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1.
Determining the atomic resolution structures of membrane proteins is of particular interest in contemporary structural biology. Helical membrane proteins constitute one-third of the expressed proteins encoded in a genome, many drugs have membrane-bound proteins as their receptors, and mutations in membrane proteins result in human diseases. Although integral membrane proteins provide daunting technical challenges for all methods of protein structure determination, nuclear magnetic resonance (NMR) spectroscopy can be an extremely versatile and powerful method for determining their structures and characterizing their dynamics, in lipid environments that closely mimic the cell membranes. Once milligram amounts of isotopically labeled protein are expressed and purified, micelle samples can be prepared for solution NMR analysis, and lipid bilayer samples can be prepared for solid-state NMR analysis. The two approaches are complementary and can provide detailed structural and dynamic information. This paper describes the steps for membrane protein structure determination using solution and solid-state NMR. The methods for protein expression and purification, sample preparation and NMR experiments are described and illustrated with examples from the FXYD proteins, a family of regulatory subunits of the Na,K-ATPase.  相似文献   

2.
Due to hydrophobicity, structural analysis of integral membrane proteins poses a formidable challenge for current mass spectrometry-based proteomics approaches. Herein, we demonstrate results from optimized sample preparation and enzymatic proteolysis procedures for the complete primary structure determination of a targeted integral membrane protein, lens aquaporin 0 (AQP0). Plasma membrane from bovine lens tissue was alkali treated and tryptic digestion was performed in optimized acetonitrile-ammonium bicarbonate solution. Full sequence coverage of AQP0 was observed as tryptic peptides using both matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and capillary liquid chromatography tandem mass spectrometry (cLC/MS/MS). An amino acid mutation of Thr to Ile/Leu at residue 199 was deduced based on MS/MS results. In a complementary effort to fully sequence the protein, peptic digestion was developed to take advantage of hydrophobic protein solubility in organic acid as well as the decreased activity of pepsin at low pH. Peptic digestion in 10% formic acid (pH 1.2) generated peptides of 500 to 3000 Da and gave 100% sequence coverage by cLC/MS/MS. In addition to post-translational modifications reported previously, a new phosphorylation site at serine 229 and two oxidation sites at tryptophan 202 and 205 were detected on the protein. These methodologies provide complementary detergent- and CNBr-free procedures for detailed analysis of this important membrane channel protein and offer promise for analysis of the integral membrane proteome.  相似文献   

3.
Integral membrane proteins have not been readily amenable to the general methods developed for mass spectrometric (or internal Edman degradation) analysis of soluble proteins. We present here a sample preparation method and high performance liquid chromatography (HPLC) separation system which permits online HPLC-electrospray ionization mass spectrometry (ESI-MS) and -tandem mass spectrometry (MS/MS) analysis of cyanogen bromide cleavage fragments of integral membrane proteins. This method has been applied to wild type (WT) bacteriorhodopsin (bR), cysteine containing mutants of bR, and the prototypical G-protein coupled receptor, rhodopsin (Rh). In the described method, the protein is reduced and the cysteine residues pyridylethylated prior to separating the protein from the membrane. Following delipidation, the pyridylethylated protein is cleaved with cyanogen bromide. The cleavage fragments are separated by reversed phase HPLC using an isopropanol/acetonitrile/aqueous TFA solvent system and the effluent peptides analyzed online with a Finnigan LCQ Ion Trap Mass Spectrometer. With the exception of single amino acid fragments and the glycosylated fragment of Rh, which is observable by matrix assisted laser desorption ionization (MALDI)-MS, this system permits analysis of the entire protein in a single HPLC run. This methodology will enable pursuit of chemical modification and crosslinking studies designed to probe the three dimensional structures and functional conformational changes in these proteins. The approach should also be generally applicable to analysis of other integral membrane proteins.  相似文献   

