Molecular Biology Biochemistry and Biophysics 24 Editors: A. Kleinzeller, Philadelphia· G. F. Springer, Evanston H. G. Wittmann, Berlin Advisory Editors: C. R. Cantor, New York· F. Cramer, Gottingen . F. Egami, Tokyo M. Eigen, Gottingen . F. Gros, Paris· H. Gutfreund, Bristol B. Hess, Dortmund· H. Jahrmiirker, Munich· R. W Jeanloz, Boston E. Katzir, Rehovot . B. Keil, Gif-sur-Yvette· M. Klingenberg, Munich I. M. Klotz, Evanston· F. Lynen, Martinsried/Munich W T. J. Morgan, London· K. Muhlethaler, Zurich· S. Ochoa, New York G. Palmer, Houston· I. Pecht, Rehovot . R. R. Porter, Oxford W Reichardt, Tiibingen . H. Tuppy, Vienna J. Waldenstrom, Malmo Chemical Relaxation in Molecular Biology Edited by I. Pecht and R. Rigler With Contributions by M. Ehrenberg· E. Grell· D. N. Hague G. Ilgenfritz. T. M. Jovin . D. Lancet· D. Magde I. Oberbaumer .1. Pecht· F. M. Pohl . D. Porschke R. Rigler· P. Schuster· G. Striker· D. Thusius K. Tortschanoff· W. Wintermeyer . P. Wolschann With 141 Figures Springer-Verlag Berlin· Heidelberg· New York 1977 Dr. ISRAEL PECHT The Weizmann Institute of Science Department of Chemical Immunology Rehovot/Israel Dr. RUDOLF RIGLER Department of Medical Biophysics . Karolinska Institutet S-10401 Stockholm 60 ISBN-13:978-3-642-81119-7 e-ISBN-13:978-3-642-81117-3 DOl: 10.1007/978-3-642-81117-3 Library of Congress Cataloging in Publication Data. Main entry under title: Chemical relaxation in molecular biology. (Molecular biology. biochemistry, and biophysics; 24) Includes bibliographical references. 1. Chemical reaction, Rate of-Addresses, essays, lectures. 2. Molecular biology-Addresses, essays, lectures. I. Pecht, I., 1937- . II. Rigler, Rudolf. III. Series. QD502.C47. 574.lf8. 77-7167 This work is subject to copyright. All rights are reserved, whether the whole oDpart of the material is concerned, specifically those of translation, reprinting, re-use of illustrations, broadcasting, reproduction by photocopying machine or similar means, and storage in data banks. Under § 54 of the German Copyright Law, where copies are made for other than private use, a fee is payable to the publisher, the amount of the fee to be determined by agreement with the publisher. © by Springer-Verlag Berlin· Heidelberg 1977. Softcover reprint of the hardcover 1st edition 1977 The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regnlations and therefore free for general use. 2131/3130-543210 Dedicated to Manfred Eigen Preface The development of an area of scientific research is a dynamic process with its own kinetic equations and its own physical mech anism. The study of fast chemical interactions and transformations is such an area, and while it is tempting to draw analogies or to speculate about the simplest model system, the lack of ade quately averaged observables is an annoying obstacle to such an undertaking. Sciences suffering from such conditions usually avoid quantitative models, be they primitive or complex. Instead, they prove their point by "case histories". Chemical relaxation kinetics started as an offspring of research in acoustics. In some aqueous ionic solutions anomalous acoustic absorption had been observed. A systematic study traced the cause of this absorption, showing that the covered frequency range and the intensity of the absorption were related in a predictable manner to the rate at which ions can interact and form structures differing in volume from the non interacting species. The step from this experimental observation and its correct, non trivial explanation to the discovery that all fast chemical pro cesses must reveal themselves quantitatively in the relaxation rate of a perturbed equilibrium state, and that perturbation para meters other than sound waves can be used for its exploitation, was made by MANFRED EIGEN in 1954. The foresightedness of K.F. BONHOEFFER in securing the early beginnings of a purposefully de lineated research program to this end was but one example of his admirable faith in the abilities of the young scientists he gath ered