Protein NMR Spectroscopy E-Book

• Herausgeber: John Wiley & Sons
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 797

Veröffentlichungsjahr: 2011

Beschreibung

Nuclear Magnetic Resonance (NMR) spectroscopy, a physical phenomenon based upon the magnetic properties of certain atomic nuclei, has found a wide range of applications in life sciences over recent decades. This up-to-date volume covers NMR techniques and their application to proteins, with a focus on practical details. Providing newcomers to NMR with practical guidance to carry out successful experiments with proteins and analyze the resulting spectra, those familiar with the chemical applications of NMR will also find it useful in understanding the special requirements of protein NMR.

Leseprobe

Contents

Cover

Title Page

Copyright

List of Contributors

Introduction

References

Chapter 1: Sample Preparation, Data Collection and Processing

1.1 Introduction

1.2 Sample Preparation

1.3 Data Collection

1.4 Data Processing

References

Chapter 2: Isotope Labelling

2.1 Introduction

2.2 Production Methods for Isotopically Labelled Proteins

2.3 Uniform Isotope Labelling of Proteins

2.4 Selective Isotope Labelling of Proteins

2.5 Segmental Labelling

2.6 SAIL Methods

2.7 Concluding Remarks

2.8 Acknowledgements

References

Chapter 3: Resonance Assignments

3.1 Introduction

3.2 Resonance Assignment of Unlabelled Proteins

3.3 -Edited Experiments

3.4 Triple Resonance

3.5 Side-Chain Assignments

References

Chapter 4: Measurement of Structural Restraints

4.1 Introduction

4.2 NOE-Based Distance Restraints

4.3 Dihedral Restraints Derived from J-Couplings

4.4 Hydrogen Bond Restraints

4.5 Orientational Restraints

4.6 Chemical Shift Structural Restraints

4.7 Solution Scattering Restraints

Acknowledgement

References

Chapter 5: Calculation of Structures from NMR Restraints

5.1 Introduction

5.2 Historical Development

5.3 Structure Calculation Algorithms

5.4 Automated NOE Assignment

5.5 Nonclassical Approaches

5.6 Fully Automated Structure Analysis

References

Chapter 6: Paramagnetic Tools in Protein NMR

6.1 Introduction

6.2 Types of Restraints

6.3 What Metals to Use?

6.4 Paramagnetic Probes

6.5 Examples

6.6 Conclusions and Perspective

References

Chapter 7: Structural and Dynamic Information on Ligand Binding

7.1 Introduction

7.2 Fundamentals of Exchange Effects on NMR Spectra

7.3 Measurement of Equilibrium and Rate Constants

7.4 Detecting Binding – NMR Screening

7.5 Mechanistic Information

7.6 Structural Information

References

Chapter 8: Macromolecular Complexes

8.1 Introduction

8.2 Spectral Simplification through Differential Isotope Labelling

8.3 Basic NMR Characterisation of Complexes

8.4 3D Structure Determination of Macromolecular Protein–Ligand Complexes

8.5 Literature Examples

References

Chapter 9: Studying Partially Folded and Intrinsically Disordered Proteins Using NMR Residual Dipolar Couplings

9.1 Introduction

9.2 Ensemble Descriptions of Unfolded Proteins

9.3 Experimental Techniques for the Characterisation of IDPs

9.4 NMR Spectroscopy of Intrinsically Disordered Proteins

9.5 Residual Dipolar Couplings

9.6 Conclusions

References

Color Plates

Index

This edition first published 2011

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Library of Congress Cataloging-in-Publication Data

Protein NMR spectroscopy: practical techniques and applications / edited by Lu-Yun Lian, Gordon Roberts.

p. cm.

Includes bibliographical references and index.

ISBN 978-0-470-72193-3

1. Proteins–Analysis. 2. Nuclear magnetic resonance spectroscopy. I.

Lian, Lu-Yun. II. Roberts, G. C. K. (Gordon Carl Kenmure)

QP551.P69725 2011

547′.7–dc22

2011010948

A catalogue record for this book is available from the British Library.

Print ISBN: 9780470721933

ePDF ISBN: 9781119972013

oBook ISBN: 9781119972006

ePub ISBN: 9781119972822

Mobi: 9781119972884

List of Contributors

Igor Barsukov, NMR Centre for Structural Biology, The University of Liverpool, School of Biological Sciences, Biosciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom

Pau Bernadó, Institute for Research in Biomedicine, c/Baldiri Reixac 10, 08028-Barcelona, Spain

Martin Blackledge, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble 38027, France

Paul Driscoll, Division of Molecular Structure, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, United Kingdom

Alex Grishaev, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 5 Memorial Drive, Bethesda, MD 20892

Stephan Grzesiek, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland

Peter Güntert, Institut für Biophysikalische Chemie, BMRZ, J.W. Goethe-Universität Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany

