NMR in Molecular Biology

685 Pages·1981·11.082 MB·English

by  Oleg Jardetzky, G. C. K. Roberts, Bernard Horecker, Nathan O. Kaplan and Julius Marmur (Auth.)

NMR
in Molecular
Biology
OLEG JARDETZKY
Stanford Magnetic Resonance Laboratory
Stanford University
Stanford, California
c.
G. K. ROBERTS
National Institute for Medical Research
The Ridgeway, MillHill
London, England
1981
ACADEMIC PRESS
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Library of Congress Cataloging in Publication Data
Jardetzky, Oleg.
NMR in molecular biology.
Bibliography: p.
Includes index.
1. Nuclear magnetic resonance spectroscopy.
2. Molecular biology—Technique. 3. Biological
chemistry—Technique. I. Roberts, G. C. K.
(Gordon Carl Kenmure) II. Title.
QP519.9.N83J37 547.19'285 80-2337
ISBN 0-12-380580-5 AACR2
PRINTED IN THE UNITED STATES OF AMERICA
81 82 83 84 9 8 7 6 5 4 3 2 1
PREFACE
This book is intended for the student or practicing scientist wishing to
gain a critical understanding of the applications of nuclear magnetic reso-
nance (NMR) to a wide range of problems in molecular biology. It is
written from the point of view of a biologist, whose principal question is
4 4 What do we know about molecular biology today that we would not have
known had magnetic resonance never been invented?" Our aim is there-
fore twofold. On one hand, it is to introduce the reader to the basic
concepts and principles of the method that are essential to a critical evalu-
ation of experimental data. On the other, it is to acquaint him in some
detail with those prototype experiments in which a definite, biologically
relevant answer has been obtained. For the most part the review of the
literature has been completed in December 1980, although some work
published in 1981 is also included.
The number and range of applications of NMR that are of some biolog-
ical interest are now such that any attempt at a comprehensive coverage is
out of the question. Faced with the necessity of a choice, we have placed
emphasis on experimental design and rules of evidence that can lead to
clear solutions of biological problems, in preference to the large body of
information that is primarily of spectroscopic interest, though this infor-
mation is valuable in its own right. We hope that this critical assessment of
the accomplishments of NMR in molecular biology will also prove useful
to our colleagues in the field.
xi
ACKNOWLEDGMENTS
Our greatest debt is to our teachers, students, and co-workers, who
through the years have provided inspiration, criticism, and enlightenment
for our own work. In the preparation of this book we have greatly ben-
efited from the advice of Dr. Berry Birdsall, Sir Arnold Burgen FRS,
Professor Mildred Cohn, Dr. James Feeney, Professor Harden McCon-
nell, and Dr. David Wemmer, who have carefully read and made critical
comments on various portions of the manuscript. We are also very grate-
ful to many of our colleagues, who have sent us manuscripts prior to
publication, alerted us to important developments, and given us permis-
sion to reproduce illustrations from their work. We are especially indebted
to Alice Walker for the compilation of the master bibliography from which
the selection of material included in the book was taken, to Drs. Anthony
and Barbara Ribeiro and Norma Wade-Jardetzky for their help in all
aspects of manuscript preparation, to Irene Godstone for typing, and to
Barbara Summey for the majority of the original drawings. Last, but not
least, we are grateful to our families whose care and support greatly eased
the burdens of our task.
We would like to thank the American Chemical Society for their gen-
erous permission to reprint the following figures and tables for which
they hold the copyright.
Figures III-5, III-7, III-8, IV-4, V-5, V-8, V-9, VI-5, VI-10, VII-5, VII-6,
VIII-5, VIII-10, VIII-21, IX-2, IX-9, X-4, X-6, XI-1, XI-3A, XI-3B,XI-10,
XII-11,XIII-1, XIII-2, XIII-4, XIII-5, XIII-6, XIII-7, XIII-11, XIII-12,
XIII-13, XIIM4, XIII-15, XIII-16, XIV-10, XIV-14, XIV-15, XIV-17.
Tables IX-4, XII-2, XII-4, XIII-1, XIII-7.
