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1.
The average hydrophobicity of a polypeptide segment is considered to be the most important factor in the formation of transmembrane helices, and the partitioning of the most hydrophobic (MH) segment into the alternative nonpolar environment, a membrane or hydrophobic core of a globular protein may determine the type of protein produced. In order to elucidate the importance of the MH segment in determining which of the two types of protein results from a given amino acid sequence, we statistically studied the characteristics of MH helices, longer than 19 residues in length, in 97 membrane proteins whose three-dimensional structure or topology is known, as well as 397 soluble proteins selected from the Protein Data Bank. The average hydrophobicity of MH helices in membrane proteins had a characteristic relationship with the length of the protein. All MH helices in membrane proteins that were longer than 500 residues had a hydrophobicity greater than 1.75 (Kyte and Doolittle scale), while the MH helices in membrane proteins smaller than 100 residues could be as hydrophilic as 0.1. The possibility of developing a method to discriminate membrane proteins from soluble ones, based on the effect of size on the type of protein produced, is discussed. 相似文献
2.
Biochemical and structural analysis of membrane proteins often critically depends on the ability to overexpress and solubilize them. To identify properties of eukaryotic membrane proteins that may be predictive of successful overexpression, we analyzed expression levels of the genomic complement of over 1000 predicted membrane proteins in a recently completed Saccharomyces cerevisiae protein expression library. We detected statistically significant positive and negative correlations between high membrane protein expression and protein properties such as size, overall hydrophobicity, number of transmembrane helices, and amino acid composition of transmembrane segments. Although expression levels of membrane and soluble proteins exhibited similar negative correlations with overall hydrophobicity, high-level membrane protein expression was positively correlated with the hydrophobicity of predicted transmembrane segments. To further characterize yeast membrane proteins as potential targets for structure determination, we tested the solubility of 122 of the highest expressed yeast membrane proteins in six commonly used detergents. Almost all the proteins tested could be solubilized using a small number of detergents. Solubility in some detergents depended on protein size, number of transmembrane segments, and hydrophobicity of predicted transmembrane segments. These results suggest that bioinformatic approaches may be capable of identifying membrane proteins that are most amenable to overexpression and detergent solubilization for structural and biochemical analyses. Bioinformatic approaches could also be used in the redesign of proteins that are not intrinsically well-adapted to such studies. 相似文献
3.
We show that the peptide backbone of an alpha-helix places a severe thermodynamic constraint on transmembrane (TM) stability. Neglect of this constraint by commonly used hydrophobicity scales underlies the notorious uncertainty of TM helix prediction by sliding-window hydropathy plots of membrane protein (MP) amino acid sequences. We find that an experiment-based whole-residue hydropathy scale (WW scale), which includes the backbone constraint, identifies TM helices of membrane proteins with an accuracy greater than 99 %. Furthermore, it correctly predicts the minimum hydrophobicity required for stable single-helix TM insertion observed in Escherichia coli. In order to improve membrane protein topology prediction further, we introduce the augmented WW (aWW) scale, which accounts for the energetics of salt-bridge formation. An important issue for genomic analysis is the ability of the hydropathy plot method to distinguish membrane from soluble proteins. We find that the method falsely predicts 17 to 43 % of a set of soluble proteins to be MPs, depending upon the hydropathy scale used. 相似文献
4.
