Molecular Biology Biochemistry and Biophysics 31 Editors: A. Kleinze(ler, 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. Guifreund, Bristol . B. Hess, Dortmund H. lahrmiirker, Munich . R. W. leanloz, Boston E. Katzir, Rehovot . B. Keil, Gif-sur-Yvette M. Klingenberg, Munich . I M. Klotz, Evanston W. T. 1. Morgan, SuttoniSurrey . K. Muhlethaler, Zurich S. Ochoa, New York' G. Palmer, Houston I Pecht, Rehovot . R. R. Porter, O:xford W. Reichardt, Tubingen . R. A. Reisfeld, La Jolla H. Tuppy, Vienna . 1. Waldstrom, Malmo Membrane Spectroscopy Edited by Ernst Grell With Contributions by D. F. Bocian S. I. Chan M. C. Foster N. P. Franks U. P. Fringeli E. Grell Hs. H. Giinthard 1. Heesemann y. K Levine M. M. Long R. C. Lord D. Marsh R. Mendelsohn N. O. Petersen H. Ruf D. W. Urry 1. Yguerabide H. P. Zingsheim With 146 Figures Springer-Verlag Berlin Heidelberg New York 1981 Dr. ERNST GRELL Max-Planck-Institut fur Biophysik Heinrich-Hoffmann-StraBe 7 6000 Frankfurt 71 ISBN-13: 978-3-642-81539-3 e-ISBN-13: 978-3-642-81537-9 DOl: 10.1007/978-3-642-81537-9 Library of Congress Cataloging in Publication Data Main entry under title: Membrane spectroscopy. (Molecular biology, biochemistry, and biophysics; 31) 1. Membranes (Biology) 2. Spectroscopy. I. Grell, Ernst, 1941- II. Series. [DNLM: 1. Membranes. 2. Spectrum analysis. WI M0195T no. 31/ QH 601 M533) QH601.M4688 574.87'5'028 80-24524 This work is subject to copyright. All rights are reserved, whether the whole or part 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 "Verwertungsgesellschaft Wort", Munich. © by Springer-Verlag Berlin Heidelberg 1981. Softcover reprint ofthe hardcover 1s t edition 1981 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 regulations and therefore free for general use. 2131/3130-543210 Foreword The last 10 years have seen an enormous growth in our understanding of the molecular organisation of biological membranes. Experimental methods have been devised to meas ure the translational and rotational mobility of lipids and proteins, thereby furnishing a quantitative basis for the concept of membrane fluidity. Likewise, the asymmetry of bi layer membranes as evidenced by the asymmetric insertion of proteins and lipids has been put on firm experimental ground. At higher molecular resolution it has been possible to provide a detailed pi2ture of the molecular conformation and dynamics of lipids and, to some extent, even of small peptides embedded in a bilayer matrix. Many of these achieve ments would not have been possible without the application of modem spectroscopic methods. Since these techniques are scattered in a variety of specialized textbooks the present monograph attempts to describe the key spectroscopic methods employed in present-day membrane research at an intermediate level. There is no question that the elusive detailed structure of the biological membrane demands a multiplicity of experi mental approaches and that no single spectroscopic method can cover the full range of physical phenomena encountered in a membrane. Much confusion in the literature has arisen by undue generalizations without considering the frequency range or other limi tations of the methods employed. It is to be hoped that the present monograph with its comprehensive description of most modem spectroscopic techniques, will contribute to- . wards a further convergence of views among the spectroscopic specialists and will enhance the understanding of membrane structure. Basel, September 1980 Joachim Seelig Preface The aim of this book is to introduce the reader to the application of spectroscopic tech niques to the study of membranes. The following principal methods are covered: mag netic resonance, optical and low-angle X-ray spectroscopy and chemical relaxation spec trometry. Each chapter summarizes the experimental and theoretical principles of a particular technique and the special applications of that technique to the investigation of mem branes. In addition, the contributions will critically review the current exploitation of the technique, from the point of the view of the authors, by considering the results ob tained on membrane constituents, simple model membranes and on biological membrane systems of a highly complex nature. A common aspect in all the contributions is the in tensive search for a detailed understanding of the structures and functions of biological membranes at a molecular level. This book will help to facilitate and to stimulate future studies in this interesting field. Frankfurt, Summer 1980 Ernst Grell Table of Contents 1. Nuclear Magnetic Resonance Studies of the Phospholipid Bilayer Membrane, S.1. Chan, D.F. Bocian, and N.O. Petersen (With 25 Figures) . 