We also acknowledge the Annual Review of Physical Chemistry for
permission to reprint Figure 11-24; © 1978 by Annual Reviews Inc.
xiii
Chapter I
IINNTTRROODDUUCTCIOTNIO N
Nuclear magnetic resonance (NMR) is a branch of spectroscopy based on
the fact that atomic nuclei oriented by a strong magnetic field absorb radia-
tion at characteristic frequencies. The parameters that can be measured on
the resulting spectral lines (line positions, intensities, line widths, multiplic-
ities, and transients in time-dependent experiments) can be interpreted in
terms of molecular structure, conformation, molecular motion, and other rate
processes. The usefulness of NMR to the chemist and the biologist stems in
large measure from the finding that nuclei of the same element in different
chemical environments give rise to distinct—"chemically shifted"—spectral
lines. This makes it possible to observe specific atoms even in complex
structures, in solution as well as in the solid state.
The history of NMR, including that of its applications to biology, still
barely spans a generation of scientists. The existence of the phenomenon was
predicted by Gorter (1936) and experimental detection was first achieved in
1945 by Felix Bloch (1946) at Stanford and Edward M. Purcell (Purcell,
Torrey, and Pound, 1946) at Harvard, almost simultaneously with the closely
related observation of electron paramagnetic resonance (EPR) by Zavoisky
(1945) in Kazan in 1944. The first theoretical formulations of the method were
given by Bloch, Hansen, and Packard (1946a,b) and Bloembergen, Purcell,
and Pound (1948). The development of techniques and theory has steadily
advanced since, and all essential formulations are summarized in the classic
treatise by Abragam (1961). The key discovery of the chemical shift, which
gave rise to all high-resolution spectroscopy anticipated by the theoretical for-
mulations of Lamb (1941) and Ramsey (1950), was reported by Knight (1949)
for metals and by Arnold, Dharmatti, and Packard (1951) for the three groups
of protons in ethanol. The discovery of the fine structure (coupling) in NMR
spectra and its theoretical explanation resulted from the work of Hahn and
Maxwell (1951) and Gutowsky, McCall, and Slichter (1951). The interpreta-
tion of coupling constants in terms of conformations was given by McConnell
(1956a) and Karplus (1959). Modern NMR technology owes much to the
research teams of Varian Associates and Oxford Instruments for the develop-
ment of high-resolution superconducting magnets and to R. R. Ernst for the
1
2 I: INTRODUCTION
concept of Fourier transform (FT) spectroscopy in its various forms (Ernst
and Anderson, 1966; Ernst, 1975). The list of other important contributions
to the method is long. Insofar as they bear on the specific topics discussed in
this book they are mentioned in the appropriate chapters.
Application of NMR to problems in biology began within a decade after
the emergence of the method. A detailed account of the history of the subject
is not within the scope of this book, but a few milestones are worth noting.
In 1954 Jacobson, Anderson, and Arnold, following earlier work on moisture
analysis by Shaw and Elksen(1950,1953), attempted to measure the hydration
of DNA by observing the broadening and decrease in the area of the water
proton signal. Although subsequent studies have shown that the results of
such measurements are not clearly interpretable, the report sparked an
interest in the method as a tool for biological research. In 1956 Jardetzky
and Wertz used the quadrupolar broadening (see Chapter II) of the 23Na
resonance to study ion binding in solutions of chelating agents and proteins,
red blood cells, and whole blood. In the same year Odeblad, Bhar, and
Lindstrom (1956) attempted to estimate the rate of water exchange in human
red blood cells.
The first successful direct observation of a biological macromolecule by
NMR was made by Saunders, Wishnia, and Kirkwood (1957), who reported
a Hl spectrum of ribonuclease at 40 MHz. Jardetzky and Jardetzky (1957)
showed that the spectrum accurately reflected the amino acid composition
of the protein, but neither the resolution nor the sensitivity at that time was
adequate to derive much additional information. Also in 1957, Davidson and
Gold first pointed out that paramagnetic ions bound to macromolecules
could be studied by their effects on water relaxation and showed that Fe 3 +
in hemoglobin was effectively inaccessible to solvent. The stereochemistry of
enzymatic reactions as reflected in the NMR spectra of the products was
first examined by Farrar, Gutowsky, Alberty, and Miller (fumarase, 1957)
and Krasna (aspartase, 1958). In 1959 Cohn reported the first 31P spectra
of ADP and ATP and inferred that Mg 2 + bound preferentially to the a and
j8, rather than the y-phosphate.
