Recent Developments in Biomolecular NMR: Volume 25 (RSC Biomolecular Sciences) - Hardcover

Hardcover

ISBN 10: 1849731209 ISBN 13: 9781849731201

Publisher: Royal Society of Chemistry, 2012

Synopsis

Edited by leading biological NMR spectroscopists, this book will cover the new developments that have occurred in biomolecular NMR over the last few years.

About this title

About the Author

Dr Marius Clore's career began at University College London where he studied for his BSc in Biochemistry, moving to University College Hospital Medical School he became an MD and finally a PhD at the National Institute for Medical Research, London. In 1984, he became head of the Biological NMR Group at Max-Planck Institute for Biochemistry and then moved in 1988 to NIH, USA. He is currently an NIH Distinguished Investigator and Chief of the Protein Nuclear Magnetic Resonance Section and has made many pioneering contributions in the development of NMR spectroscopy for structural characterization of biological macromolecules. Dr Clore has been awarded numerous prizes and honours and was ranked in the top 20 in h-index ranking of living chemists in 2009. He is also a 3rd Degree black belt in Tae Kwon Do. Dr Jennifer Potts studied at the University of Sydney before becoming a postdoctoral fellow at the University of Oxford in 1992. Staying at Oxford until 2005 as a research associate and latterly lecturer in biochemistry, she then became Anniversary Reader at the University of York where her recent work has been on fibronectin recognition domains which fold on experiencing their target.

From the Back Cover

NMR spectroscopy is widely used in biomolecular science particularly for structure determination of proteins, nucleic acids and carbohydrates. Much of the innovation within NMR spectroscopy has been within the field of protein NMR spectroscopy, an important technique in structural biology.

Filling a gap in the literature, this book draws together experts in the field to discuss the real advances in NMR methods that have occurred or have an impact on the biomolecular field in the last few years. The coverage includes recent developments in using NMR for determination of protein structures, membrane proteins, the dynamics of RNA and advances in NMR in drug discovery. Edited by leading biological NMR spectroscopists, the book is essential reference for researchers in industry and academia interested in or joining this bioanalytical field.

Excerpt. © Reprinted by permission. All rights reserved.

Recent Developments in Biomolecular NMR

By Marius Clore, Jennifer Potts

The Royal Society of Chemistry

Copyright © 2012 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-120-1

Contents

Chapter 1 Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins Michael J. Plevin and Jérôme Boisbouvier, 1, 
Chapter 2 Low-γ Nuclei Detection Experiments for Biomolecular NMR Koh Takeuchi, Maayan Gal, Ichio Shimada and Gerhard Wagner, 25, 
Chapter 3 Making the Most of Chemical Shifts R. William Broadhurst, 53, 
Chapter 4 Protein Structure Determination using Sparse NMR Data Oliver F. Lange, 84, 
Chapter 5 NMR Studies of Disordered but Functional Proteins H. Jane Dyson, 111, 
Chapter 6 Paramagnetic NMR Spectroscopy and Lowly Populated States Jesika T. Schilder, Mathias A. S. Hass, Peter H. J. Keizers and Marcellus Ubbink, 130, 
Chapter 7 NMR Relaxation Dispersion Studies of Large Enzymes in Solution Sean K. Whittier and J. Patrick Loria, 151, 
Chapter 8 Residual Dipolar Couplings as a Tool for the Study of Protein Conformation and Conformational Flexibility Loïc Salmon, Phineus Markwick and Martin Blackledge, 166, 
Chapter 9 Characterising RNA Dynamics using NMR Residual Dipolar Couplings Catherine D. Eichhorn, Shan Yang and Hashim M. Al-Hashimi, 184, 
Chapter 10 Non-Canonical Ligand-Binding Events as Detected by NMR Erik R. P. Zuiderweg, 216, 
Chapter 11 Recent Advances in Biomolecular NMR for Drug Discovery Carine Farenc and Gregg Siegal, 254, 
Chapter 12 NMR of Membrane Proteins Mark Bostock and Daniel Nietlispach, 271, 
Chapter 13 Recent Developments in Biomolecular Solid-State NMR Victoria A. Higman and Anthony Watts, 318, 
Subject Index, 335, 

CHAPTER 1

Isotope-Labelling of Methyl Groups for NMR Studies of Large Proteins

MICHAEL J. PLEVIN AND JÉRÔME BOISBOUVIER

CEA, Institut de Biologie Structurale, CNRS, Institut de Biologie Structurale Jean-Pierre and Université Joseph Fourier, Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France

