structure is the presence of two exposed heme propionates near the electron transfer site. To investigate their role of the cytochrome B5 heme propionate groups i n the interaction of the two native proteins, the stability of the complex formed between cytochrome C and dimethyl ester heme substituted cytochrome B5 (DME-cytochrome B5) has been determined under a variety of experimental conditions (Mauk et 01.. 1986). Interaction between cytochrome C and the modified cytochrome B5 was found to produce a difference spectrum in the visible range that is very similar to that generated by the interaction of the native proteins and that can be used to monitor complex formation between the two proteins. At pH 8, 25°C. 1=5 mM, DMEcytochrome B5 and cytochrome C form il 1:1 complex with an association constant KA of 3x106 M-1. This pH is the optimal pH for complex formation between these two proteins and is significantly higher than that observed for the interaction between the two native proteins. The stability of the complex formed between DMEcytochrome B5 and cytochrome C was strongly dependent on ionic strength with K A ranging from 2 . 4 ~ 1 0 7M-1 at I=1 mM to 8 . 2 ~ 1 0M-l ~ at 1=13 mM (other conditions were the same). Calculations for the native cytochrome B5 and cytochroine C confirm that the theoretical intermolecular complex described in (Salemme, 1977) describes the protein-protein orientation that is electrostatically favored at neutral pH.

98

Molecular Bioeleclronics

For direct measurement of intracomplex electron transfer froin cytochrome B5 to cytochrome C (Willie rf ol., 1992), a de novo design and synthesis of rutheniumlabelled cytochrome B5 were described. A single cysteine was substituted for Thr-65

of rat liver cytochrome B5. The single sulphydryl group on Thr65Cys cytochrome B5 was then labelled with (4-(broinoniethyl)-4'-methylbipyridine)(bisbipyridine)rutheniurn(I1) to form Ru-65-cyt B5. The ruthenium group at Cys-65 is only 12 A from the heme group of cytochrome B5 but is not located at the binding site for cytochrome C, so it only served for photoinduced electron injection into the complex, Laser excitation of the complex between Ru-65-cyt B5 and cytochrome C resulted i n electron transfer from the excited state Ru(II*) to the heme group of Ru65-cyt B5 with a rate constant greater than 106 s-1. Subsequent electron transfer from the heme group of Ru-65-cyt B5 to the heme group of cytochrome C is biphasic, with a fast-phase rate constant of 4x105 s-1 and a slow-phase rate constant of 3 x 104 s-1. This means that two different electron-transfer pathways in the complex. The reaction becomes monophasic and the rate constant decreases as the ionic strength is increased suggesting that. when there is no complex, only one of

Technologies

99

the pathways works. I t seems reasonable to consider them to be the exposed heme edgeKysl7 and the Phe82 pathways, respectively (Fig.3.36). Another approach to direct measurement of intracomplex electron-transfer is the study of the reaction (Qin and Kostic, 1994) 3

Zn -cytC/cytBS(Fe”)----;,Zn

-cytC/cytBS(Fe”)

within both self-assembling and covalently-linked complexes of zinc(I1) cytochrome C and ferricytochrome BS using laser flash photolysis (3Zn is the photoinduced triplet state of the zitic(1I)porphyriti). For the self-assembling complex, the rate constant was 3 . 5 ~ 1 0 ss-I i n aqueous solution, and the reaction kinetics is monoexponential (i.e. suggests the single electron transfer pathway). It is also independent of protein concentration and ionic strength which means the

reaction does not involve the association phase. However, the rate decreases when viscosity is raised by addition of glycerol or sucrose which means the slowest phase is not the electron transfer act itself but some conformation within the complex. In contrast, in the covalent complex, the kinetics is multiexponential suggesting at least two electron transfer pathways; from that data, a rate constant of 3 . 0 ~ 1 0 ss-1 was derived for rearrangement of the complex 3ZncytC/cytBS(Fe3+) from the initial docking configuration to a different, more reactive, configuration. One inexpensive, rapid and quantitative method for the study of ionic interactions between proteins is infrared spectroscopy utilizing the presence of the bands due to the amino acid side chains, between 1600 and 1500 cin-l, a fact commonly ignored (Holloway and Mantsch, 1989). When cytochrome B5 is mixed with cytochrome C under conditions that favour ionic complex formation, changes are seen in a band at 1562 cm-1 which is due to the side-chain carboxyl of Glu residues, rather than those of Asp residues that show a band at 1585 cm-1, and the changes in the band at IS62 ctn-1 indicate that when the two proteins interact. three ionised cwboxyl groups of

