Comparative analysis of amino acid distributions in integral membrane proteins from 107 genomes

We have performed a comparative analysis of amino acid distributions in predicted integral membrane proteins from a total of 107 genomes. A procedure for identification of membrane spanning helices was optimized on a homology-reduced data set of 170 multi-spanning membrane proteins with experimentally determined topologies. The optimized method was then used for extraction of highly reliable partial topologies from all predicted membrane proteins in each genome, and the average biases in amino acid distributions between loops on opposite sides of the membrane were calculated. The results strongly support the notion that a biased distribution of Lys and Arg residues between cytoplasmic and extra-cytoplasmic segments (the positive-inside rule) is present in most if not all organisms.     

Prediction and classification of protein subcellular location-sequence-order effect and pseudo amino acid composition

Given a protein sequence, how to identify its subcellular location? With the rapid increase in newly found protein sequences entering into databanks, the problem has become more and more important because the function of a protein is closely correlated with its localization. To practically deal with the challenge, a dataset has been established that allows the identification performed among the following 14 subcellular locations: (1) cell wall, (2) centriole, (3) chloroplast, (4) cytoplasm, (5) cytoskeleton, (6) endoplasmic reticulum, (7) extracellular, (8) Golgi apparatus, (9) lysosome, (10) mitochondria, (11) nucleus, (12) peroxisome, (13) plasma membrane, and (14) vacuole. Compared with the datasets constructed by the previous investigators, the current one represents the largest in the scope of localizations covered, and hence many proteins which were totally out of picture in the previous treatments, can now be investigated. Meanwhile, to enhance the potential and flexibility in taking into account the sequence‐order effect, the series‐mode pseudo‐amino‐acid‐composition has been introduced as a representation for a protein. High success rates are obtained by the re‐substitution test, jackknife test, and independent dataset test, respectively. It is anticipated that the current automated method can be developed to a high throughput tool for practical usage in both basic research and pharmaceutical industry. © 2003 Wiley‐Liss, Inc.     

Analysis and prediction of helix-helix interactions in membrane channels and transporters

Membrane proteins span a large variety of different functions such as cell-surface receptors, redox proteins, ion channels, and transporters. Proteins with functional pores show different characteristics of helix-helix packing as other helical membrane proteins. We found that the helix-helix contacts of 13 nonhomologous high-resolution structures of membrane channels and transporters are mainly accomplished by weakly polar amino acids (G > S > T > F) that preferably create contacts every fourth residue, typical for right-handed helix crossings. There is a strong correlation between the now available biological hydrophobicity scale and the propensities of the weakly polar and hydrophobic residues to be buried at helix-helix interfaces or to be exposed to the lipids in membrane channels and transporters. The polar residues, however, make no major contribution towards the packing of their transmembrane helices, and are therefore subsumed to be primarily exposed to the polar milieu during the folding process. The contact formation of membrane channels and transporters is therefore ruled by the solubility of the residues, which we suppose to be the driving force for the assembly of their transmembrane helices. By contrast, in 14 nonhomologous high-resolution structures of other membrane protein coils, also large and polar amino acids (D > S > M > Q) create characteristic contacts every 3.5th residues, which is a signature for left-handed helix crossings. Accordingly, it seems that dependent on the function, different concepts of folding and stabilization are realized for helical membrane proteins. Using a sequence-based matrix prediction method these differences are exploited to improve the prediction of buried and exposed residues of transmembrane helices significantly. When the sequence motifs typical for membrane channels and transporters were applied for the prediction of helix-helix contacts the quality of prediction rises by 16% to an average value of 76%, compared to the same approach when only single amino acid positions are taken into account.     

Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs.     

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.     