4.
The preparation of high quality samples is a critical challenge for the structural characterization of helical integral membrane proteins. Solving the structures of this diverse class of proteins by solution nuclear magnetic resonance spectroscopy (NMR) requires that well-resolved 2D 1H/15N chemical shift correlation spectra be obtained. Acquiring these spectra demands the production of samples with high levels of purity and excellent homogeneity throughout the sample. In addition, high yields of isotopically enriched protein and efficient purification protocols are required. We describe two robust sample preparation methods for preparing high quality, homogeneous samples of helical integral membrane proteins. These sample preparation protocols have been combined with screens for detergents and sample conditions leading to the efficient production of samples suitable for solution NMR spectroscopy. We have examined 18 helical integral membrane proteins, ranging in size from approximately 9 kDa to 29 kDa with 1–4 transmembrane helices, originating from a number of bacterial and viral genomes. 2D 1H/15N chemical shift correlation spectra acquired for each protein demonstrate well-resolved resonances, and >90% detection of the predicted resonances. These results indicate that with proper sample preparation, high quality solution NMR spectra of helical integral membrane proteins can be obtained greatly enhancing the probability for structural characterization of these important proteins.  相似文献   

5.
Recent years have seen the establishment of structural genomics centers that explicitly target integral membrane proteins. Here, we review the advances in targeting these extremely high-hanging fruits of structural biology in high-throughput mode. We observe that the experimental determination of high-resolution structures of integral membrane proteins is increasingly successful both in terms of getting structures and of covering important protein families, for example, from Pfam. Structural genomics has begun to contribute significantly toward this progress. An important component of this contribution is the set up of robotic pipelines that generate a wealth of experimental data for membrane proteins. We argue that prediction methods for the identification of membrane regions and for the comparison of membrane proteins largely suffice to meet the challenges of target selection for structural genomics of membrane proteins. In contrast, we need better methods to prioritize the most promising members in a family of closely related proteins and to annotate protein function from sequence and structure in absence of homology.  相似文献   

6.
《Molecular membrane biology》2013,30(5-8):156-178
Abstract

Solid-state NMR is unique for its ability to obtain three-dimensional structures and to measure atomic-resolution structural and dynamic information for membrane proteins in native lipid bilayers. An increasing number and complexity of integral membrane protein structures have been determined by solid-state NMR using two main methods. Oriented sample solid-state NMR uses macroscopically aligned lipid bilayers to obtain orientational restraints that define secondary structure and global fold of embedded peptides and proteins and their orientation and topology in lipid bilayers. Magic angle spinning (MAS) solid-state NMR uses unoriented rapidly spinning samples to obtain distance and torsion angle restraints that define tertiary structure and helix packing arrangements. Details of all current protein structures are described, highlighting developments in experimental strategy and other technological advancements. Some structures originate from combining solid- and solution-state NMR information and some have used solid-state NMR to refine X-ray crystal structures. Solid-state NMR has also validated the structures of proteins determined in different membrane mimetics by solution-state NMR and X-ray crystallography and is therefore complementary to other structural biology techniques. By continuing efforts in identifying membrane protein targets and developing expression, isotope labelling and sample preparation strategies, probe technology, NMR experiments, calculation and modelling methods and combination with other techniques, it should be feasible to determine the structures of many more membrane proteins of biological and biomedical importance using solid-state NMR. This will provide three-dimensional structures and atomic-resolution structural information for characterising ligand and drug interactions, dynamics and molecular mechanisms of membrane proteins under physiological lipid bilayer conditions.  相似文献   

7.
8.
Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.  相似文献   

9.
An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.  相似文献   

10.
Membrane protein structural biology is a frontier area of modern biomedical research. Twenty to thirty-five percent of the proteins encoded by an organism's genome are integral membrane proteins. Integral membrane proteins, such as channels, transporters, and receptors, are critical components of many fundamental biological processes. Also, many integral membrane proteins are important in biomedical and biotechnological applications; the majority of drug targets are integral membrane proteins. The sharp increase in the number of membrane protein structures over the last several years gives some indication that this field is poised for rather explosive growth as more and more investigators take on membrane protein projects. The purpose of this brief practical review was to take a snapshot of a field at the onset of its likely exponential growth phase, and to lay out the methods that have worked to date for obtaining membrane protein crystals suitable for structure determination by X-ray crystallography. Many of the successful experimental methods are identical to those used for soluble proteins. The major difference, and a non-trivial difference, is the necessity for inclusion of detergents above the critical micelle concentration in the purified membrane protein solution.  相似文献   

