Table Of ContentMOLECULAR BIOLOGY
An International Series of Monographs and Textbooks
Editors: BERNARD HORECKER, NATHAN O. KAPLAN, JULIUS MARMUR, AND
HAROLD A. SCHERAGA
A complete list of titles in this series appears at the end of this volume.
P r o t e in F o l d i ng
CHARIS GHELIS
JEANNINE YON
Laboratoire d'Enzymologie Physiocochimique et Moleculaire
Centre National de la Recherche Scientifique
Universite de Paris-Sud
Or say, France
1982
ACADEMIC PRESS
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Gh£lis, Charis.
Protein folding.
(Molecular biology series)
Bibliography: p.
Includes index.
1. Proteins. 2. Biochemorphology. 3. Molecular
structure. I· Yon, Jeannine, Date. II. Title.
III. Series.
QP551.G48 547.7*5 82-6830
ISBN 0-12-281520-3 AACR2
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Preface
The objective of this book is to reassemble the most important informa
tion in the field of protein folding and to understand, from the amount of
experimental data now available, the main principles that govern forma
tion of the three-dimensional structure of a protein from a nascent poly
peptide chain, and how the functional properties appear.
The interest in this field has increased considerably over the years and
information has reached a level that may allow certain generalizations. A
corollary difficulty is encountered: New pieces of information are appear
ing continually, giving us the uncomfortable impression of progressing on
shifting sand, but also the exciting feeling of moving in an important area
of interest.
The problem of protein folding has been questioned for a long time.
Before World War II Anson and Mirsky (1934, 1935), having shown the
reversibility of the denaturation process in the case of hemoglobin, eluci
dated some of its characteristics. These first studies were followed by
many works on protein denaturation. Progressively, the increased knowl
edge on protein structure allowed more precise and more significant
work. One of the most important and stimulating contributions of modern
biochemistry is undoubtedly the elucidation of the three-dimensional
structure of biological macro molecules. Certainly, the increasing number
of protein structures obtained by high resolution X-ray crystallography
represents important information to understand the mechanisms of pro
tein folding and the genesis of a biological function.
The book is divided into three parts, which follow an introduc
tory chapter where the main problems of protein folding are pre
sented and discussed at the cellular level. The conformation of native
globular proteins is described in Part I. Definitions and rules of nomencla
ture are given in Chapter 2, including characteristics of structural organi-
xi
xii Preface
zation of globular proteins deduced from X-ray crystallographic data.
Folding mechanisms were tentatively deduced from the observation of
invariants in the architecture of folded proteins. Energetics of protein
structure indicating principles of thermodynamic stability of the native
structure are found in Chapter 3. In Chapter 4, theoretical computation
studies of protein folding, structure prediction, and folding simulation are
rapidly reviewed.
Part II is a presentation of various experimental approaches. Revers
ibility of the unfolding-folding process is discussed in Chapter 5. Chapters
6 and 7 contain equilibrium studies and kinetic studies, respectively. The
different ways used to detect and characterize intermediates in protein
folding are reported in Chapter 8, omitting immunochemical approaches
which are analyzed in a separate chapter (Chapter 9). Folding and assem
bly of smaller units into a protein are examined in Chapter 10, and Chap
ter 11 treats problems specific to oligomeric proteins. Some generaliza
tions are made in the last chapter which is certainly not an exhaustive
review, but contains information that seemed to us to be most significant.
This book was written for research scientists to contain in a unique
volume information scattered throughout the literature. It was planned as
an advanced survey on protein folding, not only for specialists but for
biochemists in general. It was also written for students of biochemistry
and biology, to present more advanced knowledge in protein structure
and folding and to propose some basis for discussion.
And last, as said in the Tao Te King, "The man who speaks does not
know, the man who knows does not speak"; we can add, "The man who
writes, tries to understand." We welcome comments from the readers to
know if this goal has been reached.
Char is Ghelis
Jeannine Yon
Ac knowle dgme η t s
It is a pleasure to acknowledge Dr. George Nemethy for his help and
advice during preparation of this book and for carefully reading and criti
cizing the complete manuscript. We sincerely thank Drs. S. Bernhard, E.
Bricas, J-R Garel, M. Goldberg, G. Herve, J. Janin, M. Karplus, J-C.
Patte, D. Perahia, M. Rossmann, and A. Shechter for reading and discus
sing separate chapters or for stimulating discussions during preparation of
the manuscript. We also thank for very helpful suggestions and discus
sions Drs. R. L. Baldwin and M. Karplus.
We are indebted to Drs. N. Kellershohn and D. Verge, and also to M.
