METHODS OF EXPE R I M E NTAL PHYS IC S : C. Marton, Editor-in-Chief Volume 20 B i o p hy s i cs Edited by GERALD EHRENSTEIN and HAROLD LECAR Laboratory of Biophysics National Institutes of Health Bethesda, Maryland @ 1982 ACADEMIC PRESS A Subsidiary of Harcouc Brace Jovanovich, Publishers New York London Paris San Diego San Francisco SSo Paula Sydney Tokyo Toronto COPYRIGH@T 1 982, BY ACADEMIPCR ESSI,N C. ALL RIGHTS RESERVED. NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RE.CORDING, OR ANY INI'ORMATION STORAGI: AND RF TRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER. ACADEMIC PRESS, INC. 111 Fifth Avenue, New York, New York 10003 United Kingdom Edition published by ACADEMIC PRESS, INC. (LONDON) LTD. 24/28 Oval Road, London NW1 IDX Library of Congress Cataloging in Publication Data Main entry under title: Biophysics. (Methods of experimental physics : v. 20) Includes bibliographical references and index. 1. Biophysics. I. Lecar, H. (Harold) 11. Ehrenstein, G. (Gerald) III. Series. PH505.8472 574.19'1 02-6642 ISBN 0-12-475962-9 AACRZ PRINTED IN TIE UNITED STATES OF AMERICA 82 8.1 84 8s 9 8 I 6 5 4 3 2 1 PREFACE This volume describes three types of measurements that have been of intense interest to physicists working in biology-spectroscopy of macro- molecules, structure determination by the scattering of particles or radia- tion, and electrical measurements on cell membranes. Parts 1-4 discuss several types of spectroscopy, and are primarily con- cerned with the determination of molecular information. Parts 5-9 are concerned with methods used to visualize the structures and motions of large molecules and cells. Parts 10 and I1 discuss methods for studying membranes, either directly or by means of model systems. Almost every type of spectroscopy has been used with some success to study biological macromolecules, and any sampling of methods is some- what arbitrary. We have emphasized methods which can be used to study molecular motion and conformational changes of macromolecules. Mag- netic resonance spectroscopies have found considerable use in studies of biological systems, and the first two parts are about these methods. In nuclear magnetic resonance, the absorption frequency depends on the magnetic moment of a nucleus. Since protons have large magnetic moments and are prevalent in biological material, early work centered on the proton. With the improved sensitivity now available, other nuclei with magnetic moments are often studied. Part 1 includes an example of the use of natural abundance *TN MR,a s well as other new and more sensi- tive methods. A major theme of Part 1 is the use of nuclear magnetic resonance to measure relaxation times. Electron spin resonance (ESR)i s similar to nuclear magnetic resonance in concept, but utilizes the interaction of a magnetic field with an electron spin rather than a nuclear spin. Thus ESR spectroscopy is restricted to molecules with unpaired electron spins. Unpaired spins are present in biological molecules containing transition metals and in free radicals cre- ated as intermediates in biochemical reactions or by radiation damage. For systems without unpaired electron spins, it is sometimes possible to add covalently a group that contains an unpaired spin. This approach, called spin labeling, is the subject of detailed discussion in Part 2. Suc- cessful spin labeling requires that the label be incorporated into the de- sired part of the molecule under study and also that it not interfere with the normal functioning of that molecule. When these conditions are met, xxiii xxiv PREFACE the spectrum of the spin label can be used to determine a number of important parameters, such as molecular motions, distances between groups, and rates of reactions. A number of IR, visible, and UV spectroscopies have been successfully used for biological studies. We have chosen to include a paper on Raman spectroscopy (Part 3) because it combines several important advantages, such as the possibility of recording from small samples, the lack of inter- ference from liquid water, and sensitivity to homonuclear bonds. Raman spectroscopy has been utilized effectively in studies of heme groups, nucleic acids, and lipid membranes. Other types of spectroscopies which have been used extensively to study macromolecules, but are not de- scribed in this volume, include fluorescence, optical rotatory dispersion (ORD), and circular dichroism (CD). Fluorescence spectroscopy has been very successful because of the prevalence of chromophoric groups in biological molecules, the possibility of incorporating fluorescent markers, and the high sensitivity inherent in fluorescence measurements. ORD and CD signals, which are sensitive to helical content of macromolecules, are especially valuable spectral indicators of conformation. Picosecond laser spectroscopy, which is discussed in Part 4, is a rela- tively new method that has considerable promise for several types of biological problems. Part 4 discusses the new