11.
Rotational Alignment (RA) solid-state NMR provides the basis for a general method for determining the structures of membrane proteins in phospholipid bilayers under physiological conditions. Membrane proteins are high priority targets for structure determination, and are challenging for existing experimental methods. Because membrane proteins reside in liquid crystalline phospholipid bilayer membranes it is important to study them in this type of environment. The RA solid-state NMR approach we have developed can be summarized in five steps, and incorporates methods of molecular biology, biochemistry, sample preparation, the implementation of NMR experiments, and structure calculations. It relies on solid-state NMR spectroscopy to obtain high-resolution spectra and residue-specific structural restraints for membrane proteins that undergo rotational diffusion around the membrane normal, but whose mobility is otherwise restricted by interactions with the membrane phospholipids. High resolution spectra of membrane proteins alone and in complex with other proteins and ligands set the stage for structure determination and functional studies of these proteins in their native, functional environment.  相似文献   

12.
High stability is a prominent characteristic of integral membrane proteins of known atomic structure. But rather than being an intrinsic property, it may be due to a selection exerted by biochemical procedures prior to structure determination, since solubilization results in the transient exposure of membrane proteins to solution conditions. This may cause structural perturbations that interfere with 3D crystallization and hence with X-ray analysis. This problem also affects the preparation of samples for electron crystallography and NMR studies and may account for the fact that high-resolution structures of representatives of whole groups, such as transport proteins and signal transducers, have not been elucidated so far by any method. A knowledge of the proportion of labile proteins among membrane proteins, and of the kinetics of their denaturation, is therefore necessary. Establishing stability profiles, developing methods to maintain lateral pressure, or preventing contact with water (or both) should prove significant in establishing the structures of conformationally flexible proteins.  相似文献   

13.
The structural characterization of small integral membrane proteins pose a significant challenge for structural biology because of the multitude of molecular interactions between the protein and its heterogeneous environment. Here, the three‐dimensional backbone structure of Rv1761c from Mycobacterium tuberculosis has been characterized using solution NMR spectroscopy and dodecylphosphocholine (DPC) micelles as a membrane mimetic environment. This 127 residue single transmembrane helix protein has a significant (10 kDa) C‐terminal extramembranous domain. Five hundred and ninety distance, backbone dihedral, and orientational restraints were employed resulting in a 1.16 Å rmsd backbone structure with a transmembrane domain defined at 0.40 Å. The structure determination approach utilized residual dipolar coupling orientation data from partially aligned samples, long‐range paramagnetic relaxation enhancement derived distances, and dihedral restraints from chemical shift indices to determine the global fold. This structural model of Rv1761c displays some influences by the membrane mimetic illustrating that the structure of these membrane proteins is dictated by a combination of the amino acid sequence and the protein's environment. These results demonstrate both the efficacy of the structural approach and the necessity to consider the biophysical properties of membrane mimetics when interpreting structural data of integral membrane proteins and, in particular, small integral membrane proteins.  相似文献   

14.
Attempts at protein profiling in the alkaline pH region using isoelectric focusing have often proved difficult, greatly limiting the scope of proteome analysis. We investigated several parameters using custom pH 8-11 immobilized pH gradients to separate a Caulobacter crescentus membrane preparation. These included sample application, quenching endoosomotic flow and gel matrix composition. Among these factors, the sample application position was the predominant parameter to affect two-dimensional gel quality. Separated proteins were silver stained and profiled using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The use of a prototype MALDI-Q-Tof mass spectrometer assisted identification of several proteins by providing highly informative peptide fragmentation data from the sample digests. Thirty-two unique alkaline proteins were identified in this study, which complements our previously described C. crescentus membrane proteome. Our experiments point towards new options for proteomic researchers aiming to both extend the scope of analysis, and simplify methods of identifying proteins with high confidence.  相似文献   

15.
Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank. Solid-state Nuclear Magnetic Resonance (SSNMR) has long been considered as an attractive alternative because it allows for studies of membrane proteins in both native-like membranes composed of synthetic lipids and in cell membranes. Over the past decade, SSNMR has been rapidly developing into a major structural method, and a growing number of membrane protein structures obtained by this technique highlights its potential. Here we discuss membrane protein sample requirements, review recent progress in SSNMR methodologies, and describe recent advances in characterizing membrane proteins in the environment of a cellular membrane.  相似文献   