Malene Ringkjøbing Jensen, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble 38027, France

Masatsune Kainosho, Center for Structural Biology, Graduate School of Science, Nagoya University, Chikusa Ku, Furo Cho, Nagoya, Aichi 4648602, Japan

Peter H.J. Keizers, Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, P.O. Box 9502, 2300 RA Leiden, The Netherlands

Lu-Yun Lian, NMR Centre for Structural Biology, The University of Liverpool, School of Biological Sciences, Biosciences Building, Crown Street, Liverpool L69 7ZB, United Kingdom

Phineus Markwick, Howard Hughes Medical Institute, 9500 Gilman Drive, La Jolla, California 92093-0378, USA

Frederick W. Muskett, Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom

Gabrielle Nodet, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble 38027, France

Valéry Ozenne, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble 38027, France

Gordon Roberts, Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom

Loic Salmon, Institut de Biologie Structurale, UMR 5075 CEA-CNRS-UJF, 41 Rue Jules Horowitz, Grenoble 38027, France

Yang Shen, Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 5 Memorial Drive, Bethesda, MD 20892

Mitsuhiro Takeda, Center for Structural Biology, Graduate School of Science, Nagoya University, Chikusa Ku, Furo Cho, Nagoya, Aichi 4648602, Japan

Nico Tjandra, Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, Maryland 20892, USA

Marcellus Ubbink, Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands

Geerten Vuister, Protein Biophysics, Institute of Molecules and Materials, Radboud University Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands; and Henry Wellcome Laboratories of Structural Biology, Department of Biochemistry, University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, United Kingdom

Introduction

Lu-Yun Lian and Gordon Roberts

The nuclear magnetic resonance (NMR) method is one of the principal techniques used to obtain physical, chemical, electronic and three-dimensional structural information about molecules in solution, whether small molecules, proteins, nucleic acids, or carbohydrates. NMR is a physical phenomenon based upon the magnetic properties of certain atomic nuclei. When exposed to a very strong magnetic field (2–21.1 Tesla) these nuclei align with this field. During an NMR experiment, the alignment is perturbed using a radiofrequency signal (typically a few hundred megahertz). When the radio transmitter is turned off, the nucleus returns to equilibrium and in the process re-emits radio waves. The usefulness of this technique in biochemistry results largely from the fact that nuclei of the same element in different chemical and magnetic environments give rise to distinct spectral lines. This means that each NMR-active atom in a large molecule such as a protein can be observed and can provide information on structure, conformation, ionisation state, pKa, and dynamics. The nuclei which are most relevant to the study of biological macromolecules are shown in Table 1. The proton () is the most sensitive nucleus for NMR detection. For biological studies, and are now just as important, although enrichment with these stable isotopes is necessary.

Table 1 Properties of nuclei of interest in NMR studies of proteins.

The first published NMR spectrum of a biological macromolecule was the 40 MHz spectrum of pancreatic ribonuclease reported in 1957. Since then, the significant milestones for NMR include:

Fourier Transform NMR in the late 1960s;the development of two-dimensional NMR in the early 1970s;the development of INEPT/HMQC pulse sequences in the late 1970s;the application of NMR to solve the full three-dimensional structure of a protein in solution in the early 1980s;the introduction, in the late 1980s, of three- and four- multidimensional heteronuclear experiments for use with / isotopically labelled proteins, followed in the late 1990s by the TROSY experiments (which require protein deuteration in addition).

Each stage of these developments has been accompanied by improvements in the spectrometer hardware. In particular, the increases in magnetic field strengths, improved probeheads such as cryogenically-cooled probeheads and better electronics have together led to very substantial improvements in resolution and sensitivity. In addition, continuous advances in molecular biology and sample preparation have allowed these NMR-based improvements to be exploited, particularly in the speed with which samples of significant quantities can be produced in a cost-effective way and the ease with which stable isotope enrichment can be accomplished. Finally, the data analysis is now more streamlined and in the case of very high-quality data, the structure determination process, from resonance assignment to structure calculation, can be automated. These developments are important in order for NMR to remain a mainstream technique for high-resolution structure determination and to make significant contributions in structural biology.

For structural biology, NMR is unique in that it can be used for studies of macromolecules in both the solution and solid states and it is, furthermore, the only method that can provide information on dynamics at the atomic level. This book focuses on the use of NMR to study protein structure and interactions in solution, with the aim of providing a practical guide to users of the method. The book attempts to deal with methods, approaches and issues commonly encountered in the everyday use of NMR in structural biology. No attempt is made to provide a description of the fundamental physics of NMR, but in some chapters it is necessary to detail the theoretical aspects of the methodology in order that the methods can be appropriately applied. A full discussion of the fundamental basis of the wide range of solution NMR experiments used in structural biology can be found in [1] and other valuable introductions to modern NMR spectroscopy include [2, 3].