around him. Early progress was fast and rewarding, despite the unconventional and sometimes rather difficult experimental arrangements used. But the attack was systematic. Simple exc~ange reactions involving protons and coordination ligands yielded ~ time scale with the diffusion-controlled encounter time as a yardstick. Chemical equi libria were perturbed by electric fi·elds and sudden temperature and pressure changes. Sensitive devices for the observation of fast transient changes in almost any optical or electrical system parameter were designed. Many exotic instruments with special com binations were dreamed up and never made. Consistent and general ized theoretical treatments were worked out, dealing with an ar bitrary reaction mechanism. An increasing number of scientists became aware of the field. A highly nonlinear, cooperative growth period was the consequence. VIII The mechanism of the catalytic activity of enzymes was the first challenge where the new method had to cope with chemical proper ties and interactions between particles with a different degree of molecular organization. The potential applications in biology at the molecular level were attractive to biochemists1 at the same time they created new situations and solutions for the ex perimental approach. The development of chemical relaxation as an approach to the dy namics of systems with a high degree of complexity may not seem to proceed with the same rapid advances as the study of simpler reactions ten or more years ago, although the experimental tech niques are greatly improved. This reflects the complexity of the subject, rather than a deficiency in the method. As in chemical reactions, the coupling of fast elementary processes is not pos sible without introducing slower, mixed reaction coordinates. In molecular biology, progress in understanding dynamics and ki netic behavior cannot be uncoupled from progress in analytical and preparative procedures and from progress in obtaining infor mation on molecular structure and organization. Gottingen, April 1977 L. DE MAEYER Introduction Two decades elapsed since the introduction of the concept of chem ical relaxation for the study of chemical kinetics by EIGEN and DE MAEYER (1, 2). From the very beginning, aspects of molecular biology were in the foreground of the problems investigated by this novel approach. A wide range of reactions was characterized in terms of their detailed mechanisms and specific rates; this ranged from proton transfer processes to the allosteric control of protein function. The purpose of this monograph is to give a representative cross section of the current research activities dedicated to the anal ysis of elementary steps in biological reactions. This covers the range of the following topics: hydrogen-bond formation, nucleotide base pairing, protein folding, isomerisation of protein and nucle ic acid conformations, interactions between protein and proteins, nucleic acid,and proteins, enzymes and substrates, antibody and haptens or ionic transport through membranes. A common denominator in these studies is the search for an understanding of the laws that govern the dynamic behavior of living systems. To make this volume useful also for the nonexpert, a comprehensive introduction to the theory of chemical relaxation is given. The instrumentation used in most of the studies presented here has been reviewed in detail previously (2, 3, 4). New developments and perspectives in the methodology have been treated by the in dividual authors. We hope that this book reflecting the current state of art in the analysis of chemical kinetics of complex of biological systems will serve as an incentive for future studies of the large number of important biological reactions for which an understanding of the mechanisms of action at the molecular level is still lacking. 1. EIGEN, M.: Discussion Faraday Soc. II, ~94 (1954). 2. EIGEN, M., DE MAEYER, L.: Techniques of Organic Chemistry (ed. A. WEISSBERGER), Vol. III, part 2, p. 835. New York: Wiley 1963. 