E-mail: michael.plevin@ibs.fr or jerome.boisbouvier@ibs.fr

1.1 Introduction — Large Proteins and Solution NMR Spectroscopy

1.1.1 Isotope-Labelling and Protein NMR Spectroscopy

Solution NMR spectroscopy is a well-established technique for characterising the structure, function and dynamics of proteins at atomic resolution. Proteins are predominantly composed of carbon, nitrogen, oxygen and hydrogen. Of these four, only hydrogen has a naturally abundant, NMR-visible spin-½ nucleus and, for this reason, the proton was the major focus of early protein NMR studies. One of the major drawbacks of proton NMR spectroscopy is the inherent low dispersion of 1H chemical shifts. The narrow range of 1H resonance frequencies means that the ability to differentiate individual 1H signals becomes increasingly problematic as the size of the protein and therefore the number of potential signals increases.

The problem of low 1H signal overlap has now been largely overcome through the preparation of protein samples enriched with low natural abundance, spin-½ isotopes of carbon and/or nitrogen. Many NMR experiments have since been written that utilise the large signal dispersion of 13C or 15N nuclei to separate the signals of scalar-coupled nuclei over multiple dimensions. Furthermore, in addition to resolving spectral congestion, isotope enrichment introduces more NMR-visible probes into the molecules of interest and allows a multitude of structural and dynamic information to be accessed from their NMR signals.

Isotopic enrichment of proteins can take two forms: uniform or selective. In the most commonly used approach the recombinant target protein is over-expressed from E. coli grown in an isotopically enriched minimal-expression medium containing uniformly labelled [13C]glucose and/or [15N]ammonium chloride or sulphate, as the only carbon and nitrogen sources. The resulting protein product is isotopically enriched at the same level as the expression medium. Uniform labelling approaches were developed towards the end of the 1980s (ref. 4) and since have become routine and robust. In the last 20 years, the price of isotopically enriched reagents has decreased considerably making uniform labelling a common practice in structural biology laboratories.

Isotope-labelling of individual amino acids or groups of amino acids can also be performed. Residue-specific isotope labelling is achieved by supplementing the expression medium with isotopically enriched amino acids. This approach is somewhat limited in vivo as a result of the scrambling of the isotope-labelled sites by bacterial metabolic pathways. As an alternative, isotope-labelled amino acids can be used in combination with cell-free in vitro expression systems, which essentially alleviate isotopic dilution.

1.1.2 General Considerations for NMR Studies of Larger Proteins

Over the past 20 years an enormous array of multi-dimensional heteronuclear NMR experiments have been designed that can extract structural or dynamic information about isotopically enriched proteins. The strategy of combining isotope-labelling with tailored NMR experiments has been so successful that it has encouraged NMR spectroscopists to study larger and more complicated biomolecular systems. However, as the size of protein targets increases new problems arise.

The lifetime of the excited state in NMR spectroscopy is predominantly affected by the overall molecular tumbling rate. As molecular size increases the tumbling rate slows and this leads to an increase in the rate at which transverse magnetisation relaxes. As the linewidth of an NMR signal is proportional to the transverse relaxation rate, NMR spectra of larger molecules which tumble more slowly are characterised by broad NMR signals.

The short lifetime of transverse relaxation in large proteins severely affects the sensitivity, effectiveness and scope of NMR experiments. NMR pulse sequences frequently rely on scalar couplings to transfer magnetisation between nuclei of interest. Such transfer steps require periods in which nuclear magnetisation is the transverse plane and therefore subject to transverse relaxation. Thus, complicated pulse sequences that correlate nuclei via weak scalar couplings or that require multiple transfers mediated by scalar couplings become less effective and less sensitive for larger proteins.

Resonance assignment of proton, carbon and nitrogen nuclei in the polypeptide backbone is a critical first step in many NMR studies of protein structure, dynamics or interactions. A common starting point is a two-dimensional (2D) (1H,15N) heteronuclear correlation spectrum acquired, for example, using the Heteronuclear Single Quantum Coherence (HSQC) experiment. An in-depth assessment of NMR data requires being able to locate each NH cross-peak to a unique site in the target protein. This is achieved by determining sequence-specific resonance assignments. There are numerous experimental strategies that facilitate backbone resonance assignment, many of which make use of uniform isotope-labelling strategies and multi-dimensional heteronuclear NMR experiments. While these approaches work well for smaller proteins (<25 kDa; Figure 1.1), they cease to be applicable when the molecular weight increases as the transverse magnetisation relaxes more rapidly.

