共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
3.
A review is given of the use of nuclear magnetic resonance (NMR) spectroscopy to study bacteriorhodopsin and bovine rhodopsin. Solution and solid-state approaches are included. The studies of the bacterial proton pump examine the chromophore, the peptide backbone, and the protein side chains. The studies of the bovine visual pigment are limited to the chromophore. Various forms of each pigment are considered. Both structural and dynamic features are addressed. 相似文献
4.
Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR spectra recorded of the crude reaction mixture without concentration or chromatographic purification of the protein. We review methodological aspects of cell-free in vitro protein synthesis based on an Escherichia coli cell extract, in particular with regard to the production of 15 N-labeled proteins for analysis by NMR spectroscopy. 相似文献
5.
Denis Lacabanne Alons Lends Clément Danis Britta Kunert Marie-Laure Fogeron Vlastimil Jirasko Claire Chuilon Lauriane Lecoq Cédric Orelle Vincent Chaptal Pierre Falson Jean-Michel Jault Beat H. Meier Anja Böckmann 《Journal of biomolecular NMR》2017,69(2):81-91
We here adapted the GRecon method used in electron microscopy studies for membrane protein reconstitution to the needs of solid-state NMR sample preparation. We followed in detail the reconstitution of the ABC transporter BmrA by dialysis as a reference, and established optimal reconstitution conditions using the combined sucrose/cyclodextrin/lipid gradient characterizing GRecon. We established conditions under which quantitative reconstitution of active protein at low lipid-to-protein ratios can be obtained, and also how to upscale these conditions in order to produce adequate amounts for NMR. NMR spectra recorded on a sample produced by GRecon showed a highly similar fingerprint as those recorded previously on samples reconstituted by dialysis. GRecon sample preparation presents a gain in time of nearly an order of magnitude for reconstitution, and shall represent a valuable alternative in solid-state NMR membrane protein sample preparation. 相似文献
6.
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels.
This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen
atom positions. Potential applications of the new protocol may include production of proteins with selective isotope-labeling
of selected amino acid residues on a perdeuterated background for studies of enzyme active sites or for ligand screening in
drug discovery projects, as well as the synthesis of perdeuterated polypeptides for NMR spectroscopy with large supra-molecular
structures. As an illustration, it is demonstrated that the 800-kDa chaperonine GroEL synthesized with the D-S30 cell-free
system had a uniform deuteration level of about 95% and assembled into its biologically active oligomeric form. 相似文献
7.
Ayyalusamy Ramamoorthy 《生物化学与生物物理学报:生物膜》2007,1768(12):2947-2948
8.
Ramamoorthy A 《Biochimica et biophysica acta》2007,1768(12):2947-2948
9.
Warschawski DE Arnold AA Beaugrand M Gravel A Chartrand É Marcotte I 《Biochimica et biophysica acta》2011,1808(8):1957-1974
The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS. 相似文献
10.
Krueger-Koplin RD Sorgen PL Krueger-Koplin ST Rivera-Torres IO Cahill SM Hicks DB Grinius L Krulwich TA Girvin ME 《Journal of biomolecular NMR》2004,28(1):43-57
Structural information on membrane proteins lags far behind that on soluble proteins, in large part due to difficulties producing homogeneous, stable, structurally relevant samples in a membrane-like environment. In this study 25 membrane mimetics were screened using 2D (1)H-(15)N heteronuclear single quantum correlation NMR experiments to establish sample homogeneity and predict fitness for structure determination. A single detergent, 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-RAC-(1-glycerol)] (LPPG), yielded high quality NMR spectra with sample lifetimes greater than one month for the five proteins tested - R. sphaeroides LH1 alpha and beta subunits, E. coli and B. pseudofirmus OF4 ATP synthase c subunits, and S. aureus small multidrug resistance transporter - with 1, 2, or 4 membrane spanning alpha-helices, respectively. Site-specific spin labeling established interhelical distances in the drug transporter and genetically fused dimers of c subunits in LPPG consistent with in vivo distances. Optical spectroscopy showed that LH1 beta subunits form native-like complexes with bacteriochlorophyll a in LPPG. All the protein/micelle complexes were estimated to exceed 100 kDaltons by translational diffusion measurements. However, analysis of (15)N transverse, longitudinal and (15)N[(1)H] nuclear Overhauser effect relaxation measurements yielded overall rotational correlation times of 8 to 12 nsec, similar to a 15-20 kDalton protein tumbling isotropically in solution, and consistent with the high quality NMR data observed. 相似文献
11.