16.
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.  相似文献   

17.
Abstract

The number of structures of integral membrane proteins from higher eukaryotes is steadily increasing due to a number of innovative protein engineering and crystallization strategies devised over the last few years. However, it is sobering to reflect that these structures represent only a tiny proportion of the total number of membrane proteins encoded by a mammalian genome. In addition, the structures determined to date are of the most tractable membrane proteins, i.e., those that are expressed functionally and to high levels in yeast or in insect cells using the baculovirus expression system. However, some membrane proteins that are expressed inefficiently in these systems can be produced at sufficiently high levels in mammalian cells to allow structure determination. Mammalian expression systems are an under-used resource in structural biology and represent an effective way to produce fully functional membrane proteins for structural studies. This review will discuss examples of vertebrate membrane protein overexpression in mammalian cells using a variety of viral, constitutive or inducible expression systems.  相似文献   

18.
Membrane proteins represent up to 30% of the proteins in all organisms, they are involved in many biological processes and are the molecular targets for around 50% of validated drugs. Despite this, membrane proteins represent less than 1% of all high-resolution protein structures due to various challenges associated with applying the main biophysical techniques used for protein structure determination. Recent years have seen an explosion in the number of high-resolution structures of membrane proteins determined by NMR spectroscopy, especially for those with multiple transmembrane-spanning segments. This is a review of the structures of polytopic integral membrane proteins determined by NMR spectroscopy up to the end of the year 2010, which includes both β-barrel and α-helical proteins from a number of different organisms and with a range in types of function. It also considers the challenges associated with performing structural studies by NMR spectroscopy on membrane proteins and how some of these have been overcome, along with its exciting potential for contributing new knowledge about the molecular mechanisms of membrane proteins, their roles in human disease, and for assisting drug design.  相似文献   

19.
链霉菌是一类具有重要工业价值和复杂调控机制的微生物,天蓝色链霉菌是这个属的模式菌。已报道天蓝色链霉菌的蛋白组学的研究多采用二维聚丙烯酰胺凝胶电泳与基质辅助激光解吸电离飞行时间质谱相结合的方法,但由于膜蛋白疏水性较强,天然丰度较低,此法得到的膜蛋白很少。用蛋白酶K保护/高pH蛋白酶K法制备链霉菌膜内侧蛋白组样品,并用多维蛋白鉴别技术进行分析,得到154个可能的膜内侧蛋白(包括膜内在蛋白和膜外周蛋白),其中含跨膜区的膜内在蛋白44个,含3个以上跨膜区的膜内在蛋白有23个。此外,还鉴定了一批膜内侧蛋白的亲水性肽段及其在膜上的拓扑位置。该结果有助于对天蓝色链霉菌的膜蛋白进行拓扑学分类和功能研究。  相似文献   

20.
Membrane transporter proteins play critical physiological roles in the cell and constitute 5-10% of prokaryotic and eukaryotic genomes. High-resolution structural information is essential for understanding the functional mechanism of these proteins. A prerequisite for structural study is to overexpress such proteins in large quantities. In the last few years, over 20 bacterial membrane transporters were overexpressed at a level of 1 mg/l of culture or higher, most often in Escherichia coli. In this review, we analyzed those factors that affect the quantity and quality of the protein produced, and summarized recent progress in overexpression of membrane transporters from bacterial inner membrane. Rapid progress in genome sequencing provides opportunities for expressing several homologues and orthologues of the target protein simultaneously, while the availability of various expression vectors allows flexible experimental design. Careful optimization of cell culture conditions can drastically improve the expression level and homogeneity of the target protein. New sample preparation techniques for mass spectrometry of membrane proteins have enabled one to identity the rigid protein core, which can be subsequently overexpressed. Size-exclusion chromatography on HPLC has proven to be an efficient method in screening detergent, pH an other conditions required for maintaining the stability and monodispersity of the protein. Such high-quality preparations of membrane transporter proteins will probably lead to successful crystallization and structure determination of these proteins in the next few years.  相似文献   

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