Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy

The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. Here we present a protein structure determined by solid-state magic-angle-spinning (MAS) NMR. Almost complete (13)C and (15)N resonance assignments for a micro-crystalline preparation of the alpha-spectrin Src-homology 3 (SH3) domain formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3-(13)C]glycerol or [2-(13)C]glycerol as carbon sources. This allowed the observation of long-range distance correlations up to approximately 7 A. The calculated global fold of the alpha-spectrin SH3 domain is based on 286 inter-residue (13)C-(13)C and six (15)N-(15)N restraints, all self-consistently obtained by solid-state MAS NMR. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in bacteria.     

The CO bond angle of carboxymyoglobin determined by angular-resolved XANES spectroscopy

Our knowledge of the structure of condensed matter has been based primarily on spectroscopic methods that measure first-order pair correlations of atomic arrangements and thus provide interatomic distances (for example neutron and X-ray scattering). Bond angles are given by higher-order correlation functions, and such information can be provided by X-ray absorption near-edge structure (XANES) spectroscopy, the features of which are determined by multiple scattering of photoelectrons whose paths begin and end at the selected absorbing atom. We report here angular-resolved XANES spectroscopy of a single crystal of carboxymyoglobin (MbCO). The large dichroism of the X-ray absorption of the crystal can be fully interpreted by multiple-scattering theory which allows the determination of Fe-ligand bond angles. The analysis of the identified multiple scattering features due to CO in high signal-to-noise-ratio spectra of protein in solution has allowed the determination of the variation of CO bond angles. This opens the way to the determination of subtle structural features due to bond angle variations in proteins in solution which are relevant to an understanding of the characteristics of proteins at the atomic scale.     

Three-dimensional NMR spectroscopy of a protein in solution

The geometric information used to solve three-dimensional (3D) structures of proteins by NMR spectroscopy resides in short (less than 5 A) interproton-distance data. To obtain these distances, the 1H-NMR spectrum must first be assigned using correlation and nuclear Overhauser effect (NOE) experiments to demonstrate through-bond (scalar) and through-space connectivities, respectively. Because the NOE is proportional to r-6, distance information can then be derived. The increased resolution afforded by extending NMR experiments into a second dimension enables one to detect and interpret effects that would not be possible in one dimension owing to extensive spectral overlap and much reduced information. A number of small protein structures have previously been solved in this way. Extending this methodology to larger proteins, however, requires yet an additional improvement in resolution as overlap of cross-peaks in the two-dimensional (2D) NMR spectra present a major barrier to their unambiguous identification. One way of increasing the resolution is to extend the 2D-NMR experiments into a third dimension. We report here the applicability of three-dimensional NMR to macromolecules using the 46-residue protein alpha 1-purothionin as an example.     

Structure of the fibronectin type 1 module

The rapid accumulation of sequence data has provided insight into the evolution of proteins and led to the identification of 'mosaic proteins'. These proteins have evolved by duplication, insertion and deletion of a common pool of structural units or modules, yet their biological functions are diverse. They are involved in cell adhesion and migration, embryogenesis and the pathways of blood clotting, fibrinolysis and complement. The modular units are defined by 'consensus sequences' which often include conserved disulphide bonds. Despite the available sequence information, little is known of the tertiary structure of mosaic proteins. If, however, the 'consensus structure' of the modules were known, valuable structural information could be inferred about a wide variety of proteins and biological systems. An important mosaic protein is fibronectin, an extracellular matrix protein that consists of three types of module (see refs 3, 7 for reviews). Here we describe the structure of the fibronectin type 1 module which appears twelve times in fibronectin and is also found in factor XII and tissue plasminogen activator. The module was produced using a yeast expression system and the structure was determined in solution using 1H NMR. This methodology promises to be extremely powerful in the investigation of modules from a wide range of mosaic proteins.     