Glu become involved in salt bridge formation. This result was in good agreement with the above modelling studies. In (Northrup et a / . , 1993), reduction of wild-type and several mutants of yeast ferricytochrome C and several mutants by ferrocytochrome B5 has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of

100

Molecular Baoelectronacs

electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical obseivations of the electron-transfer rates of cytochrome C mutants. In yeast cytochrome C, as different from the horse protein, Lys72 is trimethylated, and Lysl3 is replaced by arginine. The Brownian dynamics (BD) method simulating diffusional docking was employed to predict the mutation effect on the rate constants. The electron-transfer act was assumed to happen at the Brownian trajectory points, with its probability vruying exponentially with the heme-to-heme edge distance, according to equation (3.12). The BD method was able to quantitatively predict rate constants over a considerable range of ionic strengths with the model parameter b=1.0, i n contrast to b=1.2 in (Northrup et ul., 1991) for cytochroine CSS1 self-exchange (the latter value is also accepted in (Marcus and Sutin, 1985). The theoretical rate constants for the mutant proteins were following the order observed experiinentally in the succession of rate constants: wild type > Lys79Ala > Lys72Ala > Lys72AlaLys79Ala. However, the mutation effect on the rate constants was underestimated by about 40%. The BD trajectories predict that the electrostatic steeling leads the two proteins to contact through essentially the same parts of their surfaces, the shortest distance between their haemes being around 12 A. Two predominant types of complexes were predicted, the most frequent involving the following interactions: Argl3, Lys87, Lys86, and Lys72 of cytochrome C with Glu48, GluS6, Asp60, and heme propionate of cytochrome B5, with the average electrostatic energy of -13.0 kcal mol-l. Another type of complex coincides with the one originally proposed by Salemme (see above), with the energy of -6.4 kcal mol-I. As Brownian dynamics does not allow for short-range interactions probably governing the conformation of the proteins during their immediate contact, energy minimisation of these structures in a suitable force field is definitely required. That was performed in (Guillemette et ul., 1994). One other important result is that the shape of ionic strength dependence (regardless of the value of the parameter b) of the bimolecular reaction rate was well reproduced using a dielectric model allowing for different pennittivities of the protein and solvent, but not for the a uniform dielectric model, i.e. ignoring this difference. That result is highly instructive considering the more or less spherical shape of both proteins and their electrostatic potentials in their contact areas.

101

Technologies

A particular feature of yeast iso-1-cytochrome C is the presence of an internal water molecule which is a part of a redox-state-dependent hydrogen bond network. An investigation was performed to find out whether this network affects the electron

transfer pathway(s) for the reaction with cytochrotne B5 (Whitford er ul., 1991). By site-directed mutagenesis of As1152 of cytochrome C, this water molecule was shifted with respect to the polypeptide fold or even removed altogether. Using saturation transfer 1H-NMR methods, the reverse electron transfer rate was measured within a complex fonned between either wild-type or mutant yeast iso-lcytochromes C and cytochrome B5 (Table 3.7). Because the constants measured are reverse i.e. from cytochrome C to cytochrome B5, they are very small, and the wildtype value is the smallest. The constant for Phe82Ala mutant was also measured (this is not involved in the hydrogen-bond network but may be involved in the electrontransfer pathway). Table 3.7 Reverse electroil traitsfer ralc coi~siantswithin a complex of cytochrome C and cytochromc 95 (variants of cylochroine C).

Variant

Kedux potential,

m V Rate constant, s-l

wildtype

210

0.3

Asn52-Ala

240

0.6

Asn52-lle

220

1.0

Phe82-Cly

220

0.7

Analysis of the observed cross exchange rates using Marcus theory (see equations

(3.9-3.12))shows that these changes for Asn52 mutants (but not the Phe82 mutant) can be predicted quantitatively by the shift in redox potential that accompanies mutagenesis. So, although the perturbation of the internal water molecule by mutagenesis alters both the structure and redox potential of cytochrome C (reflected in the parameters AGO and hs of the theory), it does not significantly influence the intrinsic electron transfer reactivity of the protein (parameter H A B ) . The latter, however, is affected by the Phe82 mutation suggesting its participation in the electron transfer pathway (Sivozhelezov et ul., in preparation).