technology needed to pro- duce and record pulses of light in the picosecond range and provides examples of applications to photosynthesis and vision, where previous methods have been inadequate to resolve the rapid transitions which occur immediately after the absorption of a photon. As previously mentioned, fluorescence can be a very valuable tool in the measurement of absorption spectra. The fluorescence methods pre- sented in Part 5, however, are based not on the particular frequency of absorption, but on the bleaching of fluorescent molecules and the interdif- fusion of bleached and unbleached molecules. These methods allow the determination of molecular motion within a cell membrane. Although similar information can be obtained from spin labeling (Part 2), the fluo- rescence methods are somewhat more flexible in that they can be used to determine motions over relatively long distances. Parts 6-9 describe different approaches to the problem of obtaining images of small objects. Whether the objects are macromolecules, cell organelles, or whole cells, the scattering and diffraction methods all share a common conceptual basis-the distribution of scattered radiation or particles gives the Fourier transform of the density of the object under study. Thus these parts are representative of the current emphasis on Fourier optics. Part 6 deals with x-ray diffraction, which has been the prime source of PREFACE xxv our knowledge of the detailed structure of biological macromolecules. For those macromolecules that can be crystallized, x-ray diffraction can give a complete description of the spatial molecular structure to a level of A. resolution better than 3 Much of our pictorial knowledge of the rela- tions between structure and function for proteins and for DNA comes from the x-ray crystallographic analyses of approximately 100 prototypi- cal macromolecules. Even for structures (such as fibrous macromole- cules, viruses, and ribosomes) that cannot be grown as crystals, x-ray diffraction provides a wealth of information. For example, it comple- ments the method of neutron diffraction (Part 8) to provide a good de- scription of the structure of ribosomes. The laser, because of its ability to generate coherent light, has had a strong impact on all of optical technology. Part 7 surveys the many appli- cations to cell biology of laser light scattering and emphasizes a particu- larly promising area-the interpretation of light scattering from motile bacteria. Different types of motion produce different Doppler-shift char- acteristics, and these can be distinguished by means of autocorrelation calculations for the different motions. The concepts involved in small-angle x-ray and neutron scattering, dis- cussed in Part 8, are similar to those involved in light scattering. How- ever, since the wavelength of radiation is so small (of the order of l &, scattering by macromolecules is characteristic of objects much larger than the wavelength of the incident radiation. Consequently, the scattering is confined to “small” angles near the forward direction. These short-wave- length methods provide information complementary to that obtained by crystallography. In particular, they can be used to analyze the shapes and interactions of macromolecules in solution. Part 9 emphasizes those aspects of electron microscopy that have grown out of physical considerations of the process of image formation. High-energy , high-resolution microscopy, scanning electron microscopy, and electron diffraction-techniques that provide new and striking im- ages of key supramolecular structures-are considered here. Two exam- ples are cytoskeletal architecture and membrane-protein complexes such as bacterial rhodopsin. The last two parts describe methods for studying membrane transport. Part 10 describes voltage clamping of excitable cell membranes, and Part 11 describes methods for making lipid model membranes and for deter- mining their properties. Present membrane-transport research focuses on characterizing those molecular structures in the membrane that are responsible for the various transport processes. Molecular structures of particular interest are the ion-selective channels responsible for excitability and ion “pumps ,” the xxvi PREFACE membrane-bound enzymes that use metabolic energy to maintain concen- tration gradients of ions. In electrically excitable cells, such as nerve axons, the electrical behavior is extremely nonlinear. In fact, the nonlin- earity is crucial to excitability. As discussed in Part 10, the voltage clamp has proven to be the major tool for studying such highly nonlinear sys- tems, because it enables the experimenter to control the key variable- the transmembrane potential-and thus determine the time dependence and potential dependences of membrane conductance. Without this con- trol, membrane potential and membrane conductance would vary in an extremely complex manner, making it virtually impossible to disentangle them. The concept of voltage