The majority of the early NMR applications were concerned with small
molecules of biological interest, such as amino acids (C. D. Jardetzky and
Jardetzky, 1958), nucleosides and nucleotides (O. Jardetzky and Jardetzky,
1958,1960), steroids (Shoolery and Rogers, 1958), and porphyrins (Becker and
Bradley, 1959). At the same time, key observations were being made on
macromolecular systems, providing clues to previously unobserved pheno-
mena and qualitative answers to specific questions, and defining problems
amenable to further study. Solvent relaxation enhancement by paramagnetic
ions bound to macromolecules was observed by Eisinger, Shulman, and
Blumberg for DNA (1961) and applied by Cohn and Leigh (1962) to study
INTRODUCTION 3
enzyme-substrate complexes. The usefulness of relaxation measurements to
study ligand binding to macromolecules was demonstrated by Jardetzky and
Fischer (1961). Paramagnetic shifts in heme proteins were discovered by
Kowalsky (1962, 1965). The existence of extensive internal motions in poly-
peptides, polynucleotides, and even in folded proteins became rapidly
apparent to NMR spectroscopists (Saunders and Wishnia, 1958; Bovey,
Tiers, and Filipovitch 1959; Jardetzky and Jardetzky, 1962; Jardetzky, 1964).
The fluidity of phospholipid bi- and multilayers was first clearly shown by
Chapman and Salsbury (1966) using NMR.
The major obstacles to rapid progress were the instrumental limitations of
sensitivity and resolution, which were overcome as follows:
1. The introduction of signal averaging improved the signal/noise ratio
(Klein and Barton, 1963; Jardetzky, Wade, and Fischer, 1963b). The original
method was superseded by 1970 with the advent of FT spectrometers, but
permitted a number of important studies in the meantime.
2. The manufacture of superconducting magnets permitted observations
at higher frequencies and hence higher resolution. Following the initial studies
of proteins by McDonald and Phillips (1967a) on the Varian 220 MHz
instrument, spectrometers of ever higher resolution have been constructed:
270 MHz (Oxford, 1971), 360 MHz (Stanford; Bruker 1974), 470 MHz
(Oxford, 1978), and 600 MHz (Carnegie-Mellon, 1979).
3. The introduction of selective isotopic labeling provided a method for
the simplification of macromolecular spectra (Jardetzky, 1965; Markley,
Putter, and Jardetzky 1968; Crespi, Rosenberg, and Katz 1968). The devel-
opment of mathematical data processing techniques (Ernst, 1966) allowing
the display of narrow lines in macromolecular spectra has also simplified
spectral analysis in many cases.
At present the state of NMR technology is more than adequate for a large
number of accurate measurements. The most serious methodological problem
solved only in special cases remains the assignment of individual lines in a
complex spectrum to specific chemical groups.
In the 1960s the majority of the reports reflecting the advance of biological
applications of NMR came from relatively few laboratories—M. Cohn in
Philadelphia (since 1959), O. Jardetzky, initially at Harvard and later at Merck
(since 1959), R. G. Shulman at Bell Laboratories (since 1961), W. D. Phillips
at Du Pont (since 1963), S. I. Chan at the California Institute of Technology
(since 1964), and E. M. Bradbury at Portsmouth (since 1965).
In the early 1970s many additional groups have entered the field and the
literature has grown correspondingly. Up to 1969 the number of contributions
dealing with the applications of NMR to biological problems was less than
300. At present the count is nearing 6000. In 1980 more than 900 reports
appeared and the figure for 1981 may exceed 1000. It is clearly no longer
4 I: INTRODUCTION
possible to give a complete account of the literature in a single volume. Our
emphasis is therefore on the principles of the method, the basis of inter-
pretation, and on selected prototype experiments.
To assess the role of any particular method in a field of research we must
know (1) what kinds of problems it allows us to solve, (2) how conclusive the
evidence is that can be derived from it, and (3) how it compares to other
methods that can be brought to bear on the same problems. Therefore, the
aim of this book is to discuss the contributions of NMR to molecular biology
in the light of these questions.