102


Cvtochromes C55 1 and C with azurin Cytochrome C55 1 and azurin are known to be physiological electron-transfer partners (Golberg and Pecht, 1976; Rosen et ul., 1981). For this pair the observed rate constant is 1 . 8 ~ 1 0M-Is-'. ~ For the reaction of cytochrome C with azurin (Augustin rr ul., 1983). the rate constant is over two orders of magnitude smaller than that observed for the interaction between azurin and cytochrome C551, and over three orders smaller than that between cytochrotnes C and B5 (see above). In the latter work, rate constants were detennined for the reactions of horse cytochrome C, eight 4-carboxy-2,6-dinitrophenyl(CDNP-) cytochr6mes C singly modified at lysines 7. 13, 25, 27, 60, 72, 86, or 87 with azurin. These modifications reversed the charge of the lysyl group thus diminishing the overall positive charge of the cytochrome by 2. Azurin has an overall negative charge of only about -1 to -2, and exhibits bimolecular rate constants with native ferrocytochrome C of 6.6~103M-ls-l at pH 6.1 and 4 . 0 ~ 1 0M-ls-l ~ at pH 8.6, which increase upon modification of the cytochrome C to a maximum of 4 . 1 ~ 1 0M-1s-l ~ at pH 6.1 and 2.7~104M-1s-l at pH 8.6, for the CDNP-cytochrotne C modified at lysine 72. These results showed that: in the reaction with cytochrome C, azurin behaves as a positively charged

.

reactant, the electrostatics governing to a large extent the relative reactivities of the modified cytachromes c; the interaction domain on cytochrome C is located on the 'front' surface of the protein and encompasses the solvent accessible edge of the heme prosthetic group i.e. the Cysl7 site (Figure 3.36) is the most likely pathway; and the bimolecular rate constants for azurin are orders of magnitude slower and the effects of lysine modifications far smaller than far the reactions with cytochrome

B5, which means that: (a) the electric fields around the reactants do not align them, prior to electron transfer, as effectively as for the reaction with 8 5 ; and (b) there is an absence of a precise molecular fit between cytochrotne C and azurin.

Technologies

103

Electron transfer equilibrium and kinetics between azurin from Alcaligenes faecalis (as compared to those of the Pseudoinonas azurin) and cytochrome C551 have been studied (Rosen L>r 01.. 19Xl). The equilibrium constant

K=

[Cyt(Fe3’)1 x IAz(Cu’)l [Cyt(Fe”)l x I Az(Cu”)l

(3.13)

is 0.5 at 25 O C , compared to 3.5 for the Pseudomonas azurin, i.e. the redox potentials of the two proteins are close, and the electron can proceed in both directions. Effects of site-directed mutagenesis on the electron transfer rate in the system (van de Kamp et ul., 1990, Pascher cf ul., 1989) show that Site 1 (His 117) is involved in the reaction with cytochrome C5S 1.

The P45Oscc Cvtochrome: A Uniaue Svstem for Bioelectronics

104


There is no data on self-exchange in cytochroines P450. This is related to the fact that is, unlike other cytochromes, a complex enzymatic system in which electron transfer must proceed in concert with other functions like substrate binding and oxygen activation, and is performed with the help of cofactors - the iron-sulphur protein, reductase, and, i n some cases, cytochrome B5 (Porter and Coon, 1991). Also, it follows from its 3D structure (Figure 3.37) that there is no way of bringing its possible inner electron transfer pathways close to those of another P450 molecule unless its conformation dramatically changes. Accordingly, the estimated selfexchange rate for cytochrome P450 is zero. The structure of this protein has not yet been revealed. so sequence homology modelling had to be applied to provide a model for this structure. Such a model has been developed at this Institute, but here we use, for easier reference, the model present i n the Protein Data Bank (Vijayakumx and Salerno, 1992) while we have Xray crystallography studies under way to distinguish between the two models. Accordingly, here we use the residue numbering scheme adopted in the above reference which is based on the amino acid sequence of the mature form of the protein, unlike the one used in mutagenesis studies which differs by 38 residues. Since this protein is an enzyme, its successful use in bioelectronics (involving, for instance. electron transfer enhancement by mutagenesis), demands detailed understanding how electron transfer contributes to its natural function. For this reason, we shall briefly describe here the key aspects of the enzymatic reaction catalysed by it. The key feature of cytochrome P450-catalysed reactions making it eligible as a bioelectronic inaterial is that those reactions begin with the transfer of electrons from NAD(P)H to either NADPH-cytochrome P-450 reductase in the microsomal system (P450d) or a fenedoxin reductase and a non-haern iron protein (in the case of P450scc. it is called 'adrenodoxin' i.e. adrenal ferredoxin) in the mitochondria1 (P450scc) and bacterial systems (p450cam), and then to the cytochrome P450; this leads to the activation of molecular oxygen followed by the insertion of one oxygen atom into the substrate (camphor for P450cam. cholesterol for P45Oscc). The action of cytochroines P450 comprises the following stages (White and Coon, 1980):