Mokhonova EI Mokhonov VV Akama H Nakae T 《Biochemical and biophysical research communications》2005,327(3):650-655
We developed an Escherichia coli expression system for overproduction of a highly toxic membrane protein that is impossible to overexpress by traditionally used approaches. The method is based on combination of the genetic modifications of a bicistronic expression plasmid, stabilization of a synthesized protein, and selection of a compatible expression host. This enabled us to enhance the expression level of a toxic membrane protein 30-50 times compared with expression in the native state and to obtain 3-5mg of a highly purified functionally active protein per liter of culture. We describe the method for the amplified expression of membrane proteins, using the Pseudomonas aeruginosa multidrug resistance protein, MexY, as an example. The amplified MexY was correctly folded in the cytoplasmic membrane of the E. coli without forming inclusion bodies. This method can be applicable to the large-scale expression of the other problematic membrane proteins that are otherwise extremely difficult to overproduce. 相似文献
12.
Advanced high-resolution NMR spectroscopy, including two-dimensional NMR techniques, combined with high pressure capability, represents a powerful new tool in the study of proteins. This contribution is organized in the following way. First, the specialized instrumentation needed for high-pressure NMR experiments is discussed, with specific emphasis on the design features and performance characteristics of a high-sensitivity, high-resolution, variable-temperature NMR probe operating at 500 MHz and at pressures of up to 500 MPa. An overview of several recent studies using 1D and 2D high-resolution, high-pressure NMR spectroscopy to investigate the pressure-induced reversible unfolding and pressure-assisted cold denaturation of lysozyme, ribonuclease A, and ubiquitin is presented. Specifically, the relationship between the residual secondary structure of pressure-assisted, cold-denatured states and the structure of early folding intermediates is discussed. 相似文献
13.
Dominique P Frueh Andrew C Goodrich Subrata H Mishra Scott R Nichols 《Current opinion in structural biology》2013,23(5):734-739
- Download : Download high-res image (383KB)
- Download : Download full-size image
14.
Olga Vinogradova Frank Sönnichsen Charles R. Sanders II 《Journal of biomolecular NMR》1998,11(4):381-386
Translational diffusion coefficients and catalytic activities were measured for the integral membrane protein diacylglycerol kinase (DAGK) in a variety of types of detergent micelles. Despite the structural diversity of the detergents examined, the translational diffusion coefficients observed for DAGK spanned a fairly limited range of values: 2.7 to 4.7 (× 10-7cm2/s). No general correlation was observed between the diffusion coefficients for the detergent-DAGK aggregates and the sizes of the corresponding protein-free micelles. These results indicate that the effective molecular weights of the DAGK-detergent aggregates were determined more by the structural properties of the protein than by the properties of the detergents. The catalytic activity of DAGK in detergents having medium-length alkyl chains such as dodecylphosphocholine or decylmaltoside was usually observed to be substantially higher than in short-chain detergents such as octylphosphocholine or octylglucoside. Taken together, the diffusion and activity results indicate that medium-chain detergents are generally preferred for use in NMR studies of complex membrane proteins because they are no worse than short-chained detergents in terms of increasing the effective molecular weight of the protein of interest while they are considerably better at maintaining native-like protein conformation. Among the 10 detergents examined, only sodium dodecylsulfate was observed to be unable to support DAGK activity under any conditions examined, suggesting that this well-known protein denaturant should be used with care in studies of complex membrane proteins. 相似文献
15.
