ATP synthases: insights into their motor functions from sequence and structural analyses

ATP synthases are motor complexes comprised of F₀ and F₁ parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F₁. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F₁ to the rotor of F₀. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.     

Protein family alignment annotation

For bioscientists studying protein structure and function, the Protein Family Alignment Annotation Tool (Pfaat) is a useful and simple program for annotating collections of proteins. This open-source software includes methods for viewing and aligning protein families, and for annotating sequence structure and residues with known functions. It offers new options to aid the study of proteins, and an extensible annotation tool for bioinformatics developers.     

The role of pattern databases in sequence analysis

In the wake of the numerous now-fruitful genome projects, we are entering an era rich in biological data. The field of bioinformatics is poised to exploit this information in increasingly powerful ways, but the abundance and growing complexity both of the data and of the tools and resources required to analyse them are threatening to overwhelm us. Databases and their search tools are now an essential part of the research environment. However, the rate of sequence generation and the haphazard proliferation of databases have made it difficult to keep pace with developments. In an age of information overload, researchers want rapid, easy-to-use, reliable tools for functional characterisation of newly determined sequences. But what are those tools? How do we access them? Which should we use? This review focuses on a particular type of database that is increasingly used in the task of routine sequence analysis--the so-called pattern database. The paper aims to provide an overview of the current status of pattern databases in common use, outlining the methods behind them and giving pointers on their diagnostic strengths and weaknesses.     

Structure-based prediction of binding peptides to MHC class I molecules: application to a broad range of MHC alleles

Specific binding of antigenic peptides to major histocompatibility complex (MHC) class I molecules is a prerequisite for their recognition by cytotoxic T-cells. Prediction of MHC-binding peptides must therefore be incorporated in any predictive algorithm attempting to identify immunodominant T-cell epitopes, based on the amino acid sequence of the protein antigen. Development of predictive algorithms based on experimental binding data requires experimental testing of a very large number of peptides. A complementary approach relies on the structural conservation observed in crystallographically solved peptide-MHC complexes. By this approach, the peptide structure in the MHC groove is used as a template upon which peptide candidates are threaded, and their compatibility to bind is evaluated by statistical pairwise potentials. Our original algorithm based on this approach used the pairwise potential table of Miyazawa and Jernigan (Miyazawa S, Jernigan RL, 1996, J Mol Biol 256:623-644) and succeeded to correctly identify good binders only for MHC molecules with hydrophobic binding pockets, probably because of the high emphasis of hydrophobic interactions in this table. A recently developed pairwise potential table by Betancourt and Thirumalai (Betancourt MR, Thirumalai D, 1999, Protein Sci 8:361-369) that is based on the Miyazawa and Jernigan table describes the hydrophilic interactions more appropriately. In this paper, we demonstrate how the use of this table, together with a new definition of MHC contact residues by which only residues that contribute exclusively to sequence specific binding are included, allows the development of an improved algorithm that can be applied to a wide range of MHC class I alleles.     

Prediction of amino acid sequence from structure

We have developed a method for the prediction of an amino acid sequence that is compatible with a three-dimensional backbone structure. Using only a backbone structure of a protein as input, the algorithm is capable of designing sequences that closely resemble natural members of the protein family to which the template structure belongs. In general, the predicted sequences are shown to have multiple sequence profile scores that are dramatically higher than those of random sequences, and sometimes better than some of the natural sequences that make up the superfamily. As anticipated, highly conserved but poorly predicted residues are often those that contribute to the functional rather than structural properties of the protein. Overall, our analysis suggests that statistical profile scores of designed sequences are a novel and valuable figure of merit for assessing and improving protein design algorithms.     

Evaluation of PSI-BLAST alignment accuracy in comparison to structural alignments

The PSI-BLAST algorithm has been acknowledged as one of the most powerful tools for detecting remote evolutionary relationships by sequence considerations only. This has been demonstrated by its ability to recognize remote structural homologues and by the greatest coverage it enables in annotation of a complete genome. Although recognizing the correct fold of a sequence is of major importance, the accuracy of the alignment is crucial for the success of modeling one sequence by the structure of its remote homologue. Here we assess the accuracy of PSI-BLAST alignments on a stringent database of 123 structurally similar, sequence-dissimilar pairs of proteins, by comparing them to the alignments defined on a structural basis. Each protein sequence is compared to a nonredundant database of the protein sequences by PSI-BLAST. Whenever a pair member detects its pair-mate, the positions that are aligned both in the sequential and structural alignments are determined, and the alignment sensitivity is expressed as the percentage of these positions out of the structural alignment. Fifty-two sequences detected their pair-mates (for 16 pairs the success was bi-directional when either pair member was used as a query). The average percentage of correctly aligned residues per structural alignment was 43.5+/-2.2%. Other properties of the alignments were also examined, such as the sensitivity vs. specificity and the change in these parameters over consecutive iterations. Notably, there is an improvement in alignment sensitivity over consecutive iterations, reaching an average of 50.9+/-2.5% within the five iterations tested in the current study.     