The success of any application of NMR depends on the correct sample preparation, the appropriate use of parameters for data acquisition and processing; these are covered in Chapter 1. Once initial data has been collected to assess if a protein system is suitable for NMR studies, the next step will depend upon whether the objective is a determination of the three-dimensional structure of a protein or its complex, or a more limited specific objective such as screening for ligand binding or the determination of pKa values. For all but the smallest proteins, isotope-labelling will be required (Chapter 2), and to go beyond purely qualitative experiments resonance assignments (Chapter 3) will be essential. Structure determination involves the acquisition and treatment of structural restraints (Chapter 4) and the use of these to obtain structural ensembles (Chapter 5). Chapter 6 describes the additional information on protein structure or complex formation which can be obtained when the protein contains a paramagnetic species – either naturally occurring or introduced specifically for the purpose. Chapter 7 describes different approaches to the study of the binding of small molecules, ranging from screening to full structure determination of the complex; this requires an understanding of the theoretical and practical aspects of the effects of chemical exchange in NMR, which is also important in many other areas of biological NMR. Chapter 8 provides a comprehensive description of the use of NMR to study macromolecular complexes; this is a challenging area and the chapter outlines the problems and approaches which can be taken to overcome these challenges. Chapter 9 focuses on the structural studies of intrinsically disordered proteins. The widespread existence and significance of these proteins are becoming increasingly recognised and NMR is currently the best method to provide detailed information on their conformational distributions.

NMR is uniquely suited for the characterisation of biomolecular dynamics. Since so many nuclei can be detected simultaneously, NMR can provide a comprehensive description of the internal motions and conformational fluctuations at atomic resolution, and NMR methods have been developed to quantify motions that occur at a wide range of timescales, from picoseconds to days and months. At the same time, consideration of dynamics and the averaging processes to which they lead is an essential part of the use of NMR to obtain structural information. As a result, several chapters in this book deal with methods for obtaining dynamic information from NMR. For additional information the reader is also directed to the following reviews [4–7].

Over the last few years there have been significant developments in the application of solid-state NMR techniques as a tool for determining the high-resolution structures of proteins, ranging from microcrystalline soluble proteins to protein fibrils and membrane proteins. It is now possible to assign the spectra of proteins larger than 100 amino acids using , –labelling [8]. However, as yet this remains an area for the expert and it is not covered in detail in this book (although some of the methods for isotope-labelling described in Chapter 2 will also be relevant to solid-state studies). For useful reviews, the reader is directed to [9, 10].

Note Added in Proof

Several valuable relevant reviews have appeared while this book was in production. In particular, two useful qualitative introductions to biomacromolecular NMR for the newcomer to the field would serve as valuable initial reading [11, 12]. Clore [13] has reviewed the use of relaxation methods (see Chapters 6 and 7) to observe species with low population, Wishart [14] has reviewed the use of chemical shifts in structure determination (see Chapters 4 and 5), and Dominguez et al. [15] have reviewed the use of NMR in the study of protein-RNA complexes (see Chapter 8).

References

1. Cavanagh, J., Fairbrother, W.J., Palmer, A.G. III et al. (2007) Protein NMR Spectroscopy: Principles and Practice, 2nd edn, Academic Press, San Diego.

2. Keeler, J. (2005) Understanding NMR Spectroscopy, John Wiley & Sons, Ltd, Chichester.

3. Levitt, M.H. (2001) Spin Dynamics: Basis of Nuclear Magnetic Resonance, John Wiley & Sons, Ltd, Chichester.

4. Mittermaier, A.K. and Kay, L.E. (2009) Observing biological dynamics at atomic resolution using NMR. TIBS, 34, 601–611.

5. Baldwin, A.J. and Kay, L.E. (2009) NMR spectroscopy brings invisible protein states into focus. Nature Chem. Biol., 5, 808–814.

6. Jarymowycz, V.A. and Stone, M.J. (2006) Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. Chem. Revs., 106, 1624–1671.

7. Igumenova, T.I., Frederick, K.K. and Wand, A.J. (2006) Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution. Chem. Revs., 106, 1672–1699.

8. Schuetz, A. et al. (2010) Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25kDa protein: HET-s(1-227). ChemBioChem., 11, 1543–1551.

9. Renault, M., Cukkemane, A. and Baldus, M. (2010) Solid-state NMR spectroscopy on complex biomolecules. Angew. Chem. Int. Ed., 49, 8346–8357.

10. McDermott, A. (2009) Structure and dynamics of membrane proteins by magic angle spinning solid-state NMR. Ann. Rev. Biophys., 38, 385–403.

11. Kwan, A.H., Mobli, Gooley, P.R. et al. (2011) Macromolecular NMR spectroscopy for the non-spectroscopist. FEBS Journal, 278, 687–703.