BATMAS30: amino acid substitution matrix for alignment of bacterial transporters

Aligned amino acid sequences of three functionally independent samples of transmembrane (TM) transport proteins have been analyzed. The concept of TM-kernel is proposed as the most probable transmembrane region of a sequence. The average amino acid composition of TM-kernels differs from the published amino acid composition of transmembrane segments. TM-kernels contain more alanines, glycines, and less polar, charged, and aromatic residues in contrast to non-TM-proteins. There are also differences between TM-kernels of bacterial and eukaryotic proteins. We have constructed amino acid substitution matrices for bacterial TM-kernels, named the BATMAS (BActerial Transmembrane MAtrix of Substitutions) series. In TM-kernels, polar and charged residues, as well as proline and tyrosine, are highly conserved, whereas there are more substitutions within the group of hydrophobic residues, in contrast to non-TM-proteins that have fewer, relatively more conserved, hydrophobic residues. These results demonstrate that alignment of transmembrane proteins should be based on at least two amino acid substitution matrices, one for loops (e.g., the BLOSUM series) and one for TM-segments (the BATMAS series), and the choice of the TM-matrix should be different for eukaryotic and bacterial proteins.     

Structure-function relationships of heterodimeric amino acid transporters

Heterodimeric amino acid transporters mediate the transfer of amino acids between organs and between different cell types. Members of this particular family of amino acid transporters are constituted by a heavy chain and an associated light chain. The heavy chain is a type II membrane protein with an intracellular amino terminus, a single transmembrane helix, and a large extracellular domain. The light chain, in contrast, is a typical helix-bundle protein with 12 putative transmembrane helices. Two different heavy chains, designated 4F2hc and rbAT, and seven different light chains have been identified to date. Deletion studies indicate that the extracellular domain of the heavy chain has two subdomains. The carboxy-terminal tip of 4F2hc is critical for recognition of certain light chains, whereas the carboxy-terminal tip of rbAT is involved in substrate transport. Sequence alignments suggest that the major part of the extracellular domain forms an α/β domain similar to bacterial α-amylases. A structural model of the rbAT extracellular domain is presented that is in agreement with experimental observations from several mutations and that aligns well with the α-amylase domain.     

lncRNAs功能注释和预测

随着测序技术的发展,在各种哺乳动物中发现越来越多的长非编码RNAs（long non-coding RNAs,lncRNAs）,但是大部分lncRNAs的功能却未知.鉴于lncRNAs在众多生物过程如免疫反应、发育和基因印迹中表现出对蛋白编码基因和其它非编码RNAs的重要调节作用,对lncRNAs的功能研究也成为生物学家和生物信息学家研究的热点. 其中,功能注释和预测是目前研究lncRNAs功能的主要方法之一.本文主要对lncRNAs功能注释和预测方法的研究进展作一综述,包括以下几个方面：基于共表达网络的方法、基于miRNAs的方法、基于蛋白质结合的方法、基于表观遗传修饰的方法以及基于ceRNA网络的方法. 为进一步研究lncRNAs的功能提供参考,同时为开发更加有效的注释或预测方法提供线索.     

The activation energy for insertion of transmembrane alpha-helices is dependent on membrane composition

The physical mechanisms that govern the folding and assembly of integral membrane proteins are poorly understood. It appears that certain properties of the lipid bilayer affect membrane protein folding in vitro, either by modulating helix insertion or packing. In order to begin to understand the origin of this effect, we investigate the effect of lipid forces on the insertion of a transmembrane alpha-helix using a water-soluble, alanine-based peptide, KKAAAIAAAAAIAAWAAIAAAKKKK-amide. This peptide binds to preformed 1,2-dioleoyl-l-alpha-phosphatidylcholine (DOPC) vesicles at neutral pH, but spontaneous transmembrane helix insertion directly from the aqueous phase only occurs at high pH when the Lys residues are de-protonated. These results suggest that the translocation of charge is a major determinant of the activation energy for insertion. Time-resolved measurements of the insertion process at high pH indicate biphasic kinetics with time constants of ca 30 and 430 seconds. The slower phase seems to correlate with formation of a predominantly transmembrane alpha-helical conformation, as determined from the transfer of the tryptophan residue to the hydrocarbon region of the membrane. Temperature-dependent measurements showed that insertion can proceed only above a certain threshold temperature and that the Arrhenius activation energy is of the order of 90 kJ mol(-1). The kinetics, threshold temperature and the activation energy change with the mole fraction of 1,2-dioleoyl-l-alpha-phosphatidylethanolamine (DOPE) introduced into the DOPC membrane. The activation energy increases with increasing DOPE content, which could reflect the fact that this lipid drives the bilayer towards a non-bilayer transition and increases the lateral pressure in the lipid chain region. This suggests that folding events involving the insertion of helical segments across the bilayer can be controlled by lipid forces.     

