Nuclear Magnetic Resonance (NMR) spectroscopy is the most powerful technique for characterisation of biomolecular structures at atomic resolution in the solution state. This timely book, entitled Biomolecular NMR Spectroscopy, focuses on the latest state-of-the-art NMR techniques for characterisation of biological macromolecules in the solid and solution state. The editors, Dr Andrew Dingley (University of Auckland, New Zealand) and Dr Steven Pascal (Massey University, New Zealand) have organised the book into four sections, covering the following topics: (i) sample preparation, (ii) structure and dynamics of proteins, (iii) structure and dynamics of nucleic acids and protein-nucleic acid complexes and (iv) rapid and hybrid techniques, including the latest advances in NMR data acquisition and structural analysis, and approaches that combine NMR data with data from complementary physical techniques. The book will be a valuable resource for experienced scientists in academia, government and public services and in industry. It will also be suitable for newcomers and graduate students entering the field of biomolecular NMR spectroscopy.
This book aims to highlight recent advances in characterizing biomacromolecules using a range of state-of-the-art NMR approaches. The role of NMR spectroscopy in solving biomolecular structures to atomic resolution is well-established. Novel methods now allow the extension of these studies to larger molecules and complexes. In addition, it is perhaps the ability to tailor NMR experiments to answer a broad range of specific questions at the atomic level that sets NMR apart from other techniques.
The first NMR-based structure determination of a protein was published in 1984 by the Wüthrich group. X-ray crystallographic and NMR structure determination methods were shown to arrive at similar conclusions, though by vastly different approaches. However, as the resolution and capabilities of each technique has grown, so have the distinctions. The recent extension of NMR techniques to larger biomolecules and complexes has revealed that interdomain and intermolecular contacts can be exquisitely sensitive to differences in environment, including the difference between a crystalline and solution environment. The importance of NMR in probing dynamics and folding has also continued to be evident. In addition, we now know that most proteins contain at least one region of intrinsic disorder, a state that can much more effectively be examined by NMR than by crystallography. Thus, as we enter the second decade of the 21st century, it seems clear that the complementary techniques of NMR and X-ray crystallography, with increasing cooperation from other biophysical techniques such as SAXS/SANS and computational approaches, will continue to serve hand-in-hand toward the tasks of structural biology.
The following chapters are organized into four sections. The overexpression of isotopically-enriched proteins for NMR study has become routine. Therefore, the first section entitled “Sample Preparation” focuses on the less well-established preparation of isotopically-enriched nucleic acids and on the option of cell-free protein synthesis. Section two focuses on the structure and dynamics of proteins, including large proteins, complexes and disordered states, in solution, in the solid state and in cells. The chapters in section three examine the challenges and rewards of NMR-based nucleic acid structure and dynamics studies. Finally, recent advances to speed NMR data acquisition and structural analysis, along with procedures that efficiently combine NMR data with data from complementary techniques, are discussed in section four.
We are grateful to all authors for their contributions to this volume, which includes both the writing and formatting of their chapters. We would also like to thank Parvez Haris for his ongoing support and guidance in the preparation of the book, and the publisher, Peter Brown from IOS Press, for his assistance with our queries. The permission from various publishers to reproduce figures from original publications is kindly acknowledged.
Cell-based expression strategies exist for the 2H, 13C and 15N stable-isotope enrichment of target proteins for NMR studies. However, there are no guarantees that the protein will express to a reasonable level or be folded correctly. In addition, the economic cost of producing an isotope-labeled protein may be prohibitive because of the scale of production required or the cost of isotope-labeling sources. Many of these problems may be alleviated by the use of cell-free protein synthesis systems. The open nature of these systems allows the addition of molecules that can aid the folding and stabilization of the protein being produced. Same-day mg/ml production of isotope-enriched proteins can be achieved cheaply by using isotope-labeled amino acids sparingly in the reaction mix. Cell-free systems based on Escherichia coli and wheat germ extracts have been adapted for the isotope enrichment of proteins. The best expression conditions and construct design for target protein production can be screened on the microlitre scale prior to preparative production on the millilitre scale. Selective and uniform isotope enrichment is achieved by simply replacing the amino acids in the reaction mix with their equivalent labeled versions. Combinatorial selective labeling strategies have been developed to enable residue-specific and sequence-specific assignment of NMR spectra. The study of large proteins and complexes can be facilitated by cell-free deuteration of the target protein(s), or by the stereo-array isotope labeling (SAIL) approach. The open nature of the cell-free system means that subunits of a complex can be exclusively labeled by expressing them in the presence of their unlabeled binding partners. In this way, normally insoluble subunits can be rescued into soluble complexes. A number of strategies also exist for the site-specific incorporation of non-natural amino acids. In the future, these methods will enable the incorporation of novel amino acids that can aid NMR assignment or investigations of functional activity by NMR.
