Table Of ContentBiological Magnetic Resonance
Volume 20
Protein NMR for
the Millennium
A Continuation Order Plan is available for this series. A continuation order will bring delivery of each
new volume immediately upon publication. Volumes are billed only upon actual shipment. For further
information please contact the publisher.
Biological Magnetic Resonance
Volume 20
Protein NMR for
the Millennium
Edited by
N. Rama Krishna
University of Alabama at Birmingham
Birmingham, Alabama
and
Lawrence J. Berliner
University of Denver
Denver, Colorado
KLUWER ACADEMIC PUBLISHERS
NEW YORK,BOSTON, DORDRECHT, LONDON, MOSCOW
eBookISBN: 0-306-47936-2
Print ISBN: 0-306-47448-4
©2002 Kluwer Academic Publishers
NewYork, Boston, Dordrecht, London, Moscow
Print ©2003 Kluwer Academic/Plenum Publishers
New York
All rights reserved
No part of this eBook maybe reproducedor transmitted inanyform or byanymeans,electronic,
mechanical, recording, or otherwise, without written consent from the Publisher
Created in the United States of America
Visit Kluwer Online at: http://kluweronline.com
and Kluwer's eBookstore at: http://ebooks.kluweronline.com
Contributors
Charles R. Babu Johnson Research Foundation and Department of
Biochemistry & Biophysics, University of Pennsylvania, Philadelphia,
PA 19104-6059
John L. Battiste Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115
Thorsten Biet Institute of Chemistry, Medical University of Luebeck,
Luebeck, Germany
Ulrich K. Blaschke Laboratories of Synthetic Protein Chemistry,
The Rockefeller University, 1230 York Avenue, New York, NY 10021
Florence Cordier Department of Structural Biology, Biozentrum,
University of Basel, 4056 Basel, Switzerland
David Cowburn Laboratory of Physical Biochemistry, The Rockefeller
University, 1230 York Avenue, New York, NY 10021, and New York
Structural Biology Center, c/o Box 163, 1230 York Avenue, New York, NY
10021
Andrew J. Dingley Institute of Physical Biology, Heinrich-Heine-
Universität, 40225 Düsseldorf, Germany, and Institute of Structural Biology,
IBI-2, Forschungszentrum Jülich, 52425 Jülich, Germany
Peter F. Flynn Johnson Research Foundation and Department of
Biochemistry & Biophysics, University of Pennsylvania, Philadelphia,
PA 19104-6059
David Fushman Dept. of Chemistry and Biochemistry, University of
Maryland, College Park, MD 20742
v
vi CONTRIBUTORS
ChristianGriesinger Max Planck Institute of Biophysical Chemistry,
Group 030, Am Fassberg 11, 37077 Göttingen, Germany
Angela M. Gronenborn Laboratory of Chemical Physics, NIDDK, NIH,
Bethesda, MD
John D. Gross Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115
Stephan Grzesiek Department of Structural Biology, Biozentrum,
University of Basel, 4056 Basel, Switzerland
Lars Herfurth Institute of Chemistry, Medical University of Luebeck,
Luebeck, Germany
Ann McDermott Columbia University, Department of Chemistry, New
York, NY
Jens Meiler Max Planck Institute of Biophysical Chemistry, Group 030,
Am Fassberg 11, 37077 Göttingen, Germany
Mark J. Milton Johnson Research Foundation and Department of
Biochemistry & Biophysics, University of Pennsylvania, Philadelphia,
PA 19104-6059
Tom W. Muir Laboratories of Synthetic Protein Chemistry, The
Rockefeller University, 1230 York Avenue, New York, NY 10021
Jennifer J. Ottesen Laboratory of Synthetic Protein Chemistry, The
RockefellerUniversity, 1230 York Avenue, New York, NY 10021
Konstantin V. Pervushin Laboratorium für Physikalische Chemie,
Eidgenössische Technische Hochschule CH-8092 Zürich, Switzerland
Thomas Peters Institute of Chemistry, Medical University of Luebeck,
Luebeck, Germany
Wolfgang Peti Max Planck Institute of Biophysical Chemistry, Group
030, Am Fassberg 11, 37077 Göttingen, Germany
Gerhard Wagner Department of Biological Chemistry and Molecular
Pharmacology, Harvard Medical School, Boston, MA 02115
A. Joshua Wand Johnson Research Foundation and Department of
Biochemistry & Biophysics, University of Pennsylvania, Philadelphia,
PA 19104-6059
Preface
Volume 20 is the third in a special topic series devoted to the latest
developments in protein NMR under the Biological Magnetic Resonance
series. Previous volumes in this series included 16 (Modern Techniques in
Protein NMR) and 17 (Structure Computation and Dynamics in Protein
NMR). Once again, we are indeed honored to have brought together in
Volume 20 some of the world’s foremost experts who have provided broad
leadership in advancing the protein NMR field. This volume is broadly
divided into three major sections: (I) Toward Larger Proteins in Solution and
Solid State; (II) Structure Refinement; and (III) NMR Methods for Screening
Bioactive Ligands.
