Prediction of partial membrane protein topologies using a consensus approach

We have developed a method to reliably identify partial membrane protein topologies using the consensus of five topology prediction methods. When evaluated on a test set of experimentally characterized proteins, we find that approximately 90% of the partial consensus topologies are correctly predicted in membrane proteins from prokaryotic as well as eukaryotic organisms. Whole-genome analysis reveals that a reliable partial consensus topology can be predicted for approximately 70% of all membrane proteins in a typical bacterial genome and for approximately 55% of all membrane proteins in a typical eukaryotic genome. The average fraction of sequence length covered by a partial consensus topology is 44% for the prokaryotic proteins and 17% for the eukaryotic proteins in our test set, and similar numbers are found when the algorithm is applied to whole genomes. Reliably predicted partial topologies may simplify experimental determinations of membrane protein topology.     

A simple method for predicting transmembrane alpha helices with better accuracy.

The prediction of a protein's structure from its amino acid sequence has been a long-standing goal of molecular biology. In this work, a new set of conformational parameters for membrane spanning alpha helices was developed using the information from the topology of 70 membrane proteins. Based on these conformational parameters, a simple algorithm has been formulated to predict the transmembrane alpha helices in membrane proteins. A FORTRAN program has been developed which takes the amino acid sequence as input and gives the predicted transmembrane alpha-helices as output. The present method correctly identifies 295 transmembrane helical segments in 70 membrane proteins with only two overpredictions. Furthermore, this method predicts all 45 transmembrane helices in the photosynthetic reaction center, bacteriorhodopsin and cytochrome c oxidase to an 86% level of accuracy and so is better than all other methods published to date.     

The distribution of charged amino acids in mitochondrial inner-membrane proteins suggests different modes of membrane integration for nuclearly and mitochondrially encoded proteins.

We have analyzed the amino acid distribution in seven nuclearly encoded and five mitochondrially encoded inner membrane proteins with experimentally well characterized topologies. The mitochondrially encoded proteins conform to the 'positive inside' rule, i.e. they have many more positively charged residues in their non-translocated as compared to translocated domains. However, most of the nuclearly encoded proteins do not show such a bias but instead have a surprisingly skewed distribution of Glu residues with an almost ten times higher frequency in the intermembrane space than in the matrix domains. These findings suggest that some, but possibly not all, nuclearly encoded inner membrane proteins may insert into the membrane by a mechanism that does not depend on the distribution of positively charged amino acids.     

随机森林方法预测膜蛋白类型

膜蛋白的类型与其功能是密切相关的,因此膜蛋白类型的预测是研究其功能的重要手段,从蛋白质的氨基酸序列出发对膜蛋白的类型进行预测有重要意义。文章基于蛋白质的氨基酸序列,将组合离散增量和伪氨基酸组分信息共同作为预测参数,采用随机森林分类器,对8类膜蛋白进行了预测。在Jackknife检验下的预测精度为86.3%,独立检验的预测精度为93.8%,取得了好于前人的预测结果。     

Discrimination of outer membrane proteins using support vector machines

MOTIVATION: Discriminating outer membrane proteins from other folding types of globular and membrane proteins is an important task both for dissecting outer membrane proteins (OMPs) from genomic sequences and for the successful prediction of their secondary and tertiary structures. RESULTS: We have developed a method based on support vector machines using amino acid composition and residue pair information. Our approach with amino acid composition has correctly predicted the OMPs with a cross-validated accuracy of 94% in a set of 208 proteins. Further, this method has successfully excluded 633 of 673 globular proteins and 191 of 206 alpha-helical membrane proteins. We obtained an overall accuracy of 92% for correctly picking up the OMPs from a dataset of 1087 proteins belonging to all different types of globular and membrane proteins. Furthermore, residue pair information improved the accuracy from 92 to 94%. This accuracy of discriminating OMPs is higher than that of other methods in the literature, which could be used for dissecting OMPs from genomic sequences. AVAILABILITY: Discrimination results are available at http://tmbeta-svm.cbrc.jp.     