Despite its relatively simple primary sequence, ribonucleic acids (RNA) fold into complex three-dimensional structures that, often together with tightly bound protein partners, play important roles within the cell. It is therefore of major importance to decipher the three dimensional structures of these fascinating molecules at an atomic scale. NMR has often proven the method of choice in structure determination of nucleic acids. 42.4% of all nucleic acid structures deposited in the PDB database by July 2009 were solved by NMR. This chapter will focus on in vitro transcription and purification of RNA samples for investigation by NMR. Crucial parameters for the success of a transcription reaction will be discussed and strategies to avoid and detect RNase contamination will be outlined. The problem of distinguishing between a hairpin and a duplex RNA will be considered briefly, as this is a recurring issue when pursuing structural studies with RNA. Often it is desirable to obtain RNAs with 100% homogeneous ends. This can be achieved by self-cleaving ribozymes at each end of the transcript, a strategy that will be discussed herein. This chapter aims to provide a toolkit for successful sample preparation in your own laboratory and provide you the opportunity to immerse yourself into the RNA world.
Sybren S. Wijmenga, Frank H.T. Nelissen, Hans A. Heus, Marco Tessari
36 - 58
Isotope labeling has contributed markedly to the success of multi-dimensional hetero-nuclear NMR spectroscopy in structure and dynamics investigations of proteins, RNAs, DNAs and their complexes. For proteins and RNAs, isotope labeling has become standard practice. In sharp contrast, isotope labeling of larger ds/ssDNAs remains a significant challenge, largely due to a lack of comparably simple, flexible and cost-efficient methods for their synthesis. Here we review isotope labeling of DNA and RNA for NMR structural studies and highlight recent progress made in the field of isotope labeling of larger ssDNA and related RNA labeling. The progress includes labeling schemes with selective deuteration as well as segmental labeling approaches.
Ming-Tao Pai, Shiou-Ru Tzeng, Charalampos G. Kalodimos
61 - 76
The use of NMR to determine the structure and dynamics of small proteins and domains up to about 20 kilodaltons has become fairly routine. Recent advances in technology and analysis have now made it possible to extend NMR studies to proteins and complexes of extreme size. It is even possible to obtain structural and dynamic information of complexes in the mega-dalton range.
Disordered states of proteins include (i) the unfolded states of folded proteins and (ii) the biologically functional intrinsically disordered proteins. Due to the highly dynamic and conformationally heterogeneous nature of disordered states, traditional methods for structural characterization are not directly applicable. Nevertheless, recent years have brought major advances in the experimental characterization of disordered states. In particular, multidimensional NMR methods have proven extremely valuable for improving our understanding of these highly flexible systems. Extensive experimental evidence now supports the idea that disordered states under non-denaturing or mildly denaturing conditions have interesting structural properties that deviate substantially from the random coil-like behavior observed for chemically denatured proteins. In this chapter, we review various experimental techniques for characterizing non-random secondary and tertiary structure in disordered states of proteins. In addition, we discuss recent attempts at combining experimental measurements with computational methods in order to build detailed atomic-level models of various unfolded and intrinsically disordered proteins.
Luciano A. Abriata, María-Eugenia Zaballa, Alejandro J. Vila
97 - 114
NMR spectroscopy of paramagnetic metalloproteins has been historically disfavored based on the fast nuclear relaxation rates induced by unpaired electrons, which leads to extreme line broadening and impairs magnetization transfer. Recent advances in the application of state-of-the-art pulse sequences are described herein, with particular emphasis on copper proteins, which present unfavorable electron relaxation times. In the case of binuclear copper centers, NMR is particularly useful for obtaining information on the electronic structure of partially populated (invisible) excited states which determine their reactivity.
