Diffraction and Related Studies. Proceedings of the International Symposium on Biomolecular Structure, Conformation, Function and Evolution, Madras, January 1978

1263 Pages·1981·62.388 MB·1263\1263

by  R. Srinivasan (Eds.)

BIOMOLECULAR STRUCTURE,
CONFORMATION, FUNCTION,
AND EVOLUTION
Proceedings of the International Symposium on Biomolecular
Structure, Conformation, Function and Evolution, Madras, January 1978
Volume 1
DIFFRACTION AND RELATED STUDIES
Editor
R. SRINIVASAN
Senior Professor, Department of Crystallography & Biophysics, University of Madras, Itidia
Associate Editors:
E. SUBRAMANIAN
Reader, Department of Crystallography & Biophysics, University of Madras, India
N. YATHINDRA
Reader, Department of Crystallography & Biophysics, University of Madras, India
PERGAMON PRESS
OXFORD · NEW YORK · TORONTO · SYDNEY · PARIS · FRANKFURT
UK Pergamon Press Ltd., Headington Hill Hall,
Oxford OX3 OBW, England
USA Pergamon Press Inc., Maxwell House, Fairview
Park, Elmsford, New York 10523, USA
CANADA Pergamon of Canada, Suite 104, 150 Consumers
Road, Willowdale, Ontario M2J 1P9, Canada
AUSTRALIA Pergamon Press (Aust.) Pty. Ltd., P.O. Box 544,
Potts Point, NSW 2011, Australia
FRANCE Pergamon Press SARL, 24 rue des Ecoles,
75240 Paris, Cedex 05, France
FEDERAL REPUBLIC Pergamon Press GmbH, 6242 Kronberg/Taunus,
OF GERMANY Hammerweg 6, Federal Republic of Germany
Copyright © 1981 Pergamon Press Ltd.
All Rights Reserved. No part of this publication may be
reproduced, stored in a retrieval system or transmitted in
any form or by any means: electronic, electrostatic,
magnetic tape, mechanical, photocopying, recording or
otherwise, without permission in writing from the
publisher.
First edition 1981
British Library Cataloguing in Publication Data
International Symposium on Biomolecular Structure,
Conformation, Function and Evolution, Madras, 1978
Biomolecular structure, conformation, function and
evolution.
1. Molecular biology — Congresses
I. Title II. Srinivasan, Ramachandran
574. 8'8 QH506 78-41185
ISBN 0-08-023187-X (2 Volume Set)
Printed in Hungary by Franklin Printing House, Budapest
PREFACE
This and the companion volume, on "aspects of biomolecules" cover, in two volumes,
more than a hundred articles dealing with current research activities in the area of "struc­
tural molecular biology". These articles represent essentially the contents of the invited
talks and papers presented at an International Symposium on Biomolecular Structure,
Conformation, Function, and Evolution, held at Madras, India, during 4-7 January
1978.
The symposium was organized as a part of the Silver Jubilee Celebrations (25 years) of
the Department of Crystallography and Biophysics of the University of Madras. When the
idea of holding an international symposium dealing with biomolecules was mooted it was
so heartily welcomed by the scientific community that it gave us a great stimulus for its
actual organization. The overwhelming response of the international scientific community
is evidenced by the fact that more than a hundred scientists of international repute actually
participated in the symposium. Nearly all the papers presented in oral sessions of the sym­
posium were invited from active and/or senior scientists working in several areas of struc­
tural molecular biology. These contributions represent not only review materials but also
the latest findings in their respective fields of specialization. Apart from these invited papers,
several "contributed papers" were also presented in poster sessions. The sheer magnitude
of all the contributions made it a difficult task for us to decide how much of the material
should be published from among the "invited" and "contributed" category. Our final deci­
sion to publish only the "invited" contributions was dictated purely by our desire to bring
out the publication in time before the materials presented get out of date. The abstracts of
the contributed papers have also been published in the volume of collected abstracts
brought out at the time of the symposium, and thus are available for purposes of citation
and record.