3. KUSTIN, K.: Fast reactions. In: Methods of Enzymology (eds. S.P. COLOWICK, N.O. KAPLAN), Vol. XVI. New York: Academic Press 1969. 4. YAPEL, A.F., Jr., LUMRY, R.: Methods in Biochemical Analysis, Vol. XX, pp. 169-350. New York: Wiley 1971. J. PECHT R. RIGLER Contents Theory and Simulation of Chemical Relaxation Spectra G. ILGENFRITZ ...........•................................. I. Introduction •.............•••....................... 1 A. The Relaxation Kinetic Progress Curve ............ 2 B. Optical Detection Signals ........................ 4 C. Theoretical Description of Relaxation Kinetics ... 7 D. Single Reaction Steps............................ 16 E. Approximations for Complex Reaction Systems ...... 19 F. Average Relaxation Times .•....................... 24 G. Computer Program for Simulation of Relaxation Spectra (FORTRAN IV).............................. 26 1. Numbering of Reacting Species ...•............. 27 2. Numbering of Individual Reaction Steps ........ 28 References •............................•............... 31 Concentration Correlation Analysis and Chemical Kinetics D. MAGDE ..•...•...........•.............•..•.•............ 43 I. Introduction ......•...........•..................... 43 II. Properties of Thermodynamic Fluctuations .....•...... 44 A. Magnitude of Occupation Number Fluctuations ...... 44 B. Dissipation of Number Fluctuations ............... 49 III. Measurement of Number Fluctuations ...•...........•.. 52 A. Fluorescence Correlation Analysis (FCA) .......... 54 1. Design of the FCA Experiment .................. 55 2. Correlation Computers ................•........ 60 3. Experimental Results •...•..................... 64 B. Resistance Correlation Analysis (RCA) ............ 68 C. Absorbance Correlation Analysis (ACA) ........•... 74 D. Quasi-Elastic Light Scattering (QELS) and Turbidity Correlation Analysis (TCA) ............. 76 E. Orientation Correlation Analysis (OCA) .....•..... 78 IV. Summary and Conclusions •......•.•................... 80 References ...••....•........................•........... 82 Dynamics of Substitution at Metal Ions D • N. HAGUE •.....•..•..•.....................•............. 84 I. Introduction 84 II. Formation of 1:1 Complexes with Small Ligands ...•... 86 III. Formation of 1:1 Complexes with Large Ligands ...... . 91 IV. The Effect of Bound Ligands ......•.................. 97 XI A. Non-Ring Systems •.•••••••.••••.•••••••.••••••••• 97 1. Outer-Sphere Complex Formation •••••.••.•••••• 98 2. Labilisation of Remaining Water Molecules •.•• 99 3. Steric and Electronic Interaction Between Ligands •••••••••••.••••.•.•.••..••••••••••.•• 103 4. Coordination Number Change at the Metal •••••• 103 B. Ring Systems-•••••.•••••••.••.•.••••••••••••••••• 103 V. S urnrnary •••••.•••.••••.•••..•••••.••..••••••••.•.••. 1 04 References •••••••.••.•.••••••••••.••.•.••.•.••.•..•.•• 105 Dynamics of Proton Transfer in Solution P. SCHUSTER, P. WOLSCHANN, and K. TORTSCHANOFF .•••••••••• 107 I. Introduction •..•••..••.•.•••.•••••••.•.••.••..••••• 107 II. Theoretical Background of Proton Transfer •.••.••••• 108 A. Proton Affinities ••.•••••••••••••..•••••.•..•.•• 108 B. Stability of Hydrogen Bonded Molecular Complexes. 109 C. Potential Curves for Proton Transfer ••••••••..•• 114 D. Dynamics of Proton Transfer in the Vapor Phase •• 115 E. Gas Phase Solvation •••••••••••.••••..••..••..••. 116 F. Theoretical Concepts and Mechanisms of Proton Transfer in Solution ••...••••..•.••.•.••••••••.. 119 III. Proton Transfer in Aqueous Solution •...•..••.••.•.• 121 A. Intermolecular Proton Transfer •••.••.•••.•..•.•• 122 B. Intramolecular Proton Transfer .••.••••••..•••••• 129 IV. Proton Transfer in Non-Aqueous Solvents .•.•••••.••. 139 A. Protic, Non-Aqueous Solvents ••••••••••.••••...•. 139 B. Aprotic Solvents................................ 142 V. Biochemical Model Studies ....••.•..•.••.•••••••.•.• 147 A. Amino Acids .••.•••••...•••••..••••.••.••••••.••• 148 B. Purines and Pyrimidines ••.••••..••.••••••...•..