Patterns of alternation of hydrophobic and polar residues are a profound aspect of amino acid sequences, but a feature not easily interpreted for soluble proteins. Here we report statistics of hydrophobicity patterns in proteins of known structure in a current protein database as compared with results from earlier, more limited structure sets. Previous studies indicated that long hydrophobic runs, common in membrane proteins, are underrepresented in soluble proteins. Long runs of hydrophobic residues remain significantly underrepresented in soluble proteins, with none longer than 16 residues observed. These long runs most commonly occur as buried alpha helices, with extended hydrophobic strands less common. Avoiding aggregation of partially folded intermediates during intracellular folding remains a viable explanation for the rarity of long hydrophobic runs in soluble proteins. Comparison between database editions reveals robustness of statistics on aqueous proteins despite an approximately twofold increase in nonredundant sequences. The expanded database does now allow us to explain several deviations of hydrophobicity statistics from models of random sequence in terms of requirements of specific secondary structure elements. Comparison to prior membrane-bound protein sequences, however, shows significant qualitative changes, with the average hydrophobicity and frequency of long runs of hydrophobic residues noticeably increasing between the database editions. These results suggest that the aqueous proteins of solved structure may represent an essentially complete sample of the universe of aqueous sequences, while the membrane proteins of known structure are not yet representative of the universe of membrane-associated proteins, even by relatively simple measures of hydrophobic patterns. 相似文献
5.
The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance; thus, little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Because detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation, and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination using a proteomics approach. 相似文献
6.
Silverman BD 《Journal of biomolecular structure & dynamics》2005,22(4):411-423
Hydropathy plots or window averages over local stretches of the sequence of residue hydrophobicity have revealed patterns related to various protein tertiary structural features. This has enabled identification of regions of the sequence that are at the surface or within the interior of globular soluble proteins, regions located within the lipid bilayer of transmembrane proteins, portions of the sequence that characterize repeating motifs, as well as motifs that usefully characterize different protein structural families. This, therefore, provides one example of the generally expressed maxim that "sequence determines structure". On the other hand, a number of previous investigations have shown the rapidly varying values of residue hydrophobicity along the sequence to be distributed approximately randomly. So one might question just how much of the sequence actually determines structure. It is, therefore, of interest to extract that part of this rapidly varying distribution of residue hydrophobicity that is responsible for the longer wavelength variations that correlate with protein tertiary structural features and to determine their prevalence within the entire distribution. This is accomplished by a finite Fourier analysis of the sequence of residue hydrophobicity and of a new measure of residue distance from the protein interior. Calculations are performed on a number of globins, immunoglobulins, cuprodoxins, and papain-like structures. The spectral power of the Fourier amplitudes of the frequencies extracted, whose inverse transforms underlie the windowed values of residue hydrophobicity is shown to be a small fraction of the total power of the hydrophobicity distribution and thereby consistent with a distribution that might appear to be predominantly random. The wide range of sequence identity between proteins having the same fold, all exhibiting similar small fractions of power amplitude that correlate with the longer wavelength inside-to-outside excursions of the amino acid residues, supports the general contention that close sequence identity is an expression of a close evolutionary relationship rather than an expression of structural similarity. Practical implications of the present analysis for protein structure prediction and engineering are also described. 相似文献
7.
B. David Silverman 《Journal of biomolecular structure & dynamics》2013,31(4):411-423
Abstract Hydropathy plots or window averages over local stretches of the sequence of residue hydrophobicity have revealed patterns related to various protein tertiary structural features. This has enabled identification of regions of the sequence that are at the surface or within the interior of globular soluble proteins, regions located within the lipid bilayer of transmembrane proteins, portions of the sequence that characterize repeating motifs, as well as motifs that usefully characterize different protein structural families. This, therefore, provides one example of the generally expressed maxim that “sequence determines structure”. On the other hand, a number of previous investigations have shown the rapidly varying values of residue hydrophobicity along the sequence to be distributed approximately randomly. So one might question just how much of the sequence actually determines structure. It is, therefore, of interest to extract that part of this rapidly varying distribution of residue hydrophobicity that is responsible for the longer wavelength variations that correlate with protein tertiary structural features and to determine their prevalence within the entire distribution. This is accomplished by a finite Fourier analysis of the sequence of residue hydrophobicity and of a new measure of residue distance from the protein interior. Calculations are performed on a number of globins, immunoglobulins, cuprodoxins, and papain-like structures. The spectral power of the Fourier amplitudes of the frequencies extracted, whose inverse transforms underlie the windowed values of residue hydrophobicity is shown to be a small fraction of the total power of the hydrophobicity distribution and thereby consistent with a distribution that might appear to be predominantly random. The wide range of sequence identity between proteins having the same fold, all exhibiting similar small fractions of power amplitude that correlate with the longer wavelength inside-to- outside excursions of the amino acid residues, supports the general contention that close sequence identity is an expression of a close evolutionary relationship rather than an expression of structural similarity. Practical implications of the present analysis for protein structure prediction and engineering are also described. 相似文献
8.