Technoloaies

105

1) Substrate binding which perturbs the spin equilibrium of the iron of cytochrome heme and facilitates the transfer of the fiist electron. 2) Oxygen binding to the reduced P450 at the sixth position of the iron; if, instead of oxygen, CO is bound to the heme iron, the chuacteristic absorbance band appears at around 450 nm. 3) Transfer of the second electron through P450. Note that, in liver microsomal P450s, this electron, can be obtained from cytochrome B5 (Pompon and Coon, 1984). 4) Splitting of the oxygen-oxygen bond for which two protons are required, followed by dissociation of the product from the cytochrome. This stage is the less studied, but, as with the final stage of electron transfer in smaller metalloproteins, it can be disregarded for applications in bioelectronics. while the first three stages can be utilized.

Although the reported electron transfer characteristic time observed spectrophotometrically for interaction with adrenodoxin is only around sec (Tsubaki et ul,, 1989). its use as a material for bioelectronics is justified by its exceptional ability to form films (Nicolini er ul., 1995b; Erokhin el ul., in preparation), as well as a variety of factors that can be monitored, controlled and engineered that govern its functions (including that of electron transfer), the most important being substrate or cofactor binding and related changes in spectral characteristics (Guengerich, 1991). Cytochrome P45Oscc, like other P-450s involved in steroid transformations, is very selective for its substrate which makes it very promising for biosensor applications (Gilles-Gonzalez et ul.. 1994). On the level of the whole cells, the idea was put forward (D. Pompon, private communication) to use recombinant cells caifying specific receptors to capture and amplify the primary chemical signals to finally control specific gene expression while other possibilities include engineered P450 serving as signal translator provided that a tight control of the two types of electron transfers involved in the catalytic cycle could be controlled independently. In particular, some external signal e.g. a magnetic field could be captured and converted to control a number of important biological functions. The main feature of the structure is the residue Cys357 of P450cam (Cys423 in P45Oscc) which contains the fifth heme ligand (a thiolate anion) determining the

106


spectral and functional characteristics of cytochrome P-450 including electron transfer (Poulos et a/., 1985). The peptide containing this invariant residue is the single most highly conserved P-450 segment and can be readily recognised i n P450s from all organisms, including P45Oscc (Black and Coon, 1987; Gonzales, 1988; Kalb and Loper, 1988). Possible electron transfer pathways of the protein (belowj should be near this site. The sixth coordination position (on the opposite side of the heme) is filled by an exchangeable water molecule easily replaced by oxygen or CO when the iron is reduced. Previous crystallographic and site-directed inutagenesis studies (Porter and Coon, 1991, and refs. therein) have identified residues critical to both substrate and oxygen binding at the active site of P450carn. The substrate-binding pocket of the camphor protein is lined with hydrophobic residues and is buried within the protein; access is gained via a small. dynamic solvent channel leading to the distal face of the heme. The substrate is bound by van der Waals contacts and a single sterically important hydrogen bond with TyiV6 in P450cam (the closest aromatic residue, by the sequence, is Trp109 in P450scc, proposed as one of the electron transfer pathways). It is also i n close contact to Leu244 and Val247 which are a part of the largely hydrophobic helix in the 240-260 residue number region (in P450, that helix is in the region 280-300). The oxygen-binding pocket is centered on Thr-252 (P45Oscc analogue: Thr291, mutation to Ile291 available and under study at this Institute (Eldarov o f al., 1995). When this threonine, in camphor cytochrome P450, is replaced with Ala or Val, cytochroine P450 starts to produce H202 instead of monooxygenating the substrate, which is called 'uncoupling' (lmai and Shimada.