The role of two branched‐chain amino acid transporters in Staphylococcus aureus growth,membrane fatty acid composition and virulence

The branched‐chain amino acids (BCAAs) are vital to both growth and virulence of the human pathogen Staphylococcus aureus. In addition to supporting protein synthesis, the BCAAs serve as precursors for branched‐chain fatty acids (BCFAs), which are predominant membrane fatty acids, and, in association with the global regulatory protein CodY, the BCAAs are key co‐regulators of virulence factors. Despite these critical functions, S. aureus represses Leu and Val synthesis, instead preferring to acquire them from the extracellular milieu. We previously identified BrnQ1 as a BCAA transporter, yet a brnQ1 mutant remained capable of BCAA acquisition. Here, we describe BcaP as an additional BCAA transporter, and determine that it plays a secondary role to BrnQ1 during S. aureus growth in a chemically defined medium. Furthermore, membrane fatty acid composition analysis revealed that BrnQ1, and not BcaP, is required for transporting Leu and Val to be used for iso‐BCFA synthesis. Despite a predominant role for BrnQ1 in vitro, both BrnQ1 and BcaP are required for S. aureus fitness in vivo in a hematogenous spread infection model and a nasal colonisation model. These data demonstrate the importance of BrnQ1 and BcaP for growth, environmental adaptation and virulence of S. aureus.     

Comprehensive analysis of the numbers,lengths and amino acid compositions of transmembrane helices in prokaryotic,eukaryotic and viral integral membrane proteins of high-resolution structure

We report a comprehensive analysis of the numbers, lengths and amino acid compositions of transmembrane helices in 235 high-resolution structures of integral membrane proteins. The properties of 1551 transmembrane helices in the structures were compared with those obtained by analysis of the same amino acid sequences using topology prediction tools. Explanations for the 81 (5.2%) missing or additional transmembrane helices in the prediction results were identified. Main reasons for missing transmembrane helices were mis-identification of N-terminal signal peptides, breaks in α-helix conformation or charged residues in the middle of transmembrane helices and transmembrane helices with unusual amino acid composition. The main reason for additional transmembrane helices was mis-identification of amphipathic helices, extramembrane helices or hairpin re-entrant loops. Transmembrane helix length had an overall median of 24 residues and an average of 24.9 ± 7.0 residues and the most common length was 23 residues. The overall content of residues in transmembrane helices as a percentage of the full proteins had a median of 56.8% and an average of 55.7 ± 16.0%. Amino acid composition was analysed for the full proteins, transmembrane helices and extramembrane regions. Individual proteins or types of proteins with transmembrane helices containing extremes in contents of individual amino acids or combinations of amino acids with similar physicochemical properties were identified and linked to structure and/or function. In addition to overall median and average values, all results were analysed for proteins originating from different types of organism (prokaryotic, eukaryotic, viral) and for subgroups of receptors, channels, transporters and others.     