The major source of relaxation for 1H nuclei in higher molecular weight proteins is the large number of dipolar interactions with neighbouring protons. For most heteronuclei (15N or 13C), however, the dominant factor is the direct dipolar interaction(s) with covalently bound proton(s). To overcome this limitation, proteins can be expressed in perdeuterated expression medium. Protons are then re-introduced at labile sites (e.g., HN) by purifying (or, if necessary, refolding) the protein in H2O-based buffers. This approach ensures that backbone-directed NMR experiments that utilise the amide proton are still applicable. Perdeuteration reduces proton density by introducing deuterium at all non-labile sites and therefore reduces the transverse relaxation rates of the remaining protons. The consequent narrowing of 1H signal line-widths can make a dramatic difference to NMR spectra of larger proteins. Furthermore, deuteration of aliphatic [13C] sites considerably extends the lifetime of transverse coherences which is critical when applying three-dimensional (3D) or four-dimensional (4D) NMR experiments to proteins larger than 20–30 kDa.

Using a [U-2H,13C,15N]-labelled amide-reprotonated sample it is possible to obtain backbone resonance assignments for proteins and protein complexes up to 100–150 kDa. To date the largest single chain protein for which near complete backbone resonance assignments have been acquired is the 723 residue, 82 kDa, bacterial enzyme, malate synthase G (MSG; ref. 8; Figure 1.1). Backbone resonance assignments of larger systems have been determined, but only in cases where the protein target exists as a homooligomer (e.g., refs. 9 and 10).

1.1.3 NMR Experiments Designed for Larger Systems

By isolating each proton from other protons of the protein, a high level of deuteration is an efficient way to narrow the linewidths of the remaining 1H spins. Nevertheless, 1H/2H substitution has only a moderate effect on the NMR signal of heteronuclei (15N or 13C) that are directly bonded to the remaining 1H spins. As the acquisition of high-quality 2D (1H, 15N) or (1H, 13C) NMR spectra is a prerequisite for the NMR study of a large protein, considerable effort has been spent during the last 15 years to develop new NMR tools that optimise the relaxation of the NMR signals of both 1H and covalently bonded 15N or 13C spins. This concept is known as Transverse Relaxation Optimised SpectroscopY (TROSY). In an isolated two-spin system involving covalently bonded nuclei, e.g., a 1H–15N or 1H–13C pair, the main spin interactions are dipolar interactions between nuclei and the chemical shift anisotropy (CSA) of each spin. As the same molecular motions modulate these interactions they can give rise to interference effects. Such effects, also called cross-correlated relaxation, modulate the relaxation of the different NMR observable transitions. The so-called TROSY experiments enhance resolution and sensitivity of NMR experiments of large biomolecules by selecting transitions(s) with more favourable relaxation properties. Since the development of NMR pulse schemes that allow selective spin-state excitation or transfer, several TROSY experiments have been developed for different spins systems. To date, TROSY experiments have been described for optimised observation of 15N–1H amide groups, aromatic 13C–1H sites or 13C1H2 methylene groups. For 13C1H3 methyl groups, the simple 2D HMQC experiment has been shown to preserve the slowly relaxing methyl group coherences independently from the more rapidly relaxing component of the signal. In much larger perdeuterated proteins the rapidly relaxing component disappears during the course of the pulse sequence leaving only the slowly relaxing component to be detected. 2D (1H,13C) HMQC (also called methyl-TROSY) spectra of large perdeuterated selectively methyl-protonated proteins show a high level of sensitivity and signal resolution (Figure 1.2). In recent years, the combination of methyl-TROSY spectroscopy with residue-type-specific methyl labelling has allowed solution NMR studies of very large protein systems of several hundreds of kDa. The aim of this chapter is to present an overview of recently developed isotopic-labelling methods that allow such large biomolecular systems to be investigated by NMR spectroscopy.

1.2 Using Methyl Groups as Probes for NMR Spectroscopy

1.2.1 Why the Methyl Group?

As molecular size increases it becomes increasingly difficult to rely on NH-based NMR spectra. If [U-2H,13C]glucose has been used as the sole carbon source, a perdeuterated labile site-reprotonated (e.g., all NH, OH, etc.) protein will still have a protonation level around 20 %. This level of protonation becomes detrimental for proteins larger than 100 kDa. For such proteins, the methyl group has become the NMR probe of choice. Each methyl group comprises three protons which rotate rapidly around the methyl symmetry axis. The consequent three-fold degeneracy of the chemical shifts of the methyl protons greatly increases sensitivity compared to the backbone amide proton. Furthermore, methyl groups are often located at the end of long amino acid side-chains and are generally more dynamic than backbone amide protons.