《FEBS letters》1997,400(2-3):151-155
Sucrose synthase (SuSy) plays an important role in sucrose degradation and occurs both as a soluble and as a membrane-associated enzyme in higher plants. We show that membrane association can vary in vivo in response to gravistimulation, apparently involving SuSy dephosphorylation, and is a reversible process in vitro. Phosphorylation of SuSy has little effect on its activity but decreases its surface hydrophobicity as reported with the fluorescent probe bis-ANS. We postulate that phosphorylation of SuSy (and perhaps other membrane proteins) is involved in the release of the membrane-bound enzyme in part as a result of decreased surface hydrophobicity. 相似文献
9.
In spite of the overwhelming numbers and critical biological functions of membrane proteins, only a few have been characterized by high-resolution structural techniques. From the structures that are known, it is seen that their transmembrane (TM) segments tend to fold most often into alpha-helices. To evaluate systematically the features of these TM segments, we have taken two approaches: (1) using the experimentally-measured residence behavior of specifically designed hydrophobic peptides in RP-HPLC, a scale was derived based directly on the properties of individual amino acids incorporated into membrane-interactive helices: and (2) the relative alpha-helical propensity of each of the 20 amino acids was measured in the organic non-polar environment of n-butanol. By combining the resulting hydrophobicity and helical propensity data, in conjunction with consideration of the 'threshold hydrophobicity' required for spontaneous membrane integration of protein segments, an approach was developed for prediction of TM segments wherein each must fulfill the dual requirements of hydrophobicity and helicity. Evaluated against the available high-resolution structural data on membrane proteins, the present combining method is shown to provide accurate predictions for the locations of TM helices. In contrast, no segment in soluble proteins was predicted as a 'TM helix'. 相似文献
10.
Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome. 相似文献
11.
Silva AF Carvalho G Soares R Coelho AV Barreto Crespo MT 《Applied microbiology and biotechnology》2012,95(3):767-776
Extracellular polymeric substances (EPS) are keys in biomass aggregation and settleability in wastewater treatment systems. In membrane bioreactors (MBR), EPS are an important factor as they are considered to be largely responsible for membrane fouling. Proteins were shown to be the major component of EPS produced by activated sludge and to be correlated with the properties of the sludge, like settling, hydrophobicity and cell aggregation. Previous EPS proteomic studies of activated sludge revealed several problems, like the interference of other EPS molecules in protein analysis. In this study, a successful strategy was outlined to identify the proteins from soluble and bound EPS extracted from activated sludge of a lab-scale MBR. EPS samples were first subjected to pre-concentration through lyophilisation, centrifugal ultrafiltration or concentration with a dialysis membrane coated by a highly absorbent powder of polyacrylate-polyalcohol, preceded or not by a dialysis step. The highest protein concentration factors were achieved with the highly absorbent powder method without previous dialysis step. Four protein precipitation methods were then tested: acetone, trichloroacetic acid (TCA), perchloric acid and a commercial kit. Protein profiles were compared in 4–12 % sodium dodecyl sulphate polyacrylamide gel electrophoresis gels. Both acetone and TCA should be applied for the highest coverage for soluble EPS proteins, whereas TCA was the best method for bound EPS proteins. All visible bands of selected profiles were subjected to mass spectrometry analysis. A high number of proteins (25–32 for soluble EPS and 17 for bound EPS) were identified. As a conclusion of this study, a workflow is proposed for the successful proteome characterisation of soluble and bound EPS from activated sludge samples. 相似文献
12.