Biosynthesis and structural composition of gap junction intercellular membrane channels

Gap junction channels assemble as dodecameric complexes, in which a hexameric connexon (hemichannel) in one plasma membrane docks end-to-end with a connexon in the membrane of a closely apposed cell to provide direct cell-to-cell communication. Synthesis, assembly, and trafficking of the gap junction channel subunit proteins referred to as connexins, largely appear to follow the general secretory pathway for membrane proteins. The connexin subunits can assemble into homo-, as well as distinct hetero-oligomeric connexons. Assembly appears to be based on specific signals located within the connexin polypeptides. Plaque formation by the clustering of gap junction channels in the plane of the membrane, as well as channel degradation are poorly understood processes that are topics of current research. Recently, we tagged connexins with the autofluorescent reporter green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants and combined this reporter technology with single, and dual-color, high resolution deconvolution microscopy, computational volume rendering, and time-lapse microscopy to examine the detailed organization, structural composition, and dynamics of gap junctions in live cells. This technology provided for the first time a realistic, three-dimensional impression of gap junctions as they appear in the plasma membranes of adjoining cells, and revealed an excitingly detailed structural organization of gap junctions never seen before in live cells. Here, I summarize recent progress in areas encompassing the synthesis, assembly and structural composition of gap junctions with a special emphasis on the recent results we obtained using cell-free translation/ membrane-protein translocation, and autofluorescent reporters in combination with live-cell deconvolution microscopy.     

Ion channels and amino acid transporters support the growth and invasion of primary brain tumors

The malignant growth of glial support cells causes gliomas, highly invasive, primary brain tumors that are largely resistant to therapy. Individual tumor cells spread by active cell migration, invading diffusely into the normal brain. This process is facilitated by Cl⁻ channels that endow glioma cells with an enhanced ability to quickly adjust their shape and cell volume to fit the narrow and tortuous extracellular brain spaces. Once satellite tumors enlarge, their growth is limited by the spatial constraints imposed by the bony cavity of the skull and spinal column. Glioma cells circumvent this limitation by active destruction of peritumoral neural tissue through the release of glutamate, inducing peritumoral seizures and ultimately excitotoxic neuronal cell death. Hence, primary brain tumors support their unusual biology by taking advantage of ion channels and transporters that are designed to support ion homeostatic functions in normal brain.     

Phospholipid and fatty acid composition of tetrodotoxin receptor-rich membrane fragments from Electrophorus electricus

To study the interaction of voltage-sensitive Na⁺-channels with membrane lipids, the phospholipid and fatty acid composition of highly purified membrane fragments from the remarkably differentiated plasma membrane of Electrophorus electricus has been analyzed. After density gradient fractionation and carrier free electrophoresis, fractions with up to 30 pmol tetrodotoxin binding/mg protein can be obtained, which may correspond to a 50% pure preparation of the extrasynaptic part of the excitable face. Phospholipid classes and cholesterol are separated by one-dimensional thin-layer chromatography in acidic and alkaline solvent systems. The following mean molar contents are found: 40% phosphatidylcholine, 23% phosphatidylserine, 30% phosphatidylethanolamine and 7% sphingomyelin. In a series of 11 animals, significant deviations from these mean values have been observed. The fatty acid composition of the phospholipids has been determined by gas chromatography. Phosphatidylcholine contains more than 50% 16:0, and about 20% unsaturated fatty acids in the C-18 group. Compared to other plasma membrane fractions, this phospholipid is the least differentiated. By contrast, phosphatidylethanolamine and phosphatidylserine show many characteristics in different membrane fractions, especially in their unsaturated components representing more than 50%. 22:6, as the major constituent in these fractions, accounts for a quarter to a third of all fatty acids in these fractions. 18:0 is the main saturated component in these two phospholipids with abundances of typically a quarter or less of all fatty acids. Knowledge of the lipid composition of these excitable membranes may help to conserve binding and structural properties when analyzing lipid-sensitive Na⁺-channels in vitro. It is also useful as a guideline for systematic reconstitution studies.     