Transmembrane helices predicted at 95% accuracy.

We describe a neural network system that predicts the locations of transmembrane helices in integral membrane proteins. By using evolutionary information as input to the network system, the method significantly improved on a previously published neural network prediction method that had been based on single sequence information. The input data were derived from multiple alignments for each position in a window of 13 adjacent residues: amino acid frequency, conservation weights, number of insertions and deletions, and position of the window with respect to the ends of the protein chain. Additional input was the amino acid composition and length of the whole protein. A rigorous cross-validation test on 69 proteins with experimentally determined locations of transmembrane segments yielded an overall two-state per-residue accuracy of 95%. About 94% of all segments were predicted correctly. When applied to known globular proteins as a negative control, the network system incorrectly predicted fewer than 5% of globular proteins as having transmembrane helices. The method was applied to all 269 open reading frames from the complete yeast VIII chromosome. For 59 of these, at least two transmembrane helices were predicted. Thus, the prediction is that about one-fourth of all proteins from yeast VIII contain one transmembrane helix, and some 20%, more than one.     

Characteristics affecting expression and solubilization of yeast membrane proteins

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.     

Prediction of Membrane Protein Topology Utilizing Multiple Sequence Alignments

A technique for prediction of protein membrane toplogy (intra- and extraceullular sidedness) has been developed. Membrane-spanning segments are first predicted using an algorithm based upon multiply aligned amino acid sequences. The compositional differences in the protein segments exposed at each side of the membrane are then investigated. The ratios are calculated for Asn, Asp, Gly, Phe, Pro, Trp, Tyr, and Val, mostly found on the extracellular side, and for Ala, Arg, Cys, and Lys, mostly occurring on the intracellular side. The consensus over these 12 residue distributions is used for sidedness prediction. The method was developed with a set of 42 protein families for which all but one were correctly predicted with the new algorithm. This represents an improvement over previous techniques. The new method, applied to a set of 12 membrane protein families different from the test set and with recently determined topologies, performed well, with 11 of 12 sidedness assignments agreeing with experimental results. The method has also been applied to several membrane protein families for which the topology has yet to be determined. An electronic prediction service is available at the E-mail address tmap@embl-heidelberg.de and on WWW via http://www.emblheidelberg.de.     

Proportion of membrane proteins in proteomes of 15 single-cell organisms analyzed by the SOSUI prediction system

A software system, SOSUI, was previously developed for discriminating between soluble and membrane proteins and predicting transmembrane regions (Hirokawa et al., Bioinformatics, 14 (1998) 378-379). The performance of the system was 99% for the discrimination between two types of proteins and 96% for the prediction of transmembrane helices. When all of the amino acid sequences from 15 single-cell organisms were analyzed by SOSUI, the proportion of predicted polytopic membrane proteins showed an almost constant value of 15-20%, irrespective of the total genome size. However, single-cell organisms appeared to be categorized in terms of the preference of the number of transmembrane segments: species with small genomes were characterized by a significant peak at a helix number of approximately six or seven; species with large genomes showed a peak at 10 or 11 helices; and species with intermediate genome sizes showed a monotonous decrease of the population of membrane proteins against the number of transmembrane helices.     

Negatively charged residues in the IgM stop-transfer effector sequence regulate transmembrane polypeptide integration

A non-hydrophobic sequence that contributes to the biogenesis of a transmembrane protein is termed a stop-transfer effector (STE). To examine the mechanism of STE-mediated stop-transfer, a series of fusion proteins were constructed containing variants of a putative STE from murine IgM fused to an otherwise translocated hydrophobic sequence. Unexpectedly, the fraction of molecules adopting transmembrane topology was insensitive to many amino acid substitutions within the STE sequence but varied directly with the number of negative charges. Furthermore, when present at the amino terminus of a reporter, mutants were observed that adopted type I (amino terminus lumenal) and type II (amino terminus cytoplasmic) transmembrane topologies, demonstrating that the STE sequence can be located at either side of the endoplasmic reticulum membrane. Our results suggest that recognition of a broad structural feature formed primarily by negatively charged residues within the STE halts translocation and triggers membrane integration, even when the negative charges end up on the cytoplasmic side of the membrane. Since functional STE sequences photocross-link to two membrane proteins not previously identified at the translocon, these unique proteins are presumably involved in recognizing STE sequences and/or facilitating STE function.     