The hundred and odd papers that go to make the volumes have been expediently divided
into two broad categories : (i) those concerned with the application of diffraction and related
techniques, and (ii) those concerned with the application of many physico-chemical and
theoretical methods. That this division puts roughly the same number of papers in both
categories should surprise no one since the application of diffraction methods as the final
arbiter of structure determination has dominated much of contemporary biochemistry and
molecular biology. Though this way of dividing the subject matter may appear to be arbi­
trary, we did consider other possible ways of classifying the material, but finally settled on
the above classification as being more optimal, by way of presenting the studies on various
aspects of biomolecules, than any other.
It may be added here that this way of division of volumes also brings certain cohesiveness
for each volume in that different types of biological molecules are found in each volume.
This stands in contrast to a possible textbook approach where one volume could perhaps
xiii
xiv t>REFACÊ
contain all papers dealing with, say, proteins and nucleic acids, while a second volume deals
with the rest. Such a division would have brought all the different techniques used in study­
ing a given type of molecule into each volume, and has not been preferred.
Thus Volume 1 deals with diffraction and related methods while Volume 2 deals with
physico-chemical and related methods. Articles in Volume 1 have been grouped on the basis
of various classes of biomolecules : (i) proteins; (ii) nucleic acids and constituents; (iii) pro­
tein-nucleic acid interactions; (iv) polysaccharides; (v) viruses; and (vi) peptides, drugs,
and other biomolecules. In each class, further subclassification has been attempted along the
following lines; (a) structure and conformation; (b) folding, assembly, and models; (c) struc­
ture and function ; (d) structure and evolution ; etc.
Volume 2 follows more or less similar classification as in Volume 1. The major divisions
of articles in this volume are as follows: (i) proteins; (ii) nucleic acids and constituents;
(iii) biomolecular interactions; (iv) ribosomes; (v) membranes; (vi) peptides, drugs, and
other biomolecules; and (vii) biomolecular evolution.
Thus the Parts in each volume are somewhat similar although not exactly the same. For
instance, Part III in Volume 1 is "Protein-nucleic acid interactions" while Part III in Volume
2 is "Biomolecular interactions". Parts IV and V in Volume 1 deal with polysaccharides
and viruses, respectively, while they are on ribosomes and membranes respectively in Volume
2. A few papers on biomolecular evolution find their place in Part VII of Volume 2.
It is our hope that these two volumes will benefit the scientific community as a reference
material reporting the progress of research in the area "Structural Molecular Biology"
during the seventies.
Lastly, we wish to take this opportunity to thank the international scientific community,
and especially those scientists who so readily responded to our invitations to come and
participate in the symposium, and to express our gratitude for their promptness in providing
the texts of their papers for publication in these volumes.
We should also like to express our special sense of gratitude to Professor Dorothy Crow­
foot Hodgkin, FRS, NL, who presided over the symposium and spent a brief period as the
Sir C. V. Raman Visiting Professor at the University of Madras during which time the
symposium was planned. We also wish to thank the members of the Organizing Committee
who gave valuable advice and assistance in organizational matters.
We wish to record our sincere thanks to the following National and International Organi­
zations for their generous financial support: the Government of Tamil Nadu; the Univer­
sity Grants Commission; the Council of Scientific and Industrial Research; the Department
of Science and Technology, Government of India; the Indian Council of Medical Research;
the Indian Council for Cultural Relations; the Bhabha Atomic Research Centre; the Indian
National Science Academy; the University of Madras; the National Science Foundation,
USA; Fogarty International Centre, USA, International Union of Crystallography; Inter­
national Union of Pure and Applied Biophysics; Royal Society, UK; the British Council,
UK; and UNESCO. We wish to thank Dr. Vasantha Pattabhi, Dr. K. Chandrastkhar and
Mr. V. Ravichandran for their assistance in proof reading and preparation of indices.
Madras, January, 1979 R. SRINIVASAN
E. SUBRAMANIAN
N. YATHINDRA
Presidential Address
BIOMOLECULAR STRUCTURE,
CONFORMATION, FUNCTION, AND
EVOLUTION
D. C. HODGKIN
I MUST begin by thanking most warmly the Vice-Chancellor of the University of Madras,
Dr. Malcolm S. Adiseshiah, and the Minister of Education of Tamil-Nadu, Mr. Arangana-
yakam, for their welcome on this very happy occasion. I feel myself to be very particularly
honoured and moved to be here as visiting Raman Professor in Physics, following Sir Law­
rence Bragg and Professor Linus Pauling as President of this, the third symposium to be
held in the University of Madras on biopolymers.