1989). Should the mutations perfonned at this Institute confirm the 280-300 region to be the substrate-binding domain of P45Oscc, the same consideration should apply to this protein. If, however, the threonine in question is replaced by bulky isoleucine of phenylalanine residues, it may cause steric hindrance to the oxygen or CO binding (Figure 3.38). This inay also happen upon introduction of bulky residues into the neighbouring Asn290 position. We have found this to actually happen in the Asn290Ile inutant in which the 450 iim band is absent, in contrast to the recombinant protein.

Technologies

107

F i l u r c 3.38 Ellcci of niulalions Thr/lle 291 on cytochroine P450scc.

The substrates of P450s are strongly hydrophobic (cholesterol is one example). There is no evidence for charge interactions take part in substrate binding to the cytochrome (Porter and Coon, 1991, and refs. therein). Accordingly, the subswatebinding helix in cytochrome P450scc may be identified as the highly hydrophobic helix between the residues 280 to 300 (this also follows from an analogy with cytochrome P450cain in the above paragraph). Mutants were obtained in this Institute in that region (Ala286, Gln290, and Thr291 to Ile or Phe, and are currently under study to test this assumption which is vitally important for subsequent applications because, if the model structure is confirmed, it will provide a basis for subsequent predictions of mutations to improve electron transfer and film formation properties of cytochroine P4SOscc. The rationale for performing these mutations is therefore based, most importantly, on the necessity to confirm the model structure for successful strategy planning for subsequent mutagenesis studies. Other reasons for choosing this site for mutations are dependent on this confinnation: if the helix is indeed close to heme, introduction of hydrophobic residues (both Phe or Ile) should increase the protein redox potential without disturbing any of the electron transfer pathways (in the model used, they are

M o l e c d a r Baoelectronics

108

at the opposite side of the heme - compare Figures 3.39 and 3.38). This should lead to enhancing electron transfer. As the site is located within the hydrophobic core and near the heme, those mutations should also increase the thermostability of the protein. That, however, has already been achieved by incorporating the P45Oscc into thin films.

As of now, while the work on attachment and subsequent localization of the heme

by sequence analysis (Pikuleva et ul., 1992) is i n agreement with the structure, studies with a 'suicide' substrate (Tsujita and Ichikawa, 1993) indicate that the substrate-binding region is at the N terminus which is, in itself, unlikely, because this region is known to be responsible primarily for anchoring to the membrane (Bernhardt et ul., 1994). Besides, our preliminary results indicate that the model structure used is, at least i n tenns of protein fold, correct (see above). Three possible electron tl'ansfer pathways exist i n the structure of P450scc: viaa the Trpl09 mentioned; via the solvent-exposed Tyr418; and via Phe416 which is closest to the heme.

Technologies

109

For cytochrome P450 main redox partner, adrenodoxin, although its structure is also yet to be revealed. the pathway has been already identified as His56 (Miura ef al., 1991). The first pathway, coininonly related to the so-called 'covalent switching'

hypothesis (Baldwin er al., 1991), was dismissed in recent studies (Beckert et af., 1994; Munro et al., 1994); at least for liver and bacterial proteins, where it was substituted by phenylalanine. leucine, and seiine, with no effect on its function. The most promising of the remaining two is Phe410 which is well within the adrenodoxin binding site previously identified by covalent cross-linking of P450scc complex with adrenodoxin (Chashchin et af., 1989) (see Fig. 3.40). Although it is not exposed in the structure itself, it may be accessible in the complex with adrenodoxin provided the nearby Arg412 is involved in a charge interaction. This makes Phe416 a plausible target for further mutagenesis studies.

Figure 3.40 Adrenodoxin binding site in the structure of cytochrome P450scc.