NMR analysis of a 900K GroEL GroES complex

Biomacromolecular structures with a relative molecular mass (M(r)) of 50,000 to 100,000 (50K 100K) have been generally considered to be inaccessible to analysis by solution NMR spectroscopy. Here we report spectra recorded from bacterial chaperonin complexes ten times this size limit (up to M(r) 900K) using the techniques of transverse relaxation-optimized spectroscopy and cross-correlated relaxation-enhanced polarization transfer. These techniques prevent deterioration of the NMR spectra by the rapid transverse relaxation of the magnetization to which large, slowly tumbling molecules are otherwise subject. We tested the resolving power of these techniques by examining the isotope-labelled homoheptameric co-chaperonin GroES (M(r) 72K), either free in solution or in complex with the homotetradecameric chaperonin GroEL (M(r) 800K) or with the single-ring GroEL variant SR1 (M(r) 400K). Most amino acids of GroES show the same resonances whether free in solution or in complex with chaperonin; however, residues 17 32 show large chemical shift changes on binding. These amino acids belong to a mobile loop region of GroES that forms contacts with GroEL. This establishes the utility of these techniques for solution NMR studies that should permit the exploration of structure, dynamics and interactions in large macromolecular complexes.     

Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

Many biochemical processes proceed through the formation of functionally significant intermediates. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.     

Solid-state NMR determination of the secondary structure of Samia cynthia ricini silk

Silks are fibrous proteins that form heterogeneous, semi-crystalline solids. Silk proteins have a variety of physical properties reflecting their range of functions. Spider dragline silk, for example, has high tensile strength and elasticity, whereas other silks are better suited to making housing, egg sacs or the capture spiral of spiders' webs. The differing physical properties arise from variation in the protein's primary and secondary structure, and their packing in the solid phase. The high mechanical performance of spider dragline silk, for example, is probably due to a beta-sheet conformation of poly-alanine domains, embedded as small crystallites within the fibre. Only limited structural information can be obtained from diffraction of silks, so further characterization requires spectroscopic studies such as NMR. However, the classical approach to NMR structure determination fails because the high molecular weight, repetitive primary structure and structural heterogeneity of solid silk means that signals from individual amino-acid residues cannot be resolved. Here we adapt a recently developed solid-state NMR technique to determine torsion angle pairs (phi, psi) in the protein backbone, and we study the distribution of conformations in silk from the Eri silkworm, Samia cynthia ricini. Although the most probable conformation in native fibres is an anti-parallel beta-sheet, film produced from liquid directly extracted from the silk glands appears to be primarily alpha-helical.     

Quantitative dynamics and binding studies of the 20S proteasome by NMR

The machinery used by the cell to perform essential biological processes is made up of large molecular assemblies. One such complex, the proteasome, is the central molecular machine for removal of damaged and misfolded proteins from the cell. Here we show that for the 670-kilodalton 20S proteasome core particle it is possible to overcome the molecular weight limitations that have traditionally hampered quantitative nuclear magnetic resonance (NMR) spectroscopy studies of such large systems. This is achieved by using an isotope labelling scheme where isoleucine, leucine and valine methyls are protonated in an otherwise highly deuterated background in concert with experiments that preserve the lifetimes of the resulting NMR signals. The methodology has been applied to the 20S core particle to reveal functionally important motions and interactions by recording spectra on complexes with molecular weights of up to a megadalton. Our results establish that NMR spectroscopy can provide detailed insight into supra-molecular structures over an order of magnitude larger than those routinely studied using methodology that is generally applicable.     

三铟九钨磷酸盐的合成与表征

报道了三铟取代杂多钨磷酸钾盐、铯盐及有机胺盐的合成，并对其元素分析、红外光谱、＾１８３Ｗ核磁共振谱、氧化还原性等进行了研究，钾盐化合物的＾１８３Ｗ核磁共振谱由２个峰组成，强度比１：２，表明其阴离子仍具有Ａ型Ｋｅｇ－ｇｉｎ结构，即３个ＩｎＯ６八面体是解顶共用的。     

Size-dependent control of the binding of biotinylated proteins to streptavidin using a polymer shield