The first section is entirely devoted to recent advances in further
pushing the limits of protein size amenable to solution and solid state NMR
methods. The opening chapter of Volume 20 deals with Transverse
Relaxation Optimized Spectroscopy (TROSY) by Konstantin Pervushin.
TROSY is one of the most significant developments that has extended the
limit of molecular size that can be studied by solution NMR to in excess of
100 kDa. It exploits the line-narrowing resulting from cross-correlation
effects between chemical shift anisotropy relaxation at high magnetic fields
and the dipolar relaxation. Konstantin Pervushin describes the theory of
TROSY, the pulse sequences, and some applications of this technique. The
next two chapters deal with advancements in the study of multidomain
proteins. Chapter 2 by David Cowburn, Tom Muir and their collaborators
deals with strategies for segmental isotopic labeling in multidomain proteins.
In Chapter 3, Fushman and Cowburn describe techniques based on relaxation
to characterize inter-domain orientations in solution state. They also give an
example where a difference in the interdomain orientations has been
observed between solution and crystal structures. In Chapter 4 Gerhard
Wagner and his associates describe the site-directed spin labeling approach
to study the global folds of large proteins. In Chapter 5 Ann McDermott
summarizes the recent advances in the study of uniformly isotopically
labeled proteins by solid state NMR methods. A novel approach for
vii
viii PREFACE
achieving narrowing of NMR lines and, hence, extend the size limit of
proteins, has been described by Joshua Wand and his collaborators in
Chapter 6. Their approach utilizes the combined use of low viscosity solvents
and encapsulation of proteins in reverse micelles; an interesting extension of
this method is to the study of membrane proteins.
The section on Structure Refinement starts with a chapter by Christian
Griesinger and his collaborators on the use of angular restraints derived from
residual dipolar couplings in proteins and oligosaccharides weakly aligned in
high magnetic fields. Griesinger also discusses the impact of dipolar
couplings in the area of structural genomics. This is followed by Chapter 8,
wherein Angela Gronenborn discusses methods for protein structure
refinement based on residual dipolar couplings. Next, Stephan Grzesiek and
his associates describe developments involving the detection of scalar
couplings across hydrogen bonds in proteins and nucleic acids, and the use of
these couplings as structural constraints. The third section deals with a
review by Thomas Peters and his associates on the various NMR techniques
for the screening of the binding of ligands to target proteins and the further
characterization of bioactive ligands.
We are extremely proud of this compilation of excellent contributions
from leading investigators describing significant recent advances in the
biomolecular NMR field. Because the new publisher of this series has
required the authors to prepare their chapters as camera-ready manuscripts
using a template supplied by the publisher, there are some inevitable stylistic
variations from chapter to chapter. The editors have tried their best to correct
these variations, and they take full responsibility for any irregularities that
might still remain in the finished volume. As always, the editors welcome
suggestions for topics to be covered in future volumes.
N. Rama Krishna
Lawrence J. Berliner
Contents
Section I. Toward Larger Proteins in Solution and Solid State
Chapter 1
Transverse Relaxation Optimized Spectroscopy
Konstantin V. Pervushin
1. TROSY Scope 3
2. TROSY: How does it work? 5
3. Direct Heteronuclear Chemical Shift Correlations 11
4. Suppression of Conformational Exchange Line Broadening 18
5. Backbone Resonance Assignment 21
6. NOE Spectroscopy of Large Biomolecules 26
7. Other Applications 27
8. Conclusions 30
9. References 30
Chapter 2
Segmental Isotopic Labeling: Prospects for a New Tool to Study the
Structure-function Relationships in Multi-domain Proteins
Jennifer J. Ottesen, Ulrich K. Blaschke, David Cowburn,
and Tom W. Muir
1. Introduction 35
2. Protein Ligation Techniques 38
3. NMR Techniques 43
4. Conclusions 49
ix
x CONTENTS
5. References 50
Chapter 3
Characterization of Inter-Domain Orientations in Solution Using the
NMR Relaxation Approach
David Fushman and David Cowburn
1. Introduction 53
2. Theory 56
3. Practical Example 68
4. Possible Limitations of the Approach 72
5. Conclusions 75
6. References 76
Chapter 4
Global Fold Determination of Large Proteins using Site-Directed Spin
Labeling
John L. Battiste, John D. Gross, and Gerhard Wagner
1. Introduction 79
2. Sample preparation 82
3. NMR Analysis 85
4. Restraints and Molecular Modeling 91
5. Examples of Global Fold Determination 94
6. Future Directions 97
7. References 100
Chapter 5
Solid State NMR Studies of Uniformly Isotopically Enriched Proteins
Ann McDermott
1. SSNMR: A Chemically Detailed Probe of Structure and
Function 103
2. A Strategy and Toolkit for Assignment 109
3. Structures in the Near Future? 114
4. Probes of Dynamics for Solid State NMR of Uniformly
Labeled Materials 115
5. The Future: Structural Genomics, Structure–Function
Relations, Membrane Proteins and SSNMR? 116
6. References 117