Characterization of magnetically oriented phospholipid micelles for measurement of dipolar couplings in macromolecules

Weak alignment of solute molecules with the magnetic field can be achieved in a dilute liquid crystalline medium, consisting of an aqueous mixture of dimyristoyl-phosphatidylcholine (DMPC) and dihexanoyl-phosphatidylcholine (DHPC). For a certain range of molar ratios, DMPC and DHPC can form large, disc-shaped particles, commonly referred to as bicelles (Sanders and Schwonek, 1992), which cooperatively align in the magnetic field and induce a small degree of alignment on asymmetrically shaped solute molecules. As a result, dipolar couplings between pairs of ¹H, ¹³C or ¹⁵N nuclei are no longer averaged to zero by rotational diffusion and they can be readily measured, providing valuable structural information. The stability of these liquid crystals and the degree of alignment of the solute molecules depend strongly on experimental variables such as the DMPC:DHPC ratio and concentration, the preparation protocol of the DMPC/DHPC mixtures, as well as salt, temperature, and pH. The lower temperature limit for which the liquid crystalline phase is stable can be reduced to 20 °C by using a ternary mixture of DHPC, DMPC, and 1-myristoyl-2-myristoleoyl-sn-glycero-3-phosphocholine, or a binary mixture of DHPC and ditridecanoyl-phosphatidylcholine. These issues are discussed, with an emphasis on the use of the medium for obtaining weak alignment of biological macromolecules.     

Prediction of functional residues in water channels and related proteins.

In this paper, we present an updated classification of the ubiquitous MIP (Major Intrinsic Protein) family proteins, including 153 fully or partially sequenced members available in public databases. Presently, about 30 of these proteins have been functionally characterized, exhibiting essentially two distinct types of channel properties: (1) specific water transport by the aquaporins, and (2) small neutral solutes transport, such as glycerol by the glycerol facilitators. Sequence alignments were used to predict amino acids and motifs discriminant in channel specificity. The protein sequences were also analyzed using statistical tools (comparisons of means and correspondence analysis). Five key positions were clearly identified where the residues are specific for each functional subgroup and exhibit high dissimilar physico-chemical properties. Moreover, we have found that the putative channels for small neutral solutes clearly differ from the aquaporins by the amino acid content and the length of predicted loop regions, suggesting a substrate filter function for these loops. From these results, we propose a signature pattern for water transport.     

Recent applications of Hidden Markov Models in computational biology

This paper examines recent developments and applications of Hidden Markov Models (HMMs) to various problems in computational biology, including multiple sequence alignment, homology detection, protein sequences classification, and genomic annotation.     

茶树CsBAP1基因的克隆与非生物胁迫和激素响应分析

该研究基于茶树转录组数据,从茶树‘龙井43’cDNA中克隆获得茶树CsBAP1基因,对其蛋白质序列特征和基因表达模式以及CsBAP1在茶树不同组织、不同激素处理及不同逆境胁迫下的表达水平进行荧光定量检测。结果显示：（1）克隆得到的茶树CsBAP1基因开放阅读框长度为543 bp,编码180个氨基酸;CsBAP1蛋白为亲水性蛋白,理论相对分子质量为19 398 Da,理论等电点为9.30,含有保守的Ca²⁺依赖性的C2结构域;茶树CsBAP1蛋白二级结构由8.89％的α 螺旋、7.78％的β 转角、35.56％的β 折叠和47.78％的随机卷曲组成;三级结构分析结果显示,CsBAP1包括螺旋和折叠结构,与二级结构吻合。（2）荧光定量分析结果显示, CsBAP1基因在茶树的花、花蕾、幼叶、老叶和成熟叶中均有表达,且在老叶中的表达量最高;外源施用ABA、IAA、MeJA、GA³以及SA均能够抑制茶树CsBAP1基因的表达;在高温（38 ℃）、低温（4 ℃）、干旱（200 g·L^-1 PEG）、高盐（200 mmol·L^-1 NaCl）胁迫下,CsBAP1的表达量均有所上调,且不同时间段的表达存在显著差异。该研究为进一步研究茶树CsBAP1基因的功能奠定了基础。