Long ago, when I was lecturing to undergraduates in Oxford, I used to make a joke over
the curious geographical distribution of scientific interest in certain fields—that the use of
electron diffraction, for example, to study crystal structures was practised almost exclusively
in Moscow and in Melbourne (and you can date these lectures, approximately, from this
joke). The reason, of course, was to be found in the interest and work of particular indi­
viduals in each case. And so today, the fact that Madras is a place we think about whenever
we think of problems of the conformation of biopolymers is due primarily to the interest
and work of one person—G. N. Ramachandran. We, his scientific friends and descendants,
send him very special greetings today as we celebrate the twenty-fifth anniversary of his
setting up a biophysics unit in the Department of Physics of Madras University.
1952—the moment Ramachandran chose for a new beginning in Madras—was a moment
full of promise, on the brink of great developments; the theoretical foundations were pro­
posed, the experiments already in progress which in the next few years were to transform
our knowledge of the structure of proteins and nucleic acids. In 1949, Corey, Bransoil·, and
Pauling had proposed precise models for a helical structure for a-folded protein chains and
varieties of pleated sheet models for ß-extended protein chains. Ten years later in the three-
dimensional electron density map of myoglobin, the predicted coiled form of the a-helix
was clearly seen. But the decade brought not only the first three-dimensional X-ray analysis
of protein crystals described by W. L. Bragg in his first presidential address in Madras. The
study of the diffraction patterns of helices led rapidly to the deduction of the third common
variety of peptide chain, the structure of collagen by Ramachandran, and then to the dis-
2 D. C. HODGKIN
co very of the double helix and base-pairing in deoxyribose nucleic acid by Watson and Crick.
There were major discoveries, also, in other related fields; the first complete determination
of the chemical sequence in a protein molecule, insulin, was made by Sanger in 1956. It is
difficult to remember the state of ignorance in which we existed 25 years ago ; we now have
such a wealth of solid information on which to build.
Our subject for this series of Madras symposia has extended. I take it to be essentially the
characteristics of the material components of the living cell. These components are predomi­
nantly very large molecules, proteins, nucleic acids, and carbohydrates; we have examples of
all these types in this symposium. We propose to consider not only their structure and con­
formation but their function and evolution. We have many tools, but most of our detailed
information about structure and conformation derives necessarily from the study of ordered
structures by diffraction methods, often guided and illuminated by theoretical calculations.
Some order is natural to living cells. In the nineteenth century very small haemoglobin
crystals were first observed in the drying blood of the earthworm and very small crystals
were seen in plant cells under the light microscope by botanists such as Schimper. With the
electron microscope many more have become visible; small protein crystals and other
structures of varying degrees of complexity and order, chloroplasts, cell membranes, and
storage granules such as the ^-granules of the pancreatic islet cells, many of which appear
to contain minute insulin crystals.