Natural taxonomy of collagen based on amino acid composition

Hierarchical cluster analysis and principal components have been used to provide a more detailed separation of the collagens into natural taxonomic groupings than previously obtained. These groups strongly reflect the evolutionary development of collagen. The first component separates land- from sea-based animals, primarily based on the hydroxylation of lysine and proline, indicating that control of hydroxylation, a post-translational event, has exerted a dominant influence during evolutionary adaptation. The power of the technique is illustrated by the ability to partially separate the evolutionarily closely related main homothermic species. Furthermore, the genetically different fibrous collagens, Types I and III, are well separated from basement membrane Type IV collagen and the filamentous collagens. The technique could, therefore, in addition to providing a taxonomic grouping, classify any new collagen and provide clues to its evolutionary development.     

A structural dissection of amino acid substitutions in helical transmembrane proteins

The evolution of protein folds is under strong constraints from their surrounding environment. Although folding in water‐soluble proteins is driven primarily by hydrophobic forces, the nature of the forces that determine the folding and stability of transmembrane proteins are still not fully understood. Furthermore, the chemically heterogeneous lipid bilayer has a non‐uniform effect on protein structure. In this article, we attempt to get an insight into the nature of this effect by examining the impact of various types of local structure environment on amino acid substitution, based on alignments of high‐resolution structures of polytopic helical transmembrane proteins combined with sequences of close homologs. Compared to globular proteins, burying amino acid sidechains, especially hydrophilic ones, led to a lower increase in conservation in both the lipid‐water interface region and the hydrocarbon core region. This observation is due to surface residues in HTM proteins especially in the HC region being relatively highly conserved, suggesting higher evolutionary constraints from their specific interactions with the surrounding lipid molecules. Polar and small residues, particularly Pro and Gly, show a noticeable increase in conservation as they are positioned more towards the centre of the membrane, which is consistent with their recognized key roles in structural stability. In addition, the examination of hydrogen bonds in the membrane environment identified some exposed hydrophilic residues being better conserved when not hydrogen‐bonded to other residues, supporting the importance of lipid‐protein sidechain interactions. The conclusions presented in this study highlight the distinct features of substitution matrices that take into account the membrane environment, and their potential role in improving sequence‐structure alignments of transmembrane proteins. Proteins 2010; © 2010 Wiley‐Liss, Inc.     

Interactions of tight junctions with membrane channels and transporters

Tight junctions are unique organelles in epithelial cells. They are localized to the apico-lateral region and essential for the epithelial cell transport functions. The paracellular transport process that occurs via tight junctions is extensively studied and is intricately regulated by various extracellular and intracellular signals. Fine regulation of this transport pathway is crucial for normal epithelial cell functions. Among factors that control tight junction permeability are ions and their transporters. However, this area of research is still in its infancy and much more needs to be learned about how these molecules regulate tight junction structure and functions. In this review we have attempted to compile literature on ion transporters and channels involved in the regulation of tight junctions.     

Distinct character in hydrophobicity of amino acid compositions of mitochondrial proteins

A compact mitochondrial gene contains all essential information about the synthesis of mitochondrial proteins which play their roles in a small compartment of the mitochondrium. Almost no noncoding regions have been found through the gene, but a necessary set of tRNAs for the 20 amino acids is provided for biosynthesis, some of them coding different amino acids from those in a usual cell. Since the gene is so compact that the produced proteins would have some characteristic aspects for the mitochondrium, amino acid compositions of mitochondrial proteins (mt-proteins) were examined in the 20-dimensional composition space. The results show that compositions of proteins translated from the mitochondrial genes have a distinct character having more hydrophobic content than others, which is illustrated by a clustered distribution in the multidimensional composition space. The cluster is located at the tail edge of the global distribution pattern of a Gaussian shape for other various kinds of proteins in the space. The mt-proteins are rich in hydrophobic amino acids as is a membrane protein, but are different from other membrane proteins in a lesser content of Val. A good correlation found between the base and amino acid compositions for the mitochondria was examined in comparison to those of organisms such as thermophilic bacterium having an extreme G-C-rich base composition.