Many medical and biotechnological processes rely on controlling and manipulating the molecular-recognition capabilities of proteins. This can be achieved using small molecules capable of competing for protein binding or by changing environmental parameters that affect protein structure and hence binding. An alternative is provided by stimuli-responsive polymers that change reversibly from a water-soluble expanded coil to a water-insoluble collapsed globule upon small changes in temperature, pH or light intensity: when attached to proteins in the vicinity of their binding sites, they reversibly block and release small ligands. Here we show how this approach can be extended to achieve size-selective binding of large, macromolecular ligands. We use the thermally responsive polymer poly(N,N-diethylacrylamide) (PDEAAm), and attach it to the protein streptavidin approximately 20 A from the binding site for biotinylated proteins. Below the lower critical solution temperature of PDEAAm, the polymer is in its extended state and acts as a 'shield' to block the binding of large biotinylated proteins; above this temperature, it collapses and exposes the binding site, thereby allowing binding. We find that the degree of shielding depends on both the size of the biotinylated protein and the size of PDEAAm, suggesting that 'smart' polymer shields could be tailored to achieve a wide range of size-dependent ligand discrimination for use in affinity separations, biosensors and diagnostics technologies.     

Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy

It is generally accepted that a globular protein in its native state adopts a single, well-defined conformation. However, there have been several reports that some proteins may exist in more than one distinct folded form in equilibrium. In the case of staphylococcal nuclease, evidence for multiple conformations has come from electrophoretic and NMR studies, although there has been some controversy as to whether these are actually interconvertible forms of the same molecular species. Recently, magnetization transfer (MT)-NMR has been developed as a means of studying the kinetics of conformational transitions in proteins. In the study reported here, this approach has been extended and used to demonstrate the presence of at least two native forms of nuclease in equilibrium and to study their interconversion with the unfolded state under the conditions of the thermal unfolding transition. The experiments reveal that two distinct native forms of the protein fold and unfold independently and that these can interconvert directly as well as via the unfolded state. The spectra of the different forms suggest that they are structurally similar but the MT experiments show that the kinetics of folding and unfolding are quite different. Characterization of this behaviour will, therefore, have important implications for our understanding of the relationship between structure and folding kinetics.     

Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy

A half-century after the determination of the first three-dimensional crystal structure of a protein, more than 40,000 structures ranging from single polypeptides to large assemblies have been reported. The challenge for crystallographers, however, remains the growing of a diffracting crystal. Here we report the 4.5-A resolution structure of a 22-MDa macromolecular assembly, the capsid of the infectious epsilon15 (epsilon15) particle, by single-particle electron cryomicroscopy. From this density map we constructed a complete backbone trace of its major capsid protein, gene product 7 (gp7). The structure reveals a similar protein architecture to that of other tailed double-stranded DNA viruses, even in the absence of detectable sequence similarity. However, the connectivity of the secondary structure elements (topology) in gp7 is unique. Protruding densities are observed around the two-fold axes that cannot be accounted for by gp7. A subsequent proteomic analysis of the whole virus identifies these densities as gp10, a 12-kDa protein. Its structure, location and high binding affinity to the capsid indicate that the gp10 dimer functions as a molecular staple between neighbouring capsomeres to ensure the particle's stability. Beyond epsilon15, this method potentially offers a new approach for modelling the backbone conformations of the protein subunits in other macromolecular assemblies at near-native solution states.     

Improved molecular replacement by density- and energy-guided protein structure optimization

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2?? resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.     

Simultaneous determination of protein structure and dynamics

We present a protocol for the experimental determination of ensembles of protein conformations that represent simultaneously the native structure and its associated dynamics. The procedure combines the strengths of nuclear magnetic resonance spectroscopy--for obtaining experimental information at the atomic level about the structural and dynamical features of proteins--with the ability of molecular dynamics simulations to explore a wide range of protein conformations. We illustrate the method for human ubiquitin in solution and find that there is considerable conformational heterogeneity throughout the protein structure. The interior atoms of the protein are tightly packed in each individual conformation that contributes to the ensemble but their overall behaviour can be described as having a significant degree of liquid-like character. The protocol is completely general and should lead to significant advances in our ability to understand and utilize the structures of native proteins.     