4 BIOELECTRONIC

MATERIALS Semiconductor technology Iias changed our lives within only a few decades. Sand, the starting material, has been turned into a versatile 'high-tech' product. Techniques have been developed for the production of silicon wafers and for the modification of this raw material into the final 'high-tech' product - miniature electronic logic functions. Research now focuses on the molecular level, from the micro to the nanostructures. The rapid increase i n our knowledge of the function of biological materials has also directed interest into this area. The functionality, efficiency, and flexibility of bioinaterials is impressive; they are beyond the capabilities of synthetic chemistry. The development of gene-technological methods has opened the way to the controlled modifications of proteins, which appear already in position to allow the production of newly designed biomaterials. A first example was the modification of the retinal protein bacteriorhodopsin and the generation of a versatile media for optical processing (Hainpp and Zeisel, 1992). This example will be used to describe the principle of the approach and to give an idea of possible future developments. Gene technology as the key technique i n this new direction of materials research may be the counterpart to photolithography i n semiconductor technology. The design of complex inolecular functions is a goal extremely difficult to achieve with the classical approach of chemistry and supramolecular chemistry, whereas nature developed a large variety of such 'nanornaterials'. As shown earlier, today it is possible to begin the construction of such complex materials ab initio, by starting out from the molecules that we discover in nature and try to modify their properties and adapt them towards the deinands of technical applications. In the last few years, tools have become available that are necessary to put these ideas into practice for both metalloproteins and bacteriorliodopsin. Modification of the genetic code facilitates manipulations of organisms that can produce these new 'high-tech' materials with conventional biotechnological methods. Therefore, the piice of such products will be affordable. A n intensive screening for functional biopolyiners with technically 111

112

Molecular Bioelect~onics

relevant functions and their variation and identification will create a new class of 'biomimetic' materials. The experimental studies done with bacteriorhodopsin have shown that this new approach leads to competitive materials in selected areas of optoelectronics and optical filtering (see later); a similar route is presently being pursued with metalloproteins summarized in the previous chapter. Indeed the production of conductive LB films based on metalloproteins having multiple pathways for the electron transfer will likely find polutionary applications in the field of electronics, leading to a 'bona-fide' protein-based cell automata unmatchable by traditional inorganic semiconductors that are unidirectional in conductivity (see later). However, the materials emerging from the spontaneous or LB-induced assembly of proteins, lipids and organic compounds, have interesting properties with profound implications in many fields, giving birth to a new biopolymer-based nanotechnology which goes far beyond electronics (Nicolini, 1995a. b).

Heat-Proof and Long Range Storage Thin Protein Films One common feature of all the protein films is high thermal stability of the protein structure, both secondary and higher order, and function as shown in Table 4.1; practically all the proteins used (RC, P450, antibodies etc.) demonstrated the preservation of the secondary structure till 200 "C when organized into the LB film. The understanding and the attainment of protein thermal stability is a very important goal both from basic and applied point of view as it requires a deep knowledge of all the paixmeters involved in the structural stabilization and since it is of fundameiital importance i n all those cases where proteins are used as functional elements for biodevices and bioreactors. Indeed, in recent time, proteins, mainly those involved in the electron transport, in molecular recognition and in catalytic processes, have undergone increasing utilisation i n many technological applications, from biosensors and biotransistors to biocatdlysis. However, several of these applications require operating temperatures higher than 100 O C , a condition under which most of the proteins are known to denature, including those isolated from extreme thermophiles.

113

Bioelectronic Materials

Table 4.1. Functional and slruciural melting temperatures ("C) of proteins i n solution and in LB films

PROTEIN

Solutionsecondary

CST

60 C

A P ThioredoxinP4SOscc

6OC

G0"C

-60°C

I90"C

>loo" C

and higher order

IgC Rhodopsin Reaction Centres 60°C 75°C 55°C

structure LB secondan,

structure LB tertiaryquaternary struciuix Solution functional

activity LB functional activity Solution

dehydration LB dehydration

IS0"C

200°C

200°C

-200°C 110°C

60°C

GO C

>150°

1SOC

19OC

-75°C

-60°C

-200°C

110°C

C 50°C 100°C

The usual approach to this problem is the protein engineering which, in addition to its inherent limitations despite overemphasized claims. requires enormous efforts and needs a specific approach for each individual protein under study. Therefore, it was very amazing when Nicolini ef al. in March 1993, by foiming higher packed 2Dordered protein films by Langmuir-Blodgett (LB) technique, were able to find out a general procedure for stabilising up to 200 "C protein structure which instead was lost at 55 "C in solution and at 150°C in dried 'smears' (a kind of self-assembled films). The procedure turned out to be of general validity and was proved to be effective on both water-soluble (antibodies, cytochromes) and membrane proteins (Reaction Centres from Rhodobocrer sphaeroides and bovine rhodopsins), stabilising not only the structure but also to a certain extent the function. Comfortingly enough, these findings were confirmed by X-ray study with synchrotron radiation only 5 months later by Shen et d.for dry self-assembled films of bacteriorhodopsin and recently for dried photosynthetic membrane film by Miyake et (11. (1994). It is worth noting that thermal behaviour of dry self-assembled smears of membrane proteins such as Photosynthetic Reaction Centres (RC) and water soluble ones as, e.g. antibodies, display a behaviour which is intermediate between that of