• 150 1. Formation of Hydrogen Bonded Complexes .•••••. 151 2. Proton Transfer Reactions on Purines, Pyrimi- dines and Some Related Heterocyclic Compounds. 155 C. Coenzymes and Other Model Compounds ••••.•.•.••.• 157 D. Macromolecules ••.•••..••••.•..•..••..••..•.•..•• 160 VI. Polypeptides and Proteins •.•.•.•••.•....••••••••.•• 163 A. Oligopeptides ...•••••..•.•••••..••••.••••.•....• 163 B. High Molecular Weight Polypeptides and Proteins. 164 VII. Experimental Techniques ••..•••••••••••••••....•.... 169 VIII. Conclusion......................................... 171 IX. Other Review Articles and Books on Proton Transfer. 173 References ••.••••.•....••..•......•..•.•••••••••••••••• 174 Elementary Steps of Base Recognition and Helix-Coil Tran sitions in Nucleic Acids D. P(:)RSCHKE .••••••.•••..•.•••••••.••.•.••.••••..••••..•.• 191 I. Introduction ••.•.•.•.•..•..••...•.••.•••..•..•••••• 191 II. Elementary Steps of Bases Stacking ••.•.•.•••.•••..• 192 A. Stacking of Monomer Bases and Hydrophobic Inter- actions ••..•••••..••...••.••.••••••.•.•••••...•. 192 B. Conformation Change of Single-Stranded poly- nucleotides .•••.•......•.•••••••••••••.••••••••. 194 III. Ion Condensation to Polynucleotides •••••..•••.••.•• 196 XII IV. Recognition of Monomer Bases on a polymer Template. 197 V. Helix-Coil Transition of Oligo (A) ·Oligo(U) ••••••••• 198 A. Equilibrium Parameters According to the Coopera- ti ve Reaction Model •••.•••••••••••••...•.••...•• 198 B. Relaxation Data and Their Interpretation Accord- ing to an "Allor None" Model •.••••.•.•.••...•.• 200 C. Unzippering at Helix Ends •...••.••.•.•.•••...•.. 206 D. Chain Sliding .•.•••••..••••.••••••••.•••••••.•.. 209 VI. The Influence of GC Base Pairs •.•.•.••••••.•••••••. 210 VII. Specific Effects in Helix Loops .••..•.•••...•.••..• 210 VIII. Dynamics of Polymer Helix-Coil Transitions ••••..... 212 IX. Rate and Specificity of Genetic Information Transfer 213 X. Summary............................................ 213 References .•...••.•.••.••••.•..••••..•.•.•••...•••.•.• 216 Structural Dynamics of tRNA. A Fluorescence Relaxation Study of tRNA~~~st R. RIGLER, M.EHRENBERG, and W. WINTERMEYER .•..••.....•.•• 219 I • Introduction .•••.••..•.••••••••.•.••••.•••..••••••. 219 II. Fluorescent Probes for the Structure of tRNA •••••.• 219 III. Pulsed Fluorescence Measurements ..•••••.•.•••.•.••• 221 A. The Lifetime of Excited States of the Fluorescent Probe and the Distribution of Conformational States .•••.•••.••••••.••••..•.•.•••••.••••.•.••• 221 B. Rotational Brownian Motion and Time-Dependent Fluorescence Anisotropy ••••••••••.•••••••.•••.•. 221 C. Instrumentation •••••••••..••••••••.•••..••.••..• 222 D. Results .•.•.•••••••••.•.•.•.••.••••••••.•••••••• 223 IV. Measurements Under Stationary Excitation •....•••••. 227 V. Measurements of Chemical Rates ••••.•.•••..•..••...• 228 A. Instrumentation and Data Evaluation ••••••.•.••.. 228 B. Results •.•••••••••••••.••••..•••.•••.•.....••.•. 231 VI. A Model for Allosteric Conformations of tRNA •..•••• 233 A. Evaluation of the Parameters •••..•.••••••.•.••.. 235 VII. Conformational States of tRNA with Regard to the Biological Role of tRNA ••••••..••••••••••..•..••.•• 237 References •.•....•••••••.••••••••••..•.•.••.•.•..•••.•.. 240 Chemical Relaxation Kinetic Studies of E. aoLi RNA Poly merase Binding to poly[d(A-T)] Using Ethidium Bromide as a Fluorescence Probe T.M. JOVIN and G. STRIKER................................ 245 I. Introduction •.•••••••••.••..•• ~..................... 245 II. Experimental Procedures and Data Analysis .•..••.••• 249 A. Materials ••••••••••••••...•.••••••.••••••.••.••• 249 B. Fluorescence Temperature-Jump Measurements. Instrumentation and Conditions ••••••.•.•••.••••• 249 C. On-Line Computer Acquisition of Relaxation Data. 250 D. Analysis of Relaxation Curves by the Method of Modulating Functions .•••.••••••.•.••••.••••••.•• 251 III. Excluded Site Binding of Ethidium Bromide to Poly[d(A-T)] •.••••••••.•.••••••.•••••••.••.••••••.• 253 A. Theory for the Equilibrium State •.••••.•.•.•.••• 253