A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations. Even though some unfolded and partially folded proteins are now known to possess biological function or to be involved in aggregation phenomena associated with disease states, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a 'Trp-cage' miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR pulse-labelling experiments that involve rapid in situ protein refolding. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.     

Three-dimensional heteronuclear NMR studies of RNA.

Multidimensional heteronuclear NMR has revolutionized solution structure determinations of proteins. But this technique has not been applied to nucleic acids because of difficulties in the synthesis of isotopically (13C and/or 15N) labelled molecules. Here we report the application of three-dimensional heteronuclear NMR to the study of a uniformly 13C/15N or 15N-labelled RNA duplex of defined sequence. These experiments simplify resonance assignment and the analysis of proton-proton nuclear Overhauser effects (and therefore distance information) in the molecule. Our results show that it is now possible to determine the structures of larger and more complex RNAs using multidimensional heteronuclear NMR.     

共缩聚物序列结构的~1H-NMR研究

建立了利用１Ｈ－ＮＭＲ和计算机处理具有重叠 ＮＭＲ谱峰的分峰技术；分析计算共缩聚物序列结构的方法。该法在ＨＤＡ－ＰＰＤ－ＴＰＣ三元共聚酰胺体系序列结构研究中应用，证明具有较好的适应性和准确性。     

Two-state kinetics characterized by image analysis of nuclear magnetic resonance spectra

Nuclear magnetic resonance (NMR) spectroscopy has become an important tool in modern biological research. NMR spectra image analysis can be used to analyze the kinetics of biomacromolecular conformational changes.The relationship between the image parameters and the protein dynamics was investigated by using a small globular protein ω-conotoxin SO3 (ω-CTX SO3). The physical meanings of the image parameters were characterized from the results. Comparison of the data from the traditional integral area of specific resonance peaks method and the NMR image analysis method showed the advantages of using NMR spectra image analysis for kinetic analysis of two-state processes monitored by 1D proton NMR.     

由二氧化碳和环氧丙烷生成的聚碳酸亚丙酯的合成与降解行为

由二氧化碳和环氧丙烷成功合成了高分子量、规则分子链结构的聚碳酸亚丙酯(PPC).13C NMR谱证明所得PPC共聚物具有交替结构.PPC的降解行为通过土壤埋藏法和溶液沉浸法来研究.结果表明在6个月后土壤埋藏的PPC膜比沉浸在缓冲溶液中的膜质量损失增加得更慢.而在缓冲溶液中沉浸的膜在最初的两个月中质量损失增加的很快,达到4.59%.吸水实验也同样显示在缓冲溶液中的PPC膜比土壤埋藏测试中吸水性更强.PPC膜的降解机理和样品的形态、红外光谱以及1H NMR谱相一致.扫描电镜形态和质量损失以及吸水测量的结果一致.     

Structure of the cyclic-AMP-responsive exchange factor Epac2 in its auto-inhibited state

Epac proteins (exchange proteins directly activated by cAMP) are guanine-nucleotide-exchange factors (GEFs) for the small GTP-binding proteins Rap1 and Rap2 that are directly regulated by the second messenger cyclic AMP and function in the control of diverse cellular processes, including cell adhesion and insulin secretion. Here we report the three-dimensional structure of full-length Epac2, a 110-kDa protein that contains an amino-terminal regulatory region with two cyclic-nucleotide-binding domains and a carboxy-terminal catalytic region. The structure was solved in the absence of cAMP and shows the auto-inhibited state of Epac. The regulatory region is positioned with respect to the catalytic region by a rigid, tripartite beta-sheet-like structure we refer to as the 'switchboard' and an ionic interaction we call the 'ionic latch'. As a consequence of this arrangement, the access of Rap to the catalytic site is sterically blocked. Mutational analysis suggests a model for cAMP-induced Epac activation with rigid body movement of the regulatory region, the features of which are universally conserved in cAMP-regulated proteins.