Zhou H Wang F Wang Y Ning Z Hou W Wright TG Sundaram M Zhong S Yao Z Figeys D 《Molecular & cellular proteomics : MCP》2011,10(10):O111.008425
Despite their importance in many biological processes, membrane proteins are underrepresented in proteomic analysis because of their poor solubility (hydrophobicity) and often low abundance. We describe a novel approach for the identification of plasma membrane proteins and intracellular microsomal proteins that combines membrane fractionation, a centrifugal proteomic reactor for streamlined protein extraction, protein digestion and fractionation by centrifugation, and high performance liquid chromatography-electrospray ionization-tandem MS. The performance of this approach was illustrated for the study of the proteome of ER and Golgi microsomal membranes in rat hepatic cells. The centrifugal proteomic reactor identified 945 plasma membrane proteins and 955 microsomal membrane proteins, of which 63 and 47% were predicted as bona fide membrane proteins, respectively. Among these proteins, >800 proteins were undetectable by the conventional in-gel digestion approach. The majority of the membrane proteins only identified by the centrifugal proteomic reactor were proteins with ≥ 2 transmembrane segments or proteins with high molecular mass (e.g. >150 kDa) and hydrophobicity. The improved proteomic reactor allowed the detection of a group of endocytic and/or signaling receptor proteins on the plasma membrane, as well as apolipoproteins and glycerolipid synthesis enzymes that play a role in the assembly and secretion of apolipoprotein B100-containing very low density lipoproteins. Thus, the centrifugal proteomic reactor offers a new analytical tool for structure and function studies of membrane proteins involved in lipid and lipoprotein metabolism. 相似文献
13.
Eichacker LA Granvogl B Mirus O Müller BC Miess C Schleiff E 《The Journal of biological chemistry》2004,279(49):50915-50922
Proteomics of membrane proteins is essential for the understanding of cellular function. However, mass spectrometric analysis of membrane proteomes has been less successful than the proteomic determination of soluble proteins. To elucidate the mystery of transmembrane proteins in mass spectrometry, we present a detailed statistical analysis of experimental data derived from chloroplast membranes. This approach was further accomplished by the analysis of the Arabidopsis thaliana proteome after in silico digestion. We demonstrate that both the length and the hydrophobicity of the proteolytic fragments containing transmembrane segments are major determinants for detection by mass spectrometry. Based on a comparative analysis, we discuss possibilities to overcome the problem and provide possible protocols to shift the hydrophobicity of transmembrane segment-containing peptides to facilitate their detection. 相似文献
14.
Protein sequence and structure relationship ARMA spectral analysis: application to membrane proteins. 总被引:2,自引:0,他引:2
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If it is assumed that the primary sequence determines the three-dimensional folded structure of a protein, then the regular folding patterns, such as alpha-helix, beta-sheet, and other ordered patterns in the three-dimensional structure must correspond to the periodic distribution of the physical properties of the amino acids along the primary sequence. An AutoRegressive Moving Average (ARMA) model method of spectral analysis is applied to analyze protein sequences represented by the hydrophobicity of their amino acids. The results for several membrane proteins of known structures indicate that the periodic distribution of hydrophobicity of the primary sequence is closely related to the regular folding patterns in a protein's three-dimensional structure. We also applied the method to the transmembrane regions of acetylcholine receptor alpha subunit and Shaker potassium channel for which no atomic resolution structure is available. This work is an extension of our analysis of globular proteins by a similar method. 相似文献
15.