1 I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 II. Partially Oriented Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 3 III. Effects of Motional Restriction on NMR Spectra. . . . . . . . . . . . . . . . . 6 IV. Isotropic Motion in the Presence of Restricted Local Motion: Membrane Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 21 V. Membrane Dynamics from NMR Relaxation Measurements. . . . . . . . .. 23 VI. Merits of Various Spin Probes in Membrane Studies. . . . . . . . . . . . . .. 32 VII. Selected Applications ofNMR to Membrane Studies. . . . . . . . . . . . .. 38 VIII. Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 46 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 47 2. Electron Spin Resonance: Spin Labels, D.Marsh(With 33 Figures) . . . .. 51 I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 51 II. Experimental Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 53 III. Theoretical Basis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 67 IV. Applications to Membrane Studies. . . . . . . . . . . . . . . . . . . . . . . . .. 86 V. New Developl;l1ents and Future Trends ....................... 128 References .............................................. 137 3. Absorption and Circular Dichroism Spectroscopies, M.M. Long and D.W. Urry (With 14 Figures) .............................. 143 I. Introduction ......................................... 143 II. Solution Studies ...................................... 143 III. Suspensions of Homogeneous Particles . . . . . . . . . . . . . . . . . . . . . . . 150 IV. Suspension of Biomembranes (Heterogeneous Particles) . . . . . . . . . . . . 154 References ...... ,. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 4. Optical Spectroscopy of Monolayers, Multilayer Assemblies, and Single Model Membranes, J. Heesemann and H.P. Zingsheim (With 6 Figures) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 II. General Considerations and History . . . . . . . . . . . . . . . . . . . . . . . . . 173 x Table of Contents III. Design of Spectroscopic Probes ............................ 175 N. Concentration, Orientation, and Location of Chromophores . . . . . . . . . 178 V. Monomers, Dimers, and Aggregates .......................... 180 VI. Energy Transfer ...................................... 181 VIT. Lipoid pH-Indicators in Monolayers . . . . . . . . . . . . . . . . . . . . . . . . . 182 VIII. Lateral Diffusion in Model Membranes . . . . . . . . . . . . . . . . . . . . . . . 185 IX. Electric-Field-Induced Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194 s. Fluorescence Spectroscopy of Biological Membranes, J. Yguerabide and M.C. Foster (With 24 Figures) .......................... 199 I. Basic Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 II. Types of Fluorophores Used in the Study of Membranes ............ 209 III. Applications ......................................... 214 References ............................................... 267 6. Infrared Membrane Spectroscopy, U.P. Fringeli and Hs.H. Gtinthard (With 26 Figures) ..................................... 270 I. Introduction. ........................................ 270 II. Experimental Techniques 1 Spectrophotometric Consideration ....... 271 III. Application of IR Spectroscopy to Biological Systems ............. 272 N. Review of Results of IR Membrane Spectroscopy ................ 278 References .............................................. 327 7. Chemical Relaxation Spectrometry, H. Ruf and E. Grell (With 12 Figures) . 333 I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333 II. Experimental Methods .................................. 337 III. Theoretical Aspects .................................... 344 N. Applications to Membrane Studies .......................... 361 References .. : ........................................... 372 8. Raman Spectroscopy of Membrane Constituents and Related Molecules, R.C. Lord and R. Mendelsohn (With 18 Figures) ......... 377 I. Introduction. ........................................ 377 II. Experimental Methods .................................. 383 III. The Theoretical Basis of Raman Spectroscopy .................. 389 N. Applications to Membrane Studies .......................... 397 V. Future Trends ........................................ 424 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 426 Table of Contents XI 9. Low-Angle X-Ray Diffraction, N.P. Franks and Y.K. Levine (With 23 Figures) ......................................... 437 I. Introduction ......................................... 437 II. Experimental Methods ....... 438 A •••••••••••••••••••••••••• III. Theory............................................ 439 N. Applications to Membrane Systems ......................... 457 V. Conclusions ......................................... 482 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 Subject Index .............................................. 