Characterization of two antigenically related integral membrane proteins of the guinea pig sperm periacrosomal plasma membrane

The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35-kD and 52-kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase-separation analysis with Triton X-114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore-induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

温和过甲酸氧化辅助酶解结合液质联用技术鉴定大鼠大脑皮层质膜蛋白质

由于膜蛋白质尤其是内在膜蛋白的强疏水性,分析和鉴定质膜蛋白质仍然是以质谱为基础的蛋白质组学的方法中的一个难点.过甲酸氧化是一种应用广泛的打开二硫键的方法,温和的过甲酸试剂能完全的将半胱氨酸转化为半胱磺酸,将甲硫氨酸转化为甲硫氨酸砜,从而使目的蛋白更易溶于水介质.采用蔗糖密度梯度离心法纯化得到大鼠大脑皮层质膜,提取的质膜蛋白质经温和过甲酸氧化处理后经胰酶酶解消化得到肽段,利用LC-MS/MS对所得肽段进行质谱分析,采集的原始数据用Mascot软件进行库搜寻鉴定.此方法是研究质膜蛋白质的新方法,温和过甲酸氧化显示出很好的氧化效果却避免其它不利于鉴定的副反应.从大鼠大脑皮层膜提取物共鉴定出220种蛋白质,其中73种为整合膜蛋白,证明对质膜蛋白质直接进行温和过甲酸氧化然后酶解的方法辅助酶解可以有效的鉴定质膜蛋白质.     

Protein identification by in-gel digestion and mass spectrometric analysis

This protocol details a method for the identification of proteins that have been separated by gel electrophoresis. In-gel digestion of the protein bands with trypsin followed by quadrupole ion-trap or other triple quadrupole mass spectrometry techniques is described. The proteins can be identified by database searching of the mass fingerprint of the intact peptides and of the characteristic fragment masses produced by tandem mass spectrometry.     

Complexation of integral membrane proteins by phosphorylcholine-based amphipols

Amphiphilic macromolecules, known as amphipols, have emerged as promising candidates to replace conventional detergents for handling integral membrane proteins in water due to the enhanced stability of protein/amphipol complexes as compared to protein/detergent complexes. The limited portfolio of amphipols currently available prompted us to develop amphipols bearing phosphorylcholine-based units (PC). Unlike carboxylated polymers, PC-amphipols remain soluble in aqueous media under conditions of low pH, high salt concentration, or in the presence of divalent ions. The solubilizing properties of four PC-amphipols were assessed in the case of two membrane proteins, cytochrome b₆f and bacteriorhodopsin. The protein/PC-amphipol complexes had a low dispersity in size, as determined by rate zonal ultracentrifugation. Short PC-amphipols (<M>≈ 22 kDa) of low dispersity in length, containing ∼ 30 mol% octyl side groups, ∼ 35 mol% PC-groups, and ∼ 35 mol% isopropyl side groups, appeared best suited to form stable complexes, preserving the native state of BR over periods of several days. BR/PC-amphipol complexes remained soluble in aqueous media at pH ≥ 5, as well as in the presence of 1 M NaCl or 12 mM calcium ions. Results from isothermal titration calorimetry indicated that the energetics of the conversion of BR/detergent complexes into BR/amphipol complexes are similar for PC-amphipols and carboxylated amphiphols.     

Properties of integral membrane protein structures: derivation of an implicit membrane potential

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.     

Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin

Reassembly of OmpF porin from its denatured monomer into the sodium dodecyl sulfate-resistant species was investigated by using 27 kinds of mild surfactants. Polyethyleneoxide-type surfactants with a hydrophilic-lipophilic balance value of 10.8–14.6 induced the trimerization of denatured OmpF porin. Dimerization and trimerization were induced by non-polyethyleneoxide-type mild surfactants that are generally used for membrane protein solubilization. The dependence of surfactant concentrations on reassembly was estimated to obtain a minimal concentration required for the reassembly of the protein. Extensive reassembly (85% yield) into dimer (a putative assembly intermediate) was observed at a protein concentration of 0.05 mg/ml in 7 mg/ml n-octyl--d-glucopyranoside and 1 mg/ml sodium dodecyl sulfate. This condition will be useful for the studies of the dimer and dimerization of OmpF porin. The role of mixed micelle system on the protein renaturation was discussed.     