Computational analysis of the polymorphic membrane protein superfamily of Chlamydia trachomatis and Chlamydia pneumoniae

Whole sequence genome analysis is invaluable in providing complete profiles of related proteins and gene families. The genome sequences of the obligate intracellular bacteria Chlamydia trachomatis and Chlamydia pneumoniae both encode proteins with similarity to several 90-kDa Chlamydia psittaci proteins. These proteins are members of a large superfamily, C. trachomatis with 9 members and C. pneumoniae with 21 members. All polymorphic membrane protein (Pmp) are heterogeneous, both in amino acid sequence and in predicted size. Most proteins have apparent signal peptide leader sequences and hence are predicted to be localized to the outer membrane. The unifying features of all proteins are the conserved amino acid motifs GGAI and FXXN repeated in the N-terminal half of each protein. In both genomes, the pmp genes are clustered at various locations on the chromosome. Phylogenetic analysis suggests six related families, each with at least one C. trachomatis and one C. pneumoniae orthologue. One of these families has seen prolific expansion in C. pneumoniae, resulting in 13 protein paralogues. The maintenance of orthologues from each species suggests specific functions for the proteins in chlamydial biology.     

Membrane topology of three Xcp proteins involved in exoprotein transport by Pseudomonas aeruginosa.

Xcp proteins constitute the secretory apparatus of Pseudomonas aeruginosa. Deduced amino acid sequence of xcp genes, expression, and subcellular localization revealed unexpected features. Indeed, most Xcp proteins are found in the cytoplasmic membrane although xcp mutations lead to periplasmic accumulation of exoproteins, indicating that the limiting step is translocation across the outer membrane. To understand the mechanism by which the machinery functions and the interactions between its components, it is valuable to know their membrane organization. We report data demonstrating the N(in)-C(out) topologies of three general secretion pathway components, the XcpP, -Y, and -Z proteins.     

Co-evolving residues in membrane proteins

MOTIVATION: The analysis of co-evolving residues has been exhaustively evaluated for the prediction of intramolecular amino acid contacts in soluble proteins. Although a variety of different methods for the detection of these co-evolving residues have been developed, the fraction of correctly predicted contacts remained insufficient for their reliable application in the construction of structural models. Membrane proteins, which constitute between one-fourth and one-third of all proteins in an organism, were only considered in few individual case studies. RESULTS: We present the first general study of correlated mutations in alpha-helical membrane proteins. Using seven different prediction algorithms, we extracted co-evolving residues for 14 membrane proteins having a solved 3D structure. On average, distances between correlated pairs of residues lying on different transmembrane segments were found to be significantly smaller compared to a random prediction. Covariation of residues was frequently found in direct sequence neighborhood to helix-helix contacts. Based on the results obtained from individual prediction methods, we constructed a consensus prediction for every protein in the dataset that combines obtained correlations from different prediction algorithms and simultaneously removes likely false positives. Using this consensus prediction, 53% of all predicted residue pairs were found within one helix turn of an observed helix-helix contact. Based on the combination of co-evolving residues detected with the four best prediction algorithms, interacting helices could be predicted with a specificity of 83% and sensitivity of 42%. AVAILABILITY: http://webclu.bio.wzw.tum.de/helixcorr/     

The energy landscape of modular repeat proteins: topology determines folding mechanism in the ankyrin family

Proteins consisting of repeating amino acid motifs are abundant in all kingdoms of life, especially in higher eukaryotes. Repeat-containing proteins self-organize into elongated non-globular structures. Do the same general underlying principles that dictate the folding of globular domains apply also to these extended topologies? Using a simplified structure-based model capturing a perfectly funneled energy landscape, we surveyed the predicted mechanism of folding for ankyrin repeat containing proteins. The ankyrin family is one of the most extensively studied classes of non-globular folds. The model based only on native contacts reproduces most of the experimental observations on the folding of these proteins, including a folding mechanism that is reminiscent of a nucleation propagation growth. The confluence of simulation and experimental results suggests that the folding of non-globular proteins is accurately described by a funneled energy landscape, in which topology plays a determinant role in the folding mechanism.     