Julija Romanuka Heidi van den Bulke Robert Kaptein Rolf Boelens Gert E. Folkers 《Protein expression and purification》2009,67(2):104-112
NMR studies of structural aspects of allosteric regulation by the Lac repressor requires overexpression and isotope labeling of the protein. The size of the repressor makes it a challenging target, putting constraints on both expression conditions and sample preparation methods to overcome problems associated with studies of larger proteins by NMR. We optimized protocols for the production of deuterated functionally active thermostable dimeric Lac repressor and its core domain mutants. The Lac repressor core domain has never been obtained as a recombinant protein, possibly due to the observed toxicity to the host cells. We overcame the core domain induced toxicity by co-expression of this domain with the full length Lac repressor, combined with a stringent control of culture conditions. Significant overexpression was only obtained if during all stages of pre-culturing the bacteria were kept in their exponential growth phase at low density. The sensitivity of NMR measurements is dramatically affected by buffer conditions; we therefore used a thermofluor buffer optimization screen to determine the optimal buffer conditions. The combined thermofluor and NMR screening method yielded thermostable fully functional Lac repressor domain samples suitable for high-resolution NMR studies. The optimized procedures to adapt Escherichia coli to growth in D2O, to overcome toxicity, and to optimize protein sample conditions provides a broad range of universally applicable techniques for production of larger proteins for NMR spectroscopy. 相似文献
16.
17.
Shinji Tanaka Yasushi Kawata Gottfried Otting Nicholas E. Dixon Katsumi Matsuzaki Masaru Hoshino 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(4):866-871
A novel chaperonin-encapsulation system for NMR measurements has been designed. The single-ring variant SR398 with an ATPase deficient mutation of GroEL, also known as chaperonin, bound co-chaperonin GroES irreversibly, forming a stable cage to encapsulate a target protein. A small GroEL-binding tag made it possible to perform all steps of the encapsulation under near physiological conditions while retaining the native conformation of the target protein. About half of the SR398/GroES cages encapsulated target protein molecules. As binding only depends on the 12-residue tag sequence, this encapsulation method is applicable to a large number of proteins. Isolation of the target proteins in the molecular cage of chaperonin will allow the study of highly aggregation-prone proteins by solution NMR. 相似文献
18.
Both solution and solid state nuclear magnetic resonance (NMR) techniques for structural determination are advancing rapidly such that it is possible to contemplate bringing these techniques to bear upon integral membrane proteins having multiple transmembrane segments. This review outlines existing and emerging options for model membrane media for use in such studies and surveys the special considerations which must be taken into account when preparing larger membrane proteins for NMR spectroscopic studies. 相似文献
19.
A mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. This vector was constructed to facilitate X-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. Proteins expressed with this vector possess an N-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. Advantages of this vector are high levels of expression, simple detection and purification of expressed proteins, and reliable cleavage of the fusion protein. Cotransfection of this vector with a dihydrofolate reductase gene allows amplification of expression levels with methotrexate. Over one dozen cysteine-rich secreted proteins have been expressed in sufficient quantity for structural studies using this vector; the structure of at least one of these proteins has been determined. 相似文献
20.
Thompson LK 《Current opinion in structural biology》2002,12(5):661-669
Magic-angle spinning solid-state NMR experiments are well suited to investigating the structures and mechanisms of important proteins that are inaccessible to X-ray crystallography and solution NMR spectroscopy, including membrane proteins and disease-related protein aggregates. Good progress has been made in the development of methods for the complete structure determination of small (<20 kDa) solid proteins using uniformly 13C, 15N-labeled samples. Studies of selectively labeled proteins focusing on labeled active sites have yielded insights into the mechanisms of enzymes and of membrane proteins involved in energy and signal transduction. Studies of selectively labeled synthetic peptides have yielded structural models for biomedically important systems, including amyloid fibrils and surface-associated peptides involved in biomineralization and cell adhesion. Novel NMR and biochemical methods are being developed to target solid-state NMR experiments within large proteins and whole cells. These approaches are being used to investigate mechanisms of transmembrane signaling by membrane receptors and to characterize binding interactions between antibiotics and bacterial cell walls. Thus, solid-state NMR is proving to be a valuable biophysical tool for probing structure and dynamics in a wide range of biomolecules. 相似文献