clamping has also been useful in several recent important advances in the study of membrane ionic channels-the measurement of currents from individual channels, the use of noise analy- sis to determine the properties of the channels as they switch on and of€ at random, and the measurement of gating current (the displacement current caused by molecular conformational changes during channel opening or closing). Part 11 discusses methods for using synthetic lipid bilayers as analogs of cell membranes. This approach has contributed greatly to the currently accepted paradigm for membrane structure-a lipid bilayer matrix resem- bling a two-dimensional fluid, in which various membrane proteins are embedded. In addition, the many studies of synthetic ionophores im- planted in bilayers have yielded major insights into the transport mecha- nisms of membrane channels and carriers. For example, lipid bilayer studies provided the first measurements showing that membrane ionic current passes through discrete channels. More recently, significant pro- gress has been made in reconstituting functional entities from biological membranes into lipid bilayers or lipid vesicles. Among the systems that have been reconstituted are Na-K ATPase pumps, postsynaptic acetyl- choline-activated channels, and photosynthetic reaction centers. We hope that the sample of methods presented in this volume shows some of the technological ingenuity that biophysical problems have gener- ated. We should like to thank the editors of this series for their encourage- ment, support, and patience. Dr. Claire Marton and Dr. L. Marton, both recently deceased, have taken considerable interest in furthering interac- tions between physicists and biophysicists. We should also like to thank the staff of Academic Press for their support. Finally, we thank the au- thors for their effort and for their willingness to accommodate their indi- vidual papers to the needs of this volume. GERALDE HRENSTEIN HAROLDL ECAR CONTRl BUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin. L. MARIOA MZELD, epartment of Biophysics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 (229) FRANCISCOBE ZANILLAD,e partment of Physiology, School of Medicine, University of California, Los Angeles, California 90024 (445) MARGARERT. BUNOW,N ational Institutes of Health, Bethesda, Mary- land 20205 (123) SUNNEYI. CHANA, . A. Noyes Laboratory of Chemical Physics, Califor- nia Znstitute of Technology, Pasadena, California 91125 (I) ELLIOTL . ELSON,* Department of Chemistry, Cornell University, Zthaca, New York 14853 (197) ROBERTM . GLAESERD, epartment of Biophysics and Medical Physics, University of California, Berkeley, California 94720 (391) GILLIANM . K. HUMPHRIESS,t~a uffer Laboratory for Physical Chemis- try, Stanford University, Stanford, California 94305 (53) TAKAYOSHKIO BAYASHDI,e partment of Physics, University of Tokyo, Bunkyo-Ku, Tokyo 113 Japan (163) EATONE . LATTMAND, epartment of Biophysics, Johns Hopkins Univer- sity School of Medicine, Baltimore, Maryland 21205 (229) HARDENM . MCCONNELLS,t auffer Laboratory for Physical Chemistry, Stanford University, Stanford, California 94305 (53) PETERB . MOORED, epartments of Chemistry and Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511 (337) RALPHN OSSALN,a tional Institutes of Health, Bethesda, Maryland 20205 (299) * Present address: Department of Biological Chemistry, Washington University School of Medicine, St. Louis, Missouri 631 10. t Present address: Institute for Medical Research, San Jose, California 95128. xvii xviii CONTRIBUTORS JOSEPH SCHLESSINGDEeRpa, rtment of Chemical Immunology, The Weiz- mann Institute for Science, Rehovot, Israel (197) JOSEPH R. SCHUHA, . A. Noyes Laboratory of Chemical Physics, California Institute of Technology, Pudadena, Cal$ornia 91125 (I) G. SZABOD,e partment of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77550 (51 3) ROBERTE . TAYLORL, aboratory of Biophysics, National Institutes of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205 (445) JULIO VERGARAD, epartment of Physiology, School of Medicine, Univer- sity of Calgornia, Los Angeles, California 90024 (445) R. C. WALDBILLIGD,e partment of Physiology and Biophysics, Univer- sity of Texas Medical Branch, Galveston, Texas 77550 (513) PUBLISHER’S FOREWORD It is with sadness that we inform readers of Methods of Experimental Physics of the passing of Dr. Claire Marton. Both Claire and her late husband, Bill, were associated with Academic Press almost from its founding. Their passing is both a personal and professional loss to us. Future volumes will be edited by Dr. Robert Celotta of the Electron Physics Group, Radiation Physics Division, National Bureau of Stan- dards, Washington, D.C., and by Dr. Judah Levine of the Joint Institute for Laboratory Astrophysics, University of Colorado, Boulder, Colo- rado. Dr. Celotta and Dr. Levine had already started to work with Claire. We are fortunate to have such qualified and experienced people join us. xix