488 Nuclear Magnetic Resonance Studies of the Phospholipid Bilayer Membrane S.1. Chan, D.F. Bocian and N.O. Petersen I. Introduction The cell membrane has been the focus of much recent biochemical and biophysical re search, primarily because of its role in cellular phenomena. Numerous efforts have been directed toward determining the motional state of the lipid components of the mem brane, the motivation being the contention that the lipid bilayer is the basic matrix in which membrane proteins are embedded to form the biological membrane. As such, it is likely that such diverse phenomena as maintenance of ionic gradients and transmembrane potentials, activities of membrane-bound enzymes, interactions between membrane pro teins, transmembrane signal transmission, intercellular communication, and manifesta tions of cellular development and cell transformation all depend on the structure and fluidity of the lipid bilayer. The working hypothesis for the structure of the cell membrane is the fluid mosaic model (Singer and Nicolson, 1972), where the steady state arrangements of lipids and proteins are intrinsically dynamic in nature. Thus, the structural arrangement of mem brane components contains essential information concerning membrane function. Simi larly, understanding the dynamics of membrane components both in terms of internal freedom of motion and in terms of lateral movements of the components in the lipid . phase is important for revealing how the various time-dependent membrane functions are controlled (Edidin, 1974). It is particularly in this latter area of membrane research that various spectroscopic techniques such as magnetic resonance are useful. Biological membranes are multicomponent systems composed of many different membrane proteins and lipids. One of the primary lipid constituents of most biological membranes)s phosph~lipids, which occur naturally with a variety of headgroups and acyl chains. Aqueous dispersions of phospholipids exhibit many of the physical proper ties of natural membranes, and accordingly these dispersions have been used extensively as membrane models for magnetic resonance studies (Lee et al., 1974; James, 1975; Tiddy, 1975, 1977; Seelig, 1977; Wennerstrom and Lindblom, 1977; Bocian and Chan, 1978). Phospholipids spontaneously aggregate to form bilayer structures, and the inter actions between different bilayers result in a thermodynamically stable multilamellar superstructure (Fig. 1). Pure phospholipid dispersions exist in a number of phases depending on temperature and water content. The two phases of primary importance as biological membrane mod els are the gel and the liquid crystalline phases. In multicomponent phospholipid systems, A.A. Noyes Laboratory of Chemical Physics (Contribution No. 5853), Californialnstitute of Tech nology, Pasadena, California 91125, USA 2 S.1. Chan, D.F. Bocian and N.D. Petersen Fig. 1. A representation of the muItiiamellar superstructure formed by phospholipid/water dispersions. The individual lipid bilayers are of infinite extent and are separated by the interstitial water (Khetra pal et aI., 1975) species immiscibility and phase separations can occur and the phase diagrams become ex ceedingly complicated. This phenomenon has been investigated extensively by electron spin resonance (ESR) spin-label studies (Wu and McConnell, 1975). However, most nu clear magnetic resonance (NMR) studies have been performed on single-component sys tems and, in general, at temperatures above the gel ~ liquid crystalline phase transition temperature. In principle, NMR could be used to study multicomponent systems if the components could be suitably labeled. Also, sensitivity and abundance have made lipids the principal membrane components investigated to date by NMR. However, the direct observation of nonlipid membrane components is now becoming possible through the use of specific labeling of these components, and by the use of lipid components which do not contain the nucleus being observed (Feigenson et aI., 1977). For many years, much of our knowledge about the motional state of the bilayer has come from ESR measurements on spin-labeled lipids incorporated into the bilayer (Hub bell and McConnell, 1971; Jost et aI., 1971; Schindler and Seelig, 1974). Recent efforts have been directed toward developing spectroscopic probes which would circumvent the possible perturbations in the bilayer produced by the bulky nitroxide radical. Nuclear magnetic resonance spectroscopy is a natural tool for this purpose and there has been considerable effort using lH, 2H, l3C, and 3lp probes which are either endogenous or in corporated into the lipid molecule (Lee et aI., 1974; James, 1975; Tiddy, 1975, 1977; Seelig, 1977; Wennerstrom and Lindblom, 1977; Bocian and Chan, 1978). Both ESR