solution and of LB film. (Dubrovsky e l ul., 1994a, b, 1995; Facci et ul., 1994a; Erokhin et ul., 1994, I995a). The first question we addressed was about the generality of the preservation of the protein secondary structure in LB films under thermal treatment previously reported for membrane-proteins. Figure 4.1 depicts circular dichroism spectra of antibody LB film before and after thermal treatment, while Table 4.1 summarises siinilar findings on other water- soluble and membrane-proteins. Up to now heat-proof protein structure has been firmly established for LB films of Photosynthetic Reaction Centers (Nicolini ul., 1993; Facci et ul., 1993), glutathione-S- sfer erase (Antolini et ul., 1995a), alkaline phosphatase (Petrigliano er d., IYYS), bacterial (Erokhin et ul., 1996) and bovine (Maxia ef uI., 1995) rhodopsins, urease (Pdddeu

tJf a / . ,1995),

thioredoxins (de Chiara of ul., i n preparation), cytoclirome P45Oscc (Erokhin et al., in preparation), cytochrome C5S 1 and azurin metalloproteins. The circular dichroism data confirm that also in all above cases, protein secondary structure is not affected by temperature.

0

Figuw 4.1 CD spcciru OC 1gC LB Cilin (20 monolayer) hcfoore (dotted line) and aCter (solid line) thcrmal IrcalinciiI ai ISO" C for 10 m l .

Structure and Function The second issue was about the preservation of functional properties of proteins in LB film after heating. Figure 4.2 shows that the functional activity for RC in LB film

115

Bioelectronic Materials

is retained up to a temperature about 40 "C higher than in the case of dry smears. In case of RC in solution the functional tlieiinal stability is drastically lower. Even if the disactivation temperature does not match the thermal denaturation threshold, it represents a strong improvement of tlie protein thermal stability. 0.035 T

283

303

323

343

363

383

403

423

Temperature [K] F i g i i w 1.2 Dcxrivatiori c~ticol' photosyiiiliclic tcacIioii cciiiIc IIom Rhodobacrer. sphacroides as 3 function of tempcraiwe assayed by oplical ahsorbaiice for dried sinears (dashed line) and by surface polential ineasureineiiis for LB moiiolaycr (solid line).

Similarly the functional activity appears strikingly preserved up to 150-200 "C in LB films of a wide variety of membrane and water-soluble proteins, as shown in Table 4.1 and i n Figure 4.2 (for IgG antibodies). Only in RC protein films the lightinduced charge separation appears preserved at a lower temperature. namely I10 "C (Erokhin et ut., 199Sa), as shown earlier. Figure 4.2 represents the functional activity of antibody LB films. In this case, due to tlie structure of these molecules, the functional activity was preserved at least after heating up to 150 "C and above. The aim of this section is to draw general conclusions about the thermal stabilisation i n both structural and functional term of different proteins organised in regular 2D structures by LB technique (Erokhin et ul., 1995a; Nicolini et ul., 1993). Considering that most of the proteins utilized are water-soluble and all stem from mesophilic organisms, this is an astonishing result, quite generally applied and with far reaching implications. The temperature dependencies were always peifoimed by

116


heating the samples at a fixed temperature i n a commercial oven and performing measurements at room temperature after the samples have gained thermal equilibrium. The ainazing increase of the imrnunological activity of the antibody LB film. evident from the value of the saturation level and reaction rate (Figure 4.3), was attributed to the improvement of the film order. The improvement of the film order turned out to be a general property and was demonstrated both for RC and antibody LB films. Figure 4.4 shows STM measurements of a monolayer of RC after heating at 150 "C, which appears more ordered than at room temperature. Furthermore the whole sequence of structural alterations induced in RC protein LB film with increasing temperature points to a progressive loss of functional activity, quaternary-tertiary structure, and, only at quite higher temperature, of secondary structure (as suininarised in Table 4.1).

I

130 11°

TT

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:

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