In general, methyl groups resonate in a largely uncrowded region of the 2D (1H,13C) spectrum (Figure 1.3). Methyl-group-containing residues are usually common and well dispersed in the amino acid sequence and are present both in the hydrophobic core of proteins and at interaction sites. Using methyl-TROSY NMR experiments it is possible to acquire high-quality NMR spectra of methyl-protonated perdeuterated large proteins, potentially in as little time as 1 s (ref. 23). Thus, methyl groups are excellent probes of protein structure and dynamics, particularly for very large proteins.

1.2.2 Strategies for Selective Protonation of Methyl Groups in Perdeuterated Proteins

There are six methyl-containing amino acids found in proteins, excluding posttranslational modifications. Over the past 15 years a variety of strategies for selective labelling of methyl groups in proteins have been proposed. The objective of these labelling approaches is to produce highly deuterated (i.e., >98 %) proteins with targeted [13CH3]-labelling at residue-specific methyl sites. Carbon-labelling patterns in the rest of the side-chain can vary and be modified depending on the particular system and the question(s) being asked. The basis of many of these labelling strategies lies in the biosynthesis of methyl-group containing amino acids (Figure 1.4).

The objective of this chapter is not to provide detailed labelling protocols but rather to give an introductory summary. Relevant references are cited and the reader is encouraged to read these for a more detailed explanation. Unless otherwise stated, the procedures and examples described below refer to the over-expression of recombinant proteins from E. coli grown in 100 % deuterated minimal medium. In order to avoid a great deal of confusion with nomenclature, the chemical name used in the original publication is given together, in parentheses, with the IUPAC name and Chemical Abstract Services (CAS) number of the unlabelled molecule.

1.2.2.1 Alanine

Alanine is the smallest residue that contains a methyl group and is one of the most common amino acids found in proteins. The β-methyl group of alanine is directly connected to the polypeptide backbone and therefore it can provide information about local backbone structure using the chemical shift index as well reporting on dynamics. Alanine is frequently found on the protein surface and can thus be used to detect and characterise biomolecular interactions. Furthermore, alanine is commonly used as a replacement in mutagenesis studies.

Specific labelling of the β-methyl group of alanine can be achieved by supplementing bacterial minimal expression media with 800 mg L-1 of commercially available [13C]-labelled alanine. Different 13C isotope-labelling patterns are possible (i.e., [3-13C] or [U-13C]). [13C]-labelling of both carbon-2 and carbon-3 (i.e., Cα and Cβ) gives access to backbone chemical shift information. To ensure optimal levels of background deuteration it is necessary to use [2-2H]alanine, which can either be purchased directly or generated enzymatically from protonated alanine using tryptophan synthase.

Alanine is directly synthesised from pyruvate in a reversible transamination reaction (Figure 1.4). Consequently supplementing a bacterial expression medium with isotope-labelled alanine will result in severe isotopic scrambling to other sites in the protein, notably leucine, isoleucine and valine. A reduction in the level of scrambling has been achieved in a number of ways. Isaacson and colleagues proposed using [13C]-labelled [2-2H]alanine in concert with a rich perdeuterated expression medium which contains high levels of deuterated [12C] amino acids. Such a medium also contains deuterated [12C]-labelled alanine which is incorporated into the target protein at the expense of the supplemented alanine. This approach boosts protein expression levels due to the use of rich expression medium but, due to the use of rich media, the incorporation level of [13C]alanine is severely reduced. Scrambling of alanine can also be effectively eliminated by supplementing minimal expression media with specific deuterated metabolites (such as isoleucine and α-ketoisovalerate) that saturate and inhibit metabolic pathways that the supplemented alanine would leak into. This strategy allows near complete incorporation of labelled alanine in [2H]-based M9 medium with detectable isotopic scrambling reduced to less than 1%.

1.2.2.2 Methionine

The methyl group of methionine is a useful NMR probe as it is isolated at the end of a long amino acid side-chain. Furthermore, methionine methyl groups tend to resonate in a largely unpopulated region of the (1H,13C) correlation spectrum (Figure 1.3). The absence of a scalar-coupled carbon means that the methionine methyl group is a useful probe of protein dynamics.

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2012
• Language: English
• ISBN 10: 1849731209
• ISBN 13: 9781849731201
• Binding: Hardcover
• Number of pages: 347
• Editor: Marius Clore