Douette P Navet R Gerkens P Galleni M Lévy D Sluse FE 《Biochemical and biophysical research communications》2005,333(3):686-693
Fusing recombinant proteins to highly soluble partners is frequently used to prevent aggregation of recombinant proteins in Escherichia coli. Moreover, co-overexpression of prokaryotic chaperones can increase the amount of properly folded recombinant proteins. To understand the solubility enhancement of fusion proteins, we designed two recombinant proteins composed of uncoupling protein 1 (UCP1), a mitochondrial membrane protein, in fusion with MBP or NusA. We were able to express soluble forms of MBP-UCP1 and NusA-UCP1 despite the high hydrophobicity of UCP1. Furthermore, the yield of soluble fusion proteins depended on co-overexpression of GroEL that catalyzes folding of polypeptides. MBP-UCP1 was expressed in the form of a non-covalent complex with GroEL. MBP-UCP1/GroEL was purified and characterized by dynamic light scattering, gel filtration, and electron microscopy. Our findings suggest that MBP and NusA act as solubilizing agents by forcing the recombinant protein to pass through the bacterial chaperone pathway in the context of fusion protein. 相似文献
16.
Choksawangkarn W Edwards N Wang Y Gutierrez P Fenselau C 《Journal of proteome research》2012,11(5):3030-3034
Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins. 相似文献
17.
Véronique Cabiaux Christian Wolff Jean-Marie Ruysschaert 《International journal of biological macromolecules》1997,21(4):285-298
Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane. 相似文献
18.
We examined the variation in the solvent accessibility and hydrophobicity of the amino acids along the sequences of 58 soluble globular proteins with known tertiary structure. We found that there is a significant tendency for the accessibilities to run in clusters along the sequence but that the hydrophobicities are distributed without such nonrandom clusters. Theseresults suggest severe limitations on the power of sequence analysis tools that use average hydrophobicity scores of overlapping subsequences to predict accessibility. 相似文献
19.
Experimental folding studies of membrane proteins are more challenging than water-soluble proteins because of the higher hydrophobicity content of membrane embedded sequences and the need to provide a hydrophobic milieu for the transmembrane regions. The first challenge is their denaturation: due to the thermodynamic instability of polar groups in the membrane, secondary structures in membrane proteins are more difficult to disrupt than in soluble proteins. The second challenge is to refold from the denatured states. Successful refolding of membrane proteins has almost always been from very subtly denatured states. Therefore, it can be useful to analyze membrane protein folding using computational methods, and we will provide results obtained with simulated unfolding of membrane protein structures using the Floppy Inclusions and Rigid Substructure Topography (FIRST) method. Computational methods have the advantage that they allow a direct comparison between diverse membrane proteins. We will review here both, experimental and FIRST studies of the retinal binding proteins bacteriorhodopsin and mammalian rhodopsin, and discuss the extension of the findings to deriving hypotheses on the mechanisms of folding of membrane proteins in general. This article is part of a Special Issue entitled: Retinal Proteins—You can teach an old dog new tricks. 相似文献
20.
Hydrophobicity analyses applied to databases of soluble and transmembrane (TM) proteins of known structure were used to resolve total genomic hydrophobicity profiles into (helical) TM sequences and mainly "subhydrophobic" soluble components. This information was used to define a refined "hydrophobicity"-type TM sequence prediction scale that should approach the theoretical limit of accuracy. The refinement procedure involved adjusting scale values to eliminate differences between the average amino acid composition of populations TM and soluble sequences of equal hydrophobicity, a required property of a scale having maximum accuracy. Application of this procedure to different hydrophobicity scales caused them to collapse to essentially a single TM tendency scale. As expected, when different scales were compared, the TM tendency scale was the most accurate at predicting TM sequences. It was especially highly correlated (r = 0.95) to the biological hydrophobicity scale, derived experimentally from the percent TM conformation formed by artificial sequences passing though the translocon. It was also found that resolution of total genomic sequence data into TM and soluble components could be used to define the percent probability that a sequence with a specific hydrophobicity value forms a TM segment. Application of the TM tendency scale to whole genomic data revealed an overlap of TM and soluble sequences in the "semihydrophobic" range. This raises the possibility that a significant number of proteins have sequences that can switch between TM and non-TM states. Such proteins may exist in moonlighting forms having properties very different from those of the predominant conformation. 相似文献