As in other branches of spectroscopy, the basic observable in NMR is a
spectral line, a plot of the intensity of absorption vs. the frequency of radia-
tion. The measurable parameters of spectral lines obtained under steady state
conditions are
1. The position of their center on a frequency scale (referred to a standard
line and called the chemical shift).
2. For split lines the spacings in the multiplet (called spin coupling to
reflect the origin of the splitting). These contain information on both the
electronic structure and the conformation of the molecule.
3. The intensity, properly measured as the area under the line in a single
resonance experiment. This reflects strictly the number of nuclei in each
environment.
4. The line width, usually measured at half-height, which contains informa-
tion on rate processes, including the rates of molecular motion.
In addition, an unusually wide range of non-steady-state experiments can be
carried out using NMR, and kinetic information can be obtained from the
rates of appearance and decay of spectral lines under transient conditions.
The basic features of an NMR spectrum (*H at 360 MHz) are shown in
Fig. 1-1: for an amino acid (tyrosine), with separate multiplet peaks for oc-CH,
/?-CH, and aromatic protons. Fig. 1-2 illustrates the relative complexity of
protein spectra: (A) for a small protein fragment (N-terminal headpiece of the
/ac-repressor) MW 6,000 in random coil form, showing the superposition of
lines from different amino acids, (B) for the native headpiece, and (C) for
the intact /ac-repressor, a large protein, (MW 150,000). Comparison of
spectra A and B reveals the increasing complexity of the spectrum on
protein folding and indicates that spectrum B reflects the secondary and
tertiary as well as the primary structure. Comparison of all three tracings
illustrates the progressive broadening of lines with the formation of a folded
structure and with increasing molecular weight. This reflects in part the
increasing overlap of lines as their number increases and, in part, the slowing
of molecular motion, since generally narrow lines can be associated with rapid
motion and broad lines with slow motion. The detailed interpretation of such
at
et
bl
u
o
D
2.0 ring.
1' i nyl
1 he
1 p
1 I2,5 of the
11 ns
1 o
1I3,0 prot
1 1 (3.5) H. 2
- T'3.5 meta £-C
'- m m
1' pppp
2 5
1 1 ' ' I '4.0 ublet at 7.3.2 and 3.0
1 1 I 4.5 MHz. Doplets at
1 1 I '5.0 at 360 H. Multi
11 11 11 1 1 ' I ' I '6.0 5.5 MR spectrum of tyrosine Multiplet at 3.95 ppm a-C
1 1 11 11 11 1 ' I I ' ' II3.0 7.5 7.0 6.5 Fig. 1-1 High resolution *H N6.9 ppm ortho (2.6) ring protons.
6 I: INTRODUCTION
A
8 7 6 " 5 ' 4 3 ^ ' 2 ' i PPM
Fig. 1-2 360 MHz 1H spectra of (A) Thermally denatured (random coil) /aorepressor head-
piece, MW 6000,75°C, (B) Native /aorepressor headpiece (22°C), (C) Native whole lac- repressor
(M W150,000). The large resonance from the residual protons in the solvent (2H 0), at ~ 4.7 ppm,
2
has been omitted in (B) and (C) and suppressed by pre-irradiation in (A).
observations in terms of molecular structure and dynamics is not always an
easy task, as will become apparent in subsequent chapters. Nevertheless, the
fact that NMR is the only method, aside from X-ray and neutron diffraction,
that permits the simultaneous observation of individual atoms in complex
molecules—and furthermore permits this in solution, as well as in the solid
state—gives the method a singular importance in the study of molecular
events. It also makes the effort necessary to define the rules of interpretation
and their limits, as well as the effort of perfecting the techniques, singularly
worthwhile.
Anticipating the detailed discussion we can briefly summarize here the
salient conclusions concerning the usefulness of NMR in molecular biology
that can be drawn from the developments of the last quarter century. A large
fraction of the biological NMR literature—perhaps 70%—deals with ques-
tions of molecular structure and conformation. Nearly all types of constituents
of living systems—amino acids, nucleosides, steroids, sugars, oligo- and poly-
peptides, oligo- and polynucleotides, proteins, tRNA, mRNA, DNA, phos-