An Introduction to Biological NMR Spectroscopy

NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP).     

Achieving broad molecular insights into dynamic protein interactions by integrated structural-kinetic approaches

A network of dynamic protein interactions with their protein partners, substrates, and ligands is known to be crucial for biological function. Revealing molecular and structural-based mechanisms at atomic resolution and in real-time is fundamental for achieving a basic understanding of cellular processes. These technically challenging goals may be achieved by combining time-resolved spectroscopic and structural-kinetic tools, thus providing broad insights into specific molecular events over a wide range of timescales. Here we review representative studies utilizing such an integrated real-time structural approach designed to reveal molecular mechanisms underlying protein interactions at atomic resolution.     

NMR structures of paramagnetic metalloproteins

Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+ -binding proteins and for Ca2+ -binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein–protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the ¹H, ¹⁵N and ¹³C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.     

Weak protein-protein interactions as probed by NMR spectroscopy

Weak protein-protein interactions (PPIs) are fundamental to many cellular processes, such as reversible cell-cell contact, rapid enzyme turnover and transient assembly and/or reassembly of large signaling complexes. However, structural and functional characterizations of weak PPIs have been technically challenging and lagged behind those for strong PPIs. Here, we describe nuclear magnetic resonance (NMR) spectroscopy as a highly effective tool for unraveling the atomic details of weak PPIs. We highlight the recent advances of how NMR can be used to rapidly detect and structurally determine extremely weak PPIs (K(d)>10(-4)M). Coupled with functional approaches, NMR has the potential to look into a wide variety of biologically important weak PPIs at the detailed molecular level, thereby facilitating a thorough view of how proteins function in living cells.     

Spectroscopic methods for analysis of protein secondary structure

Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of secondary structures in proteins. NMR chemical shifts may also be used to determine the positions of secondary structure within the primary sequence of a protein. However, the chemical shifts must first be assigned to particular residues, making the technique considerably slower than the optical methods. These data, together with sophisticated molecular modeling techniques, allow for refinement of protein structural models as well as rapid assessment of conformational changes resulting from ligand binding or macromolecular interactions. A selected number of examples are given to illustrate the power of the techniques in applications of biological interest.     

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions

Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.     

Automated structure determination of proteins with the SAIL-FLYA NMR method

The labeling of proteins with stable isotopes enhances the NMR method for the determination of 3D protein structures in solution. Stereo-array isotope labeling (SAIL) provides an optimal stereospecific and regiospecific pattern of stable isotopes that yields sharpened lines, spectral simplification without loss of information, and the ability to collect rapidly and evaluate fully automatically the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as those that can be analyzed using conventional methods. Here, we describe a protocol for the preparation of SAIL proteins by cell-free methods, including the preparation of S30 extract and their automated structure analysis using the FLYA algorithm and the program CYANA. Once efficient cell-free expression of the unlabeled or uniformly labeled target protein has been achieved, the NMR sample preparation of a SAIL protein can be accomplished in 3 d. A fully automated FLYA structure calculation can be completed in 1 d on a powerful computer system.     

Solution NMR views of dynamical ordering of biomacromolecules

Background

To understand the mechanisms related to the ‘dynamical ordering’ of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells.

核磁共振波谱应用于结构生物学的研究进展

综述了核磁共振波谱在结构生物学研究中的进展。在溶液中测定生物大分子的结构,分子大小的限制正被减少,尽管新结构的测定仍然需要付出比较大的努力。核磁共振是一个有效的手段,可用于研究在许多细胞过程中存在的弱的或者瞬态的蛋白质-蛋白质相互作用。结构的柔性在蛋白质分子功能中起了中心作用。由于最近方法学的发展,使NMR可以表征蛋白质的动力学,从而可以对分子机制有新的认识。核磁共振波谱可以在原子分辨率下表征无序的蛋白质系统,可以研究折叠路径。跨膜蛋白在细胞中起了关键作用,这使它们成为药物的靶标。应用液体和固体核磁共振技术已经成功测定了跨膜蛋白质的结构。     