Christian Ader, Deepak Nand, Marie Renault, Abhishek A. Cukkemane, Marc Baldus
115 - 136
We describe recent progress in using Magic-Angle-Spinning based solid-state NMR to study structural aspects of membrane-embedded proteins. Experimental aspects such as protein production and reconstitution are reviewed. Moreover, we discuss solid-state NMR methods leading to a comprehensive description of the conformational landscape of membrane proteins and review applications to retinal proteins and a chimeric ion channel.
John D. Gehman, Marc-Antoine Sani, Frances Separovic
137 - 161
In the study of lipid systems, biological solid-state NMR has historically concentrated on characterization of relatively simple model membranes. In more recent times, peptides and proteins that interact with these membrane systems have increasingly shared the focus. Here we describe how solid-state NMR provides information about the effect of antimicrobial peptides (AMPs) on membrane bilayers, and their location relative to the membrane surface, and their structure in the context of the membrane. Aligned membrane bilayers and bicelles, and magic angle spinning techniques using unoriented bilayers are discussed, together with measurements of chemical shift anisotropy and dipolar couplings to gain structural details of AMPs in membranes. In addition to 31P, 2H and 14N NMR of phospholipid bilayers, 19F, 13C and 15N NMR of labelled AMP, gramicidin A′, magainin 2, PGLa, aurein 1.2 and protegrin-1, are reviewed.
Karl Bertrand, David S. Burz, Alexander Shekhtman, Kaushik Dutta
162 - 184
In the past few decades, biological processes such as protein-protein and protein-ligand interactions have been studied to atomic resolution under “near-physiological” conditions using in vitro NMR spectroscopy. With the advent of in-cell NMR spectroscopy, biological interactions can now be studied within a cellular environment to provide information at atomic resolution under physiological conditions. By performing NMR spectroscopy on living cells, we can begin to better understand the structural underpinnings of biological activity. This article will discuss the more recent advances in the field of in-cell NMR spectroscopy, both in prokaryotic and eukaryotic cells.
Recent developments in spin-1/2 isotope labeling and NMR pulse sequence advances have been leveraged to investigate μs – ms motions in enzyme function. These studies have identified concerted motions that occur over large regions of enzymes and often involve highly conserved amino acids. In combination with functional studies these NMR-identified motions have been implicated in partaking in the rate-determining step in the catalytic cycle. This review examines several of the more recent solution NMR studies that demonstrate the essential nature of conformational motions.
RNA exhibits considerable structural and functional diversity beyond well established roles of ribosomal, transfer and messenger RNAs, as illustrated by the discovery of ever increasing numbers of diverse RNA structures involved in gene processing and regulation. RNA molecules are often quite flexible; they can function in a genuinely unfolded form and adapt for recognition of both the shape and the charge distribution on a potential ligand with exquisite specificity. Liquid state NMR spectroscopy is uniquely suited to answer important questions concerning biophysical properties of RNA molecules including their three-dimensional shape, secondary structure distribution, and flexibility by looking at dynamic ensembles of structures. Here we review the fields of RNA sample preparation and NMR methodology that facilitate the determination of RNA structure in solution.
Lawrence J. Clos II, Samuel E. Butcher, Yun-Xing Wang
229 - 248
Investigation of large nucleic acids (> 30 kDa) challenges the current limits of NMR spectroscopy. Extended helical regions in RNA and DNA increase correlation times more so than for proteins of comparable size, resulting in line broadening and reduced sensitivity. Also, the lower proton density and poor proton spectral dispersion in nucleic acids can reduce the effectiveness of traditional solution NMR methods in studies of large nucleic acids. These limitations represent a substantial hindrance to the future investigation of biologically relevant large nucleic acid structures. Herein we describe methods that have been implemented to help overcome these challenges and extend the size limits of nucleic acid NMR spectroscopy, including construct design, selective isotopic labeling, multi-dimensional NMR experiments and complementary techniques.