Prediction of membrane protein types based on the hydrophobic index of amino acids

A new algorithm to predict the types of membrane proteins is proposed. Besides the amino acid composition of the query protein, the information within the amino acid sequence is taken into account. A formulation of the autocorrelation functions based on the hydrophobicity index of the 20 amino acids is adopted. The overall predictive accuracy is remarkably increased for the database of 2054 membrane proteins studied here. An improvement of about 13% in the resubstitution test and 8% in the jackknife test is achieved compared with those of algorithms based merely on the amino acid composition. Consequently, overall predictive accuracy is as high as 94% and 82% for the resubstitution and jackknife tests, respectively, for the prediction of the five types. Since the proposed algorithm is based on more parameters than those in the amino acid composition approach, the predictive accuracy would be further increased for a larger and more class-balanced database. The present algorithm should be useful in the determination of the types and functions of new membrane proteins. The computer program is available on request.     

Liver fatty acid binding protein enhances sterol transfer by membrane interaction

Among the large family of fatty acid binding proteins, the liver L-FABP is unique in that it not only binds fatty acids but also interacts with sterols to enhance sterol transfer between membranes. Nevertheless, the mechanism whereby L-FABP potentiates intermembrane sterol transfer is unknown. Both fluorescence and dialysis data indicate L-FABP mediated sterol transfer between L-cell fibroblast plasma membranes occurs by a direct membrane effect: First, dansylated-L-FABP (DNS-L-FABP) is bound to L-cell fibroblast plasma membranes as indicated by increased DNS-L-FABP steady state polarization and phase resolved limiting anisotropy. Second, coumarin-L-FABP (CPM-L-FABP) fluorescence lifetimes were significantly increased upon interaction with plasma membranes. Third, dialysis studies with³H-cholesterol loaded plasma membranes showed that L-FABP added to the donor compartment of the dialysis cell stimulated³H-cholesterol transfer whether or not the dialysis membrane was permeable to L-FABP. However, L-FABP mediated intermembrane sterol transfer did require a sterol binding site on L-FABP. Chemically blocking the ligand binding site also inhibited L-FABP activity in intermembrane sterol transfer. Finally, L-FABP did not act either as an aqueous carrier or in membrane fusion. The fact that L-FABP interacted with plasma membrane vesicles and required a sterol binding site was consistent with a mode of action whereby L-FABP binds to the membrane prior to releasing sterol from the bilayer.Abbreviations ³H-CHO [1,2-³H(N)]-cholesterol - ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - CF carboxyfluorescein - CHO cholesterol - CPM (coumarin maleimide) 7-diethylamino-3-(4-maleimidylphenyl)-4-methylcoumarin - cPNA cisparinaric acid - DHE (dehydroergosterol) ^5,7,9(11),22-ergostatetraen-3-ol - DMF dimethyl formamide - DMPOPOP 1,4-bis[4-methyl-5-phenyl-2-oxazolyl]benzene - DNS (dansyl chloride) 5-dimethylaminonaphthalene-1-sulfonylchloride - DPX p-xylene-bis-pyridinium bromide - FBS fetal bovine serum - fluorescamine 4-phenylspiro[furan-2(3H), 1 phthalan]-3,3-dione - L-FABP liver fatty acid binding protein - NPG p-nitrophenylglyoxal - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine - SUV small unilamellar vesicle(s) - TNM tetranitromethane This work was supported in part by the National Institutes of Health United States Public Health Service (GM31651 and DK41402) and the American Heart Association (Postdoctoral Fellowship to JKW). The helpful assistance of Dr. Scott M. Colles and Mr. Daniel R. Prows in isolating L-FABP was much appreciated.     

The GEF1 gene of Saccharomyces cerevisiae encodes an integral membrane protein; mutations in which have effects on respiration and iron-limited growth