Accurate structural correlations from maximum likelihood superpositions

The cores of globular proteins are densely packed, resulting in complicated networks of structural interactions. These interactions in turn give rise to dynamic structural correlations over a wide range of time scales. Accurate analysis of these complex correlations is crucial for understanding biomolecular mechanisms and for relating structure to function. Here we report a highly accurate technique for inferring the major modes of structural correlation in macromolecules using likelihood-based statistical analysis of sets of structures. This method is generally applicable to any ensemble of related molecules, including families of nuclear magnetic resonance (NMR) models, different crystal forms of a protein, and structural alignments of homologous proteins, as well as molecular dynamics trajectories. Dominant modes of structural correlation are determined using principal components analysis (PCA) of the maximum likelihood estimate of the correlation matrix. The correlations we identify are inherently independent of the statistical uncertainty and dynamic heterogeneity associated with the structural coordinates. We additionally present an easily interpretable method (“PCA plots”) for displaying these positional correlations by color-coding them onto a macromolecular structure. Maximum likelihood PCA of structural superpositions, and the structural PCA plots that illustrate the results, will facilitate the accurate determination of dynamic structural correlations analyzed in diverse fields of structural biology.     

NMR resonance assignments for the tetramethylrhodamine binding RNA aptamer 3 in complex with the ligand 5-carboxy-tetramethylrhodamine

RNA aptamers are used in a wide range of biotechnological or biomedical applications. In many cases the high resolution structures of these aptamers in their ligand-complexes have revealed fundamental aspects of RNA folding and RNA small molecule interactions. Fluorescent RNA-ligand complexes in particular find applications as optical sensors or as endogenous fluorescent tags for RNA tracking in vivo. Structures of RNA aptamers and aptamer ligand complexes constitute the starting point for rational function directed optimization approaches. Here, we present the NMR resonance assignment of an RNA aptamer binding to the fluorescent ligand tetramethylrhodamine (TMR) in complex with the ligand 5-carboxy-tetramethylrhodamine (5-TAMRA) as a starting point for a high-resolution structure determination using NMR spectroscopy in solution.     

Conformational fluctuations of proteins revealed by variable pressure NMR

With the high-resolution variable-pressure NMR spectroscopy, one can study conformational fluctuations of proteins in a much wider conformational space than hitherto explored by NMR and other spectroscopic techniques. This is because a protein in solution generally exists as a dynamic mixture of conformers mutually differing in partial molar volume, and pressure can select the population of a conformer according to its relative volume. In this review, we describe how variable-pressure NMR can be used to probe conformational fluctuations of proteins in a wide conformational space from the folded to the fully unfolded structures, with actual examples. Furthermore, the newly emerging technique "NMR snapshots" expresses amply fluctuating protein structures as changes in atomic coordinates. Finally, the concept of conformational fluctuation is extended to include intermolecular association leading to amyloidosis.     

Molecular dynamics simulations of the complex between human U1A protein and hairpin II of U1 small nuclear RNA and of free RNA in solution.

RNA-protein interactions are essential to a wide range of biological processes. In this paper, a 0.6-ns molecular dynamics simulation of the sequence-specific interaction of human U1A protein with hairpin II of U1 snRNA in solution, together with a 1.2-ns simulation of the free RNA hairpin, is reported. Compared to the findings in the x-ray structure of the complex, most of the interactions remained stable. The nucleotide U8, one of the seven conserved nucleotides AUUGCAC in the loop region, was unusually flexible during the simulation, leading to a loss of direct contacts with the protein, in contrast to the situation in the x-ray structure. Instead the sugar-phosphate backbone of nucleotide C15 was found to form several interactions with the protein. Compared to the NMR structure of U1A protein complexed with the 3'-untranslated region of its own pre-mRNA, the protein core kept the same conformation, and in the two RNA molecules the conserved AUUGCAC of the loop and the closest CG base pair were located in very similar positions and orientations, and underwent very similar interactions with the protein. Therefore, a common sequence-specific interaction mechanism was suggested for the two RNA substrates to bind to the U1A protein. Conformational analysis of the RNA hairpin showed that the conformational changes of the RNA primarily occurred in the loop region, which is just involved in the sites of binding to the protein and in agreement with experimental observation. Both the loop and stem of the RNA became more ordered upon binding to the protein. It was also demonstrated that the molecular dynamics method could be successfully used to simulate the dynamical behavior of a large RNA-protein complex in aqueous solution, thus opening a path for the exploration of the complex biological processes involving RNA at a molecular level.