Through X-ray analysis we now have growing and varied detail on the structure and con­
formation of some fifty protein molecules and the beginnings of detail on carbohydrates and
nucleic acids. I will illustrate the kind of information—I mean with reference to the recent
work of my own research group on insulin. (It is nice to give here some of the latest most
accurate work on the insulin crystal structure to replace long-ago errors in my premature
report in 1967.) Within the crystal structure of 2-zinc insulin, six insulin molecules are aggre­
gated around two zinc ions to form a close-packed nearly spheroidal hexamer. Within each
molecule one can trace pieces of regular secondary structure : 2\ turns of a-helix in the
B-chain, 1 in the A-chain, and extended chains at the beginning and end of the B-chain,
giving /^-structures hydrogen-bonded either within one molecule or between two molecules
to strengthen dimer formation. When the first a-helix was seen at 2 Â resolution in myoglo-
bin, it seemed remarkable how closely the electron density fitted the dimensions predicted
by Corey, Branson, and Pauling. Now that we have more accurate, higher resolution data,
in many proteins we can see significant variations from regularity. Distortions occur regu­
larly and equivalently in the B-chain helices of the two insulin molecules seen at 1.5 Â resolu­
tion and our present state of refinement. The NH-CO bonds vary from 2.76 to 3.22 Â in
length and the CO groups lean out to make contacts with water molecules in the crystal. In
the A-chain the situation is more complicated. The conformation of the 6-11 disulphide-
bridged ring is very similar in the two molecules, but the initial A-chain helical residues are
differently related to the ring owing to rotations about the bonds 6C a-NH. This rotation
is identified easily on Ramachandran plots, where it appears as a change principally in φ of
A6. As a consequence of this rotation, the hydrogen bond pattern changes; the irregular
a-helical contacts extend rather further in molecule 1 than in molecule 2, where CO 4 con­
tacts NH 9 and CO 5, NH 10 as the^r-helix (Figs. 1 and 2). The differences and distortions
are almost certainly a consequence of different extended contacts with solvent, other parts of
BIOMOLECULAR STRUCTURE, CONFORMATION, FUNCTION, AND EVOLUTION 3
( a) Molecule I
( b) Molecule 2
FIG. 1. Residues A 1-11 of the pig insulin molecules seen in the crystal structure of 2-zinc insulin. The atomic
positions are projected along the normal to the approximate plane of the 6-11 disulphide ring, outlined in
heavy lines. They show the close similarity of the rings in the two molecules and the different orientations of
the 1-5 helices. Close, possibly H-bonded, contacts are dotted.
the molecule, and other molecules. In other insulin crystals, particularly those of the most
primitive insulin yet studied, hagfish insulin, the differences between the A-chain in the two
molecules in the dimer vanish; the dimer is symmetrical. And the same is true for cubic pig
insulin crystals formed at pH 9—though here the analysis is still at rather limited resolution
for certainty. In 4-zinc insulin, on the other hand, where the crystals form in the presence
of 1 normal sodium chloride solution, there are further changes in the conformation of one
molecule of the insulin dimer compared with the other—the initial extended residues of one
of the B-chains coil up in a helical form and the A-chain moves a little in position relative to
2
2 D. C. HÔDGKIN
Ψ
II II
ο·
10
•
1 1 T r i i
y'° 6
.2
° 5° 2
•7
8' 3i
.9
^
° Molecule I
• Molecule 2
FIG. 2. Conformational map of residues 1-11 of the insulin A-chains (after Ramachandran).
the terminal extended chains. New studies in Madras on peptide chains found in proteins
are throwing light on the many factors that modify conformations and induce transforma­
tions such as these.
In the other biopolymer systems we have to consider, there is not yet the volume of ob­
served large molecules in crystals on which we can base our evaluation of conformation vari­
ations. These would be of particular importance in relation to the nucleic acid structures,
where there have been new theoretical studies on possible alternative methods of chain fold­
ing and kinking. In the very remarkable crystals of tRNA one can see regions of double-
helical character, rather short, but providing some support for the structure of the DNA
double helix; the chains bend and turn to permit further more complex interactions between
the bases. In the tobacco mosaic virus itself it is now possible to trace the likely conformation
of single strands of DNA. We may hope that these will become clearer in the many more
regular crystalline viruses, bushy stunt, southern bean mosaic, and the satellite virus, now
under detailed investigation.
BIÖMOLECULAR STRUCTURÉ, CONFORMATION, FUNCTION, AND EVOLUTION 5
The way in which the biomolecules interact with one another and exercise their natural
functions lies through the relative movements of their constituent parts. Many of these
movements have been traced in detail in systems such as that of the haemoglobins or of the
enzymes, lysozyme, and chymotrypsin, where the reaction of the molecules with oxygen or
with their substrates have been followed in the crystalline state. With other protein mole­
cules their possible functions and possible mode of action can be partly inferred from their
arrangement in the crystal as new varieties of large molecules are explored. I think, for
example, of prealbumin, where the particular form of protein chain folding suggests a pos­
sible method of interaction between the protein chain and the double helix of DNA; or the
protein obtained from a light harvesting bacterium where seven bacteriochlorophyl mole­
cules are enclosed within a sheath of ^-folded peptide chains, a remarkable system, which
would surely have been of great interest to Sir C. V. Raman.