We have isolated a new class of respiration-defective, i.e petite, mutants of the yeast Saccharomyces cerevisiae. Mutations in the GEF1 gene cause cells to grow slowly on rich media containing carbon sources utilized by respiration. This phenotype is suppressed by adding high concentrations of iron to the growth medium. Gef1 ^– mutants also fail to grow on a fermentable carbon source, glucose, when iron is reduced to low concentrations in the medium, suggesting that the GEF1 gene is required for efficient metabolism of iron during growth on fermentable as well as respired carbon sources. However, activity of the iron uptake system appears to be unaffected in gef1 ^– mutants. Fe(II) transporter activity and regulation is normal in gef1 ^– mutants. Fe(III) reductase induction during iron-limited growth is disrupted, but this appears to be a secondary effect of growth rate alterations. The wild-type GEF1 gene was cloned and sequenced; it encodes a protein of 779 amino acids, 13 possible transmembrane domains, and significant similarity to chloride channel proteins from fish and mammals, suggesting that GEF1 encodes an integral membrane protein. A gef1 ^– deletion mutation generated in vitro and introduced into wild-type haploid strains by gene transplacement was not lethal. Oxygen consumption by intact gef1 ^– cells and by mitochondrial fractions isolated from gef1 ^– mutants was reduced 25–50% relative to wild type, indicating that mitochondrial function is defective in these mutants. We suggest that GEF1 encodes a transport protein that is involved in intracellular iron metabolism.     

NMR structure of the integral membrane protein OmpX

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.     

Sodium-deoxycholate-assisted tryptic digestion and identification of proteolytically resistant proteins

Identification of proteolytically resistant proteins with compact molecular structure and/or poor water solubility is a challenge in current proteomic study. In this study, sodium deoxycholate (SDC)-assisted tryptic digestion and identification of proteolytically resistant myoglobin and integral membrane proteins were systematically investigated. When the effect of SDC up to 10% on trypsin activity was investigated, little decrease in the trypsin activity was observed in 1% SDC solution, 2-5% SDC decreased the enzyme activity only by about 13.6%, and even in the presence of 10% SDC trypsin still retained 77.4% of its activity. Matrix-assisted laser desorption ionization time of flight mass spectrometry analysis showed that SDC could be removed from sample solution with acid treatment followed by centrifugation, and the remaining SDC, if any, had little effect on mass spectrometry analysis with regard to the number and signal/noise ratio of ions in the mass spectra. Compared with urea and methanol, two other commonly used additives in addition to SDS in proteomic analysis, SDC improved more efficiently the denaturation, solubilization, and tryptic digestion of proteins, particularly proteolytically resistant myoglobin and integral membrane proteins, thereby enhancing the efficiency of their identification with regard to the number of identified proteins and unique peptides and the sequence coverage of matched proteins.     

Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification

Acrylamide concentration, urea content, and the trailing ion used for sodium dodecyl sulfate (SDS)-gels modify electrophoretic protein mobilities in a protein-dependent way. Varying these parameters we coupled two SDS-gels to a two-dimensional (2-D) electrophoresis system. Protein spots in 2-D gels are dispersed around a diagonal. Hydrophobic proteins are well separated from water-soluble proteins which is the essential advantage of the novel technique. Mass spectrometric identification of previously unaccessible hydrophobic proteins is now possible.     

包封膜蛋白的仿生细胞膜

本文概述了脂质囊泡的组成成分和制作方法以及用于膜蛋白方面研究的相关技术,包括膜蛋白整合到囊泡的方法、复合体系的表征等。脂质囊泡可以为膜蛋白提供类似体内的环境,包括疏水区和内外亲水环境,因其组分单一,可以方便地进行结构、功能、信号转导等方面的研究,因此可以模拟细胞膜作为研究膜蛋白的有力工具,目前大多是以脂质体形态作为仿生囊泡体系进行这方面研究。     

Using optimized evidence-theoretic K-nearest neighbor classifier and pseudo-amino acid composition to predict membrane protein types

Knowledge of membrane protein type often provides crucial hints toward determining the function of an uncharacterized membrane protein. With the avalanche of new protein sequences emerging during the post-genomic era, it is highly desirable to develop an automated method that can serve as a high throughput tool in identifying the types of newly found membrane proteins according to their primary sequences, so as to timely make the relevant annotations on them for the reference usage in both basic research and drug discovery. Based on the concept of pseudo-amino acid composition [K.C. Chou, Proteins: Struct. Funct. Genet. 43 (2001) 246-255; Erratum: Proteins: Struct. Funct. Genet. 44 (2001) 60] that has made it possible to incorporate a considerable amount of sequence-order effects by representing a protein sample in terms of a set of discrete numbers, a novel predictor, the so-called "optimized evidence-theoretic K-nearest neighbor" or "OET-KNN" classifier, was proposed. It was demonstrated via the self-consistency test, jackknife test, and independent dataset test that the new predictor, compared with many previous ones, yielded higher success rates in most cases. The new predictor can also be used to improve the prediction quality for, among many other protein attributes, structural class, subcellular localization, enzyme family class, and G-protein coupled receptor type. The OET-KNN classifier will be available as a web-server at http://www.pami.sjtu.edu.cn/kcchou.     

