蛋白质结构与功能研究中的分子模拟技术

分子模拟技术为蛋白质的研究提供了一种崭新的手段,在理论上解决了结构预测和功能分析以及蛋白质工程实施方面所面临的难题。它在蛋白质的结构预测和模建工作中占有举足轻重的地位,实现了生物技术与计算机技术的完美结合。本文简要阐述了该技术的基本步骤和工作原理,并以目前应用最广的生物大分子领域的商品化分子模拟软件Accelrys公司基于Linux系统开发的InsightII为例,介绍了相关程序模块的功能和作用,同时结合该技术在蛋白质的结构预测和模建、结构与功能关系分析、分子设计等过程中的开发与应用,加以具体说明和展望。     

纤维素酶家族及其催化结构域分子改造的新进展

纤维素酶的分子改造是其催化性能改进及催化效率提升的重要手段。近年来,组学技术与结构测定技术的迅速发展,人们已建立了包括糖苷水解酶(Glycoside hydrolase,GH)在内的碳水化合物活性酶组分数据库。通过对同一蛋白家族进行序列比对、分子进化分析与祖先基因重构,以结构模建分析为指导的纤维素酶分子改造,可以明显缩小序列或结构的搜索空间,加快酶分子改造的速度,增大理性设计成功的概率;同时针对催化中心活性架构的分析可以进一步阐明纤维素酶的催化机理与酶分子持续性降解机制。文中主要对纤维素酶家族及其催化结构域的分子改造取得的最新进展作了综述。在后基因组时代基于蛋白质家族中的海量数据分析,以其保守结构信息为指导的理性设计,将会成为纤维素酶分子改造的重要方向,从而推动生物质转化工艺的快速发展。     

同源建模关键步骤的研究动态

应用同源建模的蛋白质结构预测已经成为一种快速获得蛋白质结构的技术,这种技术也将成为完成结构基因组计划的有力工具.同源建模是指寻找与目标序列同源而且有实验测定结构的蛋白质作为模板,从而构建目标序列的结构模型的方法.限制这种方法的应用主要是同源建模的关键步骤,即目标与模板之间序列比对和环区建模的准确性.当模型的准确性达到令人信服的程度时,更为精确的计算机辅助药物设计和改造蛋白质,甚至设计全新功能的蛋白质将成为可能.综述了从算法和策略上提高同源建模关键步骤准确性的研究进展.     

基于SWISS-MODEL的蛋白质三维结构建模

蛋白质的三级结构预测可通过同源建模、Threading和TOPITS等方法进行,但同源建模是应用最为广泛的方法。SWISS-MODEL正是一个基于同源建模的蛋白质结构服务器。它与ExPASy网站和DeepView程序是紧密相联系的。该文重点介绍SWISS-MODEL的提交方式、建模的步骤、结果的评估和应用程序等。     

米曲霉木糖醇脱氢酶的分子模建与对接

[目的]研究米曲霉木糖醇脱氢酶基因的结构与功能.[方法]克隆测序来源于米曲霉的木糖醇脱氢酶(XDH)基因,利用Swiss-MODEL和Modeller对XDH进行三级结构模建,通过PROCHECK和Prosa2003对得到的4个目标模型进行评价,从中得到一个最佳模型.在同源建模的基础上,通过分子对接软件MolsoftICM-Pro,对辅因子进行对接,预测了XDH与NAD+、Zn2+作用的相关残基.寻找底物木糖醇与XDH结合的可能活性口袋,用Molsoft模拟XDH与木糖醇的对接,预测了酶与底物作用的关键氨基酸残基.[结果]结构分析显示,米曲霉XDH含有醇脱氢酶家族锌指纹结构和典型醇脱氢酶Rossmann折叠的辅酶结合域,属于Medium-chain脱氢酶(MDR)家族.通过对接研究,预测了XDH与NAD+之间形成氢键的氨基酸有Asp206、Arg211、Ser255、Ser301和Arg303,这些氨基酸位于结合域,与Zn2+形成氢键的氨基酸有His72和Glu73,位于催化域,与天然底物木糖醇形成氢键的氨基酸有Ile46、Ile349、Lys350和Thr351,位于催化域.[结论]所得信息对XDH分子定向改造、拓展米曲霉工业应用范围有重要意义.     

持续性催化酶类机理研究及其分子动力学模拟

酶分子催化机理研究是生命科学研究领域一个重要的问题.近80年来,过渡态理论在解释酶催化机理问题上占据了主导地位,结合热力学循环、锁钥学说、诱导契合学说以及酶活性中心柔性学说等理论,可以很好地解释多种酶分子的催化过程.近年来,随着蛋白质结构解析方法、单分子分析检测技术及计算机模拟技术的发展,人们对酶分子催化机理的认识愈加深刻.但持续性催化酶类的催化动力研究表明,过渡态理论的解释并不充分.本文对酶催化机理研究的相关进展进行了综述,并针对持续性酶类催化动态过程的特点提出了可能的研究方向及可行的研究方法.     