The course of the reactions by which insulin exercises its function is still obscure. Present
evidence suggests that to see the molecule in action it is necessary to isolate first the insulin
receptor protein from the membranes where it exists (this has been done but in very small
quantities, 1 ppm), and then to study the receptor-insulin complex in crystals. This complex
is likely to provide information on only a small part of a chain of events. There are, however,
a number of clues at least to the parts of the insulin molecule important to its action, both
from the study of insulins of different sequences derived by evolution and by the modification
of the molecule by reaction, degradation, and chemical synthesis. Among the 51-52 residues
which make up the insulin molecules found in different species, 20 have so far remained
invariant throughout evolution and a few others are highly conservative. About half of
these appear to be intimately concerned with maintaining the general form of the molecule—
the cystine and glycine residues, for example, and most of the invariant leucine residues
closely in contact in the heart of the molecule. Degradation and semi-synthesis has proved,
in the hands of the biochemists at Shanghai and Aachen, that despenta and deshexapeptide
insulin, which have lost the end residues of the B-chain, are biologically active and do not
dimerize; desheptapeptide insulin, lacking B24, is inactive. So it seems at least highly prob­
able that insulin is active as the monomer, that interaction with the receptor protein does
not require the capacity to form the extended ß-sheet of the dimer, but that at least B24
phenylalanine is involved in the process. B12, another non-polar invariant residue, is sugges­
tively near in the dimer interface; it may be important both in maintaining the insulin
structure and its function. Experiments of Cuatracasas have shown that its substitution by
a hydrophilic residue wholly inhibits the formation of the B-chain helix.
So all of the subjects of this symposium are intimately linked, the structure and confor­
mation, the function, and the evolution of biomolecules. The tools we have to study them can
already provide us with tremendous information if we use them still more extensively and
intensively. And there are new tools to come if we follow Professor Ochoa towards the
complete understanding and control of the processes by which biomolecules are made in
living cells. Again, if we look at the immensity of nature we have only so far touched a small
fraction of the problems before us—we are ready for a new take off.
2*
1.1. Structure and Conformation
MULTI-DOMAIN STRUCTURE
OF THE DIMERIC LECTIN
WHEAT GERM AGGLUTININ
C. S. WRIGHT
Department of Biochemical Sciences, Frick Chemical Laboratory,
Princeton University, Princeton, New Jersey 08540 USA
Biochemical Properties
Wheat germ agglutinin (WGA) is a member of a class of proteins, referred to as lectins,
which are now appreciated to be widespread in nature. Although differing in molecular struc­
ture and origin, these proteins share one property. They possess on their surface multicom-
bining sites for carbohydrate residues and therefore are able to attach themselves to cell
surface membranes in a highly specific manner [1]. This property enables lectins to aggluti­
nate many different types of animal cells and bacteria. Among these are malignant cells or
virally transformed cells which are more readily agglutinated than their normal counterpart
by most lectins including WGA [2]. This ability to differentiate between normal and trans­
formed cells is a property which has made great impact as a tool for studying differences in
surface structure among different cell types.
In addition to agglutinating cells, many lectins have been observed to stimulate cell divi­
sion in lymphocytes [3]. In the case of WGA, formerly thought to be nonmitogenic, recent
studies show that it does possess weak mitogenic activity [4].
Although it is not clear of what importance these lectins are in the organism from which
they are isolated, evidence points more and more toward a role in recognition processes [5].
For example, it has been shown that WGA inhibits the growth of plant fungus (Trichoderma
viride) by binding to the cell wall of hyphal tips which contain chitin, a specific inhibitor of
agglutination for WGA [6]. This study suggests a natural role for WGA, namely to protect
wheat against chitin-containing pathogens during seed development. Lectins derived from
legumes may play a direct part in the symbiotic relationship between bacterium and legume
seed [5].
Dimeric WGA (mol. wt. 35,000) is isolated as a mixture of three or four molecular species
called isolectins. These possess nearly identical amino acid compositions [7], but differ with
respect to their electrophoretic mobility at acid pH (4.0-5.0). One out of every five residues
of the polypeptide chain is a half-cystine. The heavily disulfide cross-linked protein molecule
9