MetaTM - a consensus method for transmembrane protein topology prediction

Background  

Transmembrane (TM) proteins are proteins that span a biological membrane one or more times. As their 3-D structures are hard to determine, experiments focus on identifying their topology (i. e. which parts of the amino acid sequence are buried in the membrane and which are located on either side of the membrane), but only a few topologies are known. Consequently, various computational TM topology predictors have been developed, but their accuracies are far from perfect. The prediction quality can be improved by applying a consensus approach, which combines results of several predictors to yield a more reliable result.     

DNA and amino acid sequence analysis of structural and immunity genes of colicins Ia and Ib.

The nucleotide sequences for colicin Ia and colicin Ib structural and immunity genes were determined. The two colicins each consist of 626 amino acid residues. Comparison of the two sequences along their lengths revealed that the two colicins are nearly identical in the N-terminal 426 amino acid residues. The C-terminal 220 amino acid residues of the colicins are only 60% identical, suggesting that this is the region most likely recognized by their cognate immunity proteins. The predicted proteins for the colicin immunity proteins would contain 111 amino acids for the colicin Ia immunity protein and 115 amino acids for the colicin Ib immunity protein. The colicin immunity proteins have no detectable DNA or amino acid homology but do exhibit a conservation of overall hydrophobicity. The colicin immunity genes lie distal to and in opposite orientation to the colicin structural genes. The colicin Ia immunity protein was purified to apparent homogeneity by a combination of isoelectric focusing and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified Ia immunity protein was determined and was found to be in perfect agreement with that predicted from the DNA sequence of its structural gene. The Ia immunity protein is not a processed membrane protein.     

Ultrastructural aspects and amino acid composition of the purified inner and outer membranes of human liver mitochondria as compared to rat liver mitochondria.

1. The mitochondria isolated from human or rat liver were fractionated into submitochondrial particles and purified inner and outer membrane. According to different marker enzymes the inner membranes were enriched about 5-6-fold and the outer membranes about 12-14-fold. The electron microscopical appearance of the membranes was that expected on the basis of enzymic characterization. 2. A comparison of the average amino acid composition of the membrane proteins from the two types of mitochondria has been made. In the case of submitochondrial particles there were statistically significant differences between the human and rat hydrolysates for only five amino acids. Analysing the purified mitochondrial membranes there were significant differences between the two species for nine amino acids in the case of outer membranes and for 12 amino acids in the case of inner membranes. 3. With one exception all amino acids that were increased or decreased in the outer membrane exhibited a similar trend in the inner membrane of human compared with rat liver mitochondria. It appears that liver mitochondrial membranes have a species-dependent pattern of amino acid composition of their proteins.     

Prediction by a neural network of outer membrane beta-strand protein topology.

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) beta-strand proteins. Specifically, the NN predicts the z-coordinate of Calpha atoms in a coordinate frame with the outer membrane in the xy-plane, such that low z-values indicate periplasmic turns, medium z-values indicate transmembrane beta-strands, and high z-values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z-axis. The relationship between Calpha z-values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z-values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.     

Consensus predictions of membrane protein topology

We have explored the possibility that consensus predictions of membrane protein topology might provide a means to estimate the reliability of a predicted topology. Using five current topology prediction methods and a test set of 60 Escherichia coli inner membrane proteins with experimentally determined topologies, we find that prediction performance varies strongly with the number of methods that agree, and that the topology of nearly half of all E. coli inner membrane proteins can be predicted with high reliability (>90% correct predictions) by a simple majority-vote approach.