Desmadril, M. Laurent, and M. Tempete-Gaillourdet for their help in
carefully reading particular chapters. We want to acknowledge all the
authors who sent us original figures, documents and preprints of their
manuscripts.
We are indebted to Mrs. Barthelemy and Mrs. Lavorel for typing and
retyping the manuscript, Mrs. Clais for providing documentation and for
their help during the material preparation of the manuscript.
xiii
Introduction: Cellular
Environment
and Significance
of Folding Processes
Dans le processus de structuration d'une proteine globulaire, on peut
voir a la fois Fimage microscopique et la source du developpement
epigenetique autonome de l'organisme lui-meme.
J. Monod, "Le Hasard et La Necessite"
1
1
Folding and Processing:
The Last Events in
Protein Biosynthesis
1.1 EVOLUTION OF THE MAIN CONCEPTS
OF PROTEIN FOLDING
With reference to the tremendous progress in molecular biology this last
decade, it might seem that all mechanisms by which genetic information is
transmitted have been elucidated. It might also appear that all events involved
in the formation of a biologically active protein are well known. In fact,
only duplication, transcription, and translation processes which contribute
to the formation of a linear polypeptide chain with a known sequence of
amino acids are rather well understood. But posttranslational (or cotrans-
lational) events, i.e., the ones that generate globular structure and biological
function, are not yet fully elucidated. It is possible to distinguish two different
kinds of events:
(a) Covalent processes including limited proteolysis and chemical
modifications such as glycosylation, phosphorylation, hydroxylation,
lipidation, methylation, ADP-ribosylation, and so on;
(b) Noncovalent processes such as polypeptide chain folding and
subunit assembly.
Figure 1.1 summarizes the different events possibly involved in producing
an active protein. They are of importance in generating functional prop
erties of proteins, recognition of specific ligands, catalysis, active transport,
3
4 Introduction
REPLICATION
TRANSCRIPTION
TRANSLATION
Fig. 1.1. Posttranslational and cotranslational processes.
and hormonal action. Furthermore, the biological function of a protein
appears in specific loci in cells and higher organisms, thus allowing very
precise and subtle regulations which insure the preservation and function
of living organisms.
This chapter is an introduction to the main problems of protein folding.
The term protein folding includes all the events occurring from translation
to formation of the functional structure of a protein molecule. These prob
lems must be considered at the cellular level taking into account the particular
environment, the effect of compartmentalization, (Palade, 1975), the sequence
1. Folding and Processing 5
of the different processes, with reference to their biological meaning, and
the regulatory processes by which functional structure takes place. However,
because of the difficulties encountered when attempting to study protein
folding accurately under cellular conditions, it is necessary to shift from the
cellular to the molecular aspect of these problems. Until now, mechanisms
of protein folding were studied on molecules isolated from their biological
environment. A rapid historical survey of the evolution of the main concepts
is presented.
The processes involved in the formation of the "native structure" of
proteins were questioned for a long time before the mechanism of protein
biosynthesis was known, and before the three dimensional structure and
even the primary structure of proteins were determined. With increasing
knowledge in both fields, protein structure and protein biosynthesis,
questions concerning protein folding can be asked more precisely. More
significant experimental approaches of the problem can now be attempted.
Considering the frequency and importance of the papers which appeared in
the literature on this topic, one notes three maxima with a periodicity of
about 20 years, approximately located around 1935,1955, and 1975. Indeed,
with a certain lag, each of these periods corresponds to a specific discovery.
The first significant works appeared around 1935 with the analysis of the
denaturation process by Wu (1931), with the experiments of Anson and
Mirsky (1934a,b 1935), and those of Northrop (1932). These first works
clearly outlined the correlation between the biological activity of a protein
and the so called "native structure," defined not only by the activity but also
by some physical characteristics (solubility, ability to crystallize, hydro-
dynamic properties), and later by chemical properties. In fact, very little
was known at this time about proteins. This period was principally marked
by success in protein crystallization; one has to remember that such an
event ended the old arguments of vitalists concerning the mysterious nature
of enzymes. The decrease of solubility and the loss of activity were the first
parameters used as conformational probes at the beginning of the work on
protein denaturation. Thus, the first studies on denaturation remained only
qualitative approaches. However, at this period the reversibility of the
denaturation-renaturation process was clearly shown but only for few
proteins, hemoglobin, chymotrypsinogen, trypsinogen. This represents the
first important fact concerning the understanding of protein folding.
In the next 20 years, tremendous progress was made in the study of the
physical chemistry of macromolecules and the application of new meth
odology to the study of protein conformation was extensively developed. A
better resolution of optical techniques (UV spectroscopy, spectropolari-
metry, etc.) allowed their utilization in following conformational changes of