The role of protein-RNA recognition is fundamental to many biological processes, protein-RNA interaction being at the heart of every molecular mechanisms controlling post-transcriptional gene expression. Deciphering the 3D structures of protein-RNA complexes is therefore of high significance. RNA binding proteins are very abundant in all kingdoms of life and often embed one to several small RNA binding domains. Since these domains often act as independent units, NMR spectroscopy is ideally suited to study the structure and dynamics of such domains in complex with their RNA targets. We review here how NMR spectroscopy has been used to solve the structure of more than fifty protein-RNA complexes and to understand for a few their dynamics.
Michael F. Bardaro Jr., Kari Pederson, Gary Drobny, Gabriele Varani
279 - 301
RNA and DNA can adopt highly different conformations to facilitate protein or ligand binding. These conformational changes are significant, since they often are an essential part of nucleic acid function. As a result, there is ever increasing interest in studying dynamics of nucleic acids and their connection to function. Here, we review NMR methods, in both the solid and liquid states, to study nucleic acid dynamics, including relaxation, residual dipolar couplings and lineshape analysis.
The advent of isotope labeling strategies for macromolecules has enabled the application of multidimensional heteronuclear NMR experiments to biomolecular systems of unprecedented size and complexity. The improved signal dispersion afforded by multiple dimensions; however, comes at a price: each added dimension exponentially increases the required NMR data acquisition time if the conventional approach of Fourier Transform NMR is employed. Efforts to overcome this time barrier have been given additional impetus due to technological advances such as the advent of cryogenically cooled probes, increased strength of high-field magnets, and improvements in computation. In this chapter we review the most successful methods for accelerating NMR data acquisition, highlighting relationships among the methods as well as their respective strengths and weaknesses. Our aim is to provide a practical reference guide for researchers to better understand this rapidly growing field and help identify the approach best suited to a particular problem.
Three-dimensional structures of proteins in solution can be calculated on the basis of conformational restraints derived from NMR measurements. This chapter gives an overview of the computational methods pertinent to NMR protein structure analysis. The most widely used algorithms based on simulated annealing by molecular dynamics simulation in torsion angle space and the automated assignment of NOE distance restraints are presented, as well as non-classical approaches and fully automated NMR protein structure analysis.
Structural genomics requires that accurate atomic resolution protein structures are solved in high-throughput. The Northeast Structural Genomics Consortium (NESG), USA, has established a scalable platform for NMR-based structure determination, including protocols for rapid NMR data acquisition, semi-automated data processing, data analysis and structure calculation, and structure validation. The main features of this pipeline are discussed along with (i) recent methodological advances and (ii) perspectives for future improvements and expansion to larger bio-molecular systems.
Drug discovery is a challenging endeavor with a high failure rate. This is further complicated by the fact that each disease has its own unique set of obstacles to developing a safe and effective therapy. Flexible and robust analytical methods are critical for efficiently solving these issues, where nuclear magnetic resonance (NMR) plays an important role in nearly every stage of the drug discovery process. This review will highlight recent developments in the application of NMR as a screening tool for drug discovery that includes: library design, various ligand-affinity screening techniques, rapid determination of protein-ligand co-structures, and the functional annotation of proteins to the discovery of new therapeutic targets.
Julie R. Kesting, Kenneth T. Johansen, Jerzy W. Jaroszewski
413 - 434
This chapter describes principles and applications of on-line coupling (hyphenation) of NMR spectroscopy with liquid-chromatographic and other separation methods as well as with other forms of spectroscopy. This results in highly integrated and automated systems for structure determination of components of complex mixtures at microgram and nanogram levels. Hyphenated NMR techniques provide accelerated and detailed insight into components of complex mixtures of biological origin without initial separation of the constituents.
Nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimetry (ITC), and differential scanning calorimetry (DSC) provide different, yet highly complementary views of biomolecular dynamics and energetics. NMR quantifies internal motions over a wide range of time scales with atomic resolution. Calorimetry is extremely sensitive to the energetics of conformational transitions and macromolecular interactions. Together, they can yield detailed, quantitative descriptions of biomolecular function that are inaccessible to any of the techniques alone. This Chapter outlines the ITC, DSC and several NMR dynamics methodologies, and gives an overview of selected applications that highlight the potential of combining NMR and calorimetry.
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