12. Bieri, M., Kwan, A.H., Mobli, M. et al. (2011) Macromolecular NMR spectroscopy for the non-spectroscopist: beyond macromolecular solution structure determination. FEBS Journal, 278, 704–715.

13. Clore, G.M. (2011) Exploring sparsely populated states of macromolecules by diamagnetic and paramagnetic NMR relaxation. Protein Sci., 20, 229–246.

14. Wishart, D.S. (2011) Interpreting protein chemical shift data. Prog. Nucl. Magn. Reson. Spectrosc., 58, 1–61.

15. Dominguez, C., Schubert, M., Duss, O. et al. (2011) Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy. Prog. Nucl. Magn. Reson. Spectrosc., 58, 62–87.

Chapter 1

Sample Preparation, Data Collection and Processing

Frederick W. Muskett

Purgamentum init, exit purgamentum

1.1 Introduction

The power of NMR spectroscopy for the analysis of biological macromolecules is undisputed. During the last two decades, the development of spectrometers and the experiments they perform, software, and the molecular biological techniques for the expression and purification of proteins have progressed at a formidable rate. Enrichment of molecules in the three major isotopes used in NMR (, and ) is now commonplace and the cost is no longer prohibitive. The software used to analyse the plethora of data we can generate makes spectral assignment and the extraction of data straightforward for all but the most challenging systems. With all these developments it is easy to forget some of the more fundamental requirements for obtaining good quality NMR data, namely a good sample and a well-set-up NMR experiment.

1.2 Sample Preparation

The first, and possibly one of the most important, steps before embarking on an NMR-based project is the preparation of the sample. Spending some time optimising sample conditions for concentration, ionic strength, pH and temperature before collecting large amounts of data will pay dividends, particularly if the sample is difficult and expensive to produce. Ideally, the optimised sample will not only give the best possible NMR data, but will also have long-term stability as, assuming the project requires backbone and side-chain assignments, the total acquisition time required can be in the order of several weeks.

The following sections will outline the general requirements of a biological sample that is to be used to record NMR data. The assumption has been made that full resonance assignment is required; however, these guidelines could, and probably should, be applied to all samples regardless of the intention of the experiments.

1.2.1 Initial Considerations

This optimisation can be performed in the NMR spectrometer but much can be done using other biophysical techniques such as circular dichroism or fluorescence spectroscopy. These methods require much lower concentrations and do not require isotopic enrichment. The effects of buffer composition on secondary structure content and the melting temperature of the sample give a useful starting point. Once the initial conditions have been determined, final optimisation in the spectrometer can begin. If the sample is a protein, although much can be learned from a simple one-dimensional proton experiment, by far the most useful experiment is the -edited HSQC. This type of experiment removes a great deal of resonance overlap, allowing the user to see in much more detail the effects of varying pH, ionic strength and temperature.

NMR has intrinsically poor sensitivity and, as a result, the concentration of the sample needs to be in the millimolar range. For a conventional room temperature probe ideally the sample concentration needs to be ≥1 mM but can be as low as 0.5 mM. With the development of cryogenically cooled probes this concentration can be reduced to ≥0.2 mM and given the right sample can be as low as 0.05 mM (depending on the experiments performed). Whilst the sample can be exchanged into the buffer intended for NMR experiments in the last step of purification (usually gel filtration chromatography) it is rarely at the concentration required. The two main methods used to increase concentration are lyophilisation with subsequent re-suspension in a lower volume and ultra-filtration, or a combination of the two. Unfortunately, whether a sample will survive either method cannot be predicted; in the end one must simply try and see what happens. However, lyophilisation is generally considered the more dangerous of the two. The number and type of disposable ultra-filtration devices on the market is large, each with their own characteristics regarding compatibility with a particular sample and the effective volumes with which they can be used; again, try and see what happens. As a final step, either passing the sample though a 0.2 μm filter or centrifuging in a benchtop micro-centrifuge, to remove any insoluble material or dust, will greatly help sample homogeneity.

With modern solvent suppression techniques that can effectively eliminate the 110 M protons from the water signal, dissolving the sample in would, at first, no longer seem to be required. However, such samples are still important in recording the experiments designed to allow assignment of protein side-chain resonances and for -edited nuclear Overhauser effect (NOE) experiments. Even the most efficient solvent suppression techniques still leave residual solvent signal and at the same time suppress or distort the signals of interest in that area of the spectrum. In addition, the use of allows one to carry out experiments in which the coherences are recorded in the directly detected dimension where they have the highest resolution. These two advantages alone outweigh the effort required to transfer the sample into . Methods for exchanging the solvent for are the same as for concentrating samples, either lyophilisation or repeated concentration and dilution with . Alternatively, if the sample is unlikely to survive those methods, the sample can be passed down a short de-salting gel-filtration column that has been pre-equilibrated in .