糜蛋白酶提高膜蛋白质谱分析序列覆盖率的胶内消化方法

近年来,质谱技术在膜蛋白结构与功能研究中被广泛应用。由于膜蛋白的跨膜结构域含有大量疏水性氨基酸,常常导致液质串联质谱检测的序列覆盖率较低,从而限制了质谱技术在膜蛋白结构与功能研究中的应用。文中利用人的整合膜蛋白维生素K环氧化物还原酶为模型,优化胶内消化条件,建立了一种稳定提高膜蛋白质谱序列覆盖率的糜蛋白酶胶内消化方法。通过探索钙离子浓度、pH值和缓冲体系对序列覆盖率、检测特异肽段的总数和类型以及特异肽段大小的影响,发现在5–10 mmol/L钙离子浓度、pH 8.0–8.5的Tris-HCl缓冲液中,可以兼顾序列覆盖率和肽段的多样性。该方法可以使膜蛋白的质谱覆盖率达到80%以上,将在膜蛋白结构与功能、膜蛋白相互作用位点的鉴定以及膜蛋白与小分子药物结合位点的鉴定等研究中具有广泛的应用价值。     

High-performance liquid chromatography separation and intact mass analysis of detergent-solubilized integral membrane proteins

We have developed a method for intact mass analysis of detergent-solubilized and purified integral membrane proteins using liquid chromatography–mass spectrometry (LC–MS) with methanol as the organic mobile phase. Membrane proteins and detergents are separated chromatographically during the isocratic stage of the gradient profile from a 150-mm C3 reversed-phase column. The mass accuracy is comparable to standard methods employed for soluble proteins; the sensitivity is 10-fold lower, requiring 0.2–5 μg of protein. The method is also compatible with our standard LC–MS method used for intact mass analysis of soluble proteins and may therefore be applied on a multiuser instrument or in a high-throughput environment.     

Controlling the folding efficiency of an integral membrane protein

Research into the folding mechanisms of integral membrane proteins lags far behind that of water-soluble proteins, to the extent that the term protein folding is synonymous with water-soluble proteins. Hydrophobic membrane proteins, and particularly those with transmembrane alpha-helical motifs, are frequently considered too difficult to work with. We show that the stored curvature elastic stress of lipid bilayers can be used to guide the design of efficient folding systems for these integral membrane proteins. The curvature elastic stress of synthetic phosphatidylcholine/phosphatidylethanolamine lipid bilayers can be used to control both the rate of folding and the yield of folded protein. The use of a physical bilayer property generalises this approach beyond the particular chemistry of the lipids involved.     

A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.     

Mapping the membrane proteome of Corynebacterium glutamicum

In order to avoid the specific problems with intrinsic membrane proteins in proteome analysis, a new procedure was developed which is superior to the classical two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) method in terms of intrinsic membrane proteins. For analysis of the membrane proteome from Corynebacterium glutamicum, we replaced the first separation dimension, i.e., the isoelectric focusing step, by anion-exchange chromatography, followed by sodium dodecyl sulfate (SDS)-PAGE in the second separation dimension. Enrichment of the membrane intrinsic subproteome was achieved by washing with 2.5 M NaBr which removed more than 35% of the membrane-associated soluble proteins. For the extraction and solubilization of membrane proteins, the detergent amidosulfobetaine 14 (ASB-14) was most efficient in a detailed screening procedure and proved also suitable for chromatography. 356 gel bands were spotted, and out of 170 different identified proteins, 50 were membrane-integral. Membrane proteins with one up to 13 transmembrane helices were found. Careful analysis revealed that this new procedure covers proteins from a wide pI range (3.7-10.6) and a wide mass range of 10-120 kDa. About 50% of the identified membrane proteins belong to various functional categories like energy metabolism, transport, signal transduction, protein translocation, and proteolysis while for the others a function is not yet known, indicating the potential of the developed method for elucidation of membrane proteomes in general.