蛋白质工程：从定向进化到计算设计

定向进化通过建立突变体文库与高通量筛选方法,快速提升蛋白的特定性质,是目前蛋白质工程最为常用的蛋白质设计改造策略。近十年随着计算机运算能力大幅提升以及先进算法不断涌现,计算机辅助蛋白质设计改造得到了极大的重视和发展,成为蛋白质工程新开辟的重要方向。以结构模拟与能量计算为基础的蛋白质计算设计不但能改造酶的底物特异性与热稳定性,还可从头设计具有特定功能的人工酶。近年来机器学习等人工智能技术也被应用于计算机辅助蛋白质设计改造,并取得瞩目的成绩。文中介绍了蛋白质工程的发展历程,重点评述当前计算机辅助蛋白质设计改造方面的进展与应用,并展望其未来发展方向。     

纤维素酶分子结构和功能研究进展

概述了近10年来利用结构生物学和蛋白质工程技术在纤维素酶分子结构和功能方面研究的进展,包括：酶分子结构域的拆分、催化域和纤维素结合结构域的结构和功能的研究,纤维素酶的分子折叠.并展望了该领域的研究前景.     

苏云金芽孢杆菌Ⅳ型抗癌晶体蛋白的分子模建

通过同源建模得到了抗癌晶体蛋白Parasporin-4的初始三维结构,利用分子动力学的方法对初始三维结构进行优化,同时分析了模建分子的结构,最后利用Ramachandran plot,结构匹配等方法对模型进行评价。结果显示得到的Parasporin-4结构是具有三个结构域,蛋白模型分子中的键长、键角以及二面角的分布合理,与模版蛋白的主链a碳原子的均方根差RMSD值为0．547742,在合理范围之内,表明Parasporin-4蛋白模型良好。研究结果为抗癌晶体蛋白的抗癌的分子机制及其定向改造提供了结构信息。     

耐热碱性磷酸酶(FD-TAP)的结构模型研究

 以大肠杆菌碱性磷酸酶 (BAP)为主要结构模板 ,用计算机同源结构模拟方法构建了耐热碱性磷酸酶 (FD TAP)的三维结构 ,对它们的结构特征进行了分析比较 ,并用Profile 3D和Ramachand ran图等方法分析了结构的合理性 .在此基础上 ,又构建了FD TAP 3个突变体的结构 ,用CHARMM能量计算法研究了FD TAP及其 3个突变体的能量与酶蛋白热稳定性之间的关系 ,得到了与实验完全一致的结果 .结果说明 ,如果氨基酸残基置换使蛋白质分子的总能量降低 ,疏水性增高和柔韧性减小 ,往往使蛋白质的耐热性增加     

A different look at the quality of modeled three-dimensional protein structures

Measuring the accuracy of protein three-dimensional structures is one of the most important problems in protein structure prediction. For structure-based drug design, the accuracy of the binding site is far more important than the accuracy of any other region of the protein. We have developed an automated method for assessing the quality of a protein model by focusing on the set of residues in the small molecule binding site. Small molecule binding sites typically involve multiple regions of the protein coming together in space, and their accuracy has been observed to be sensitive to even small alignment errors. In addition, ligand binding sites contain the critical information required for drug design, making their accuracy particularly important. We analyzed the accuracy of the binding sites on two sets of protein models: the predictions submitted by the top-performing CASP7 groups, and the models generated by four widely used homology modeling packages. The results of our CASP7 analysis significantly differ from the previous findings, implying that the binding site measure does not correlate with the traditional model quality measures used in the structure prediction benchmarks. For the modeling programs, the resolution of binding sites is extremely sensitive to the degree of sequence homology between the query and the template, even when the most accurate alignments are used in the homology modeling process.     

Improvement of comparative modeling by the application of conserved motifs amongst distantly related proteins as additional restraints

Protein comparative modeling has useful applications in large-scale structural initiatives and in rational design of drug targets in medicinal chemistry. The reliability of a homology model is dependent on the sequence identity between the query and the structural homologue used as a template for modeling. Here, we present a method for the utilization and conservation of important structural features of template structures by providing additional spatial restraints in comparative modeling programs like MODELLER. We show that root mean square deviation at C(alpha) positions between the model and the corresponding experimental structure and the quality of the models can be significantly improved for distantly related systems by utilizing additional spatial restraints of the template structures. We demonstrate the influence of such approaches to homology modeling during distant relationships in understanding functional properties of protein such as ligand binding using cytochrome P450 as an example.     

Molecular modeling approaches for determining gene function: application to a putative poly-A binding protein from Leishmania amazonensis (LaPABP)

The great expansion in the number of genome sequencing projects has revealed the importance of computational methods to speed up the characterization of unknown genes. These studies have been improved by the use of three dimensional information from the predicted proteins generated by molecular modeling techniques. In this work, we disclose the structure-function relationship of a gene product from Leishmania amazonensis by applying molecular modeling and bioinformatics techniques. The analyzed sequence encodes a 159 amino acids polypeptide (estimated 18 kDa) and was denoted LaPABP for its high homology with poly-A binding proteins from trypanosomatids. The domain structure, clustering analysis and a three dimensional model of LaPABP, basically obtained by homology modeling on the structure of the human poly-A binding protein, are described. Based on the analysis of the electrostatic potential mapped on the model's surface and conservation of intramolecular contacts responsible for folding stabilization we hypothesize that this protein may have less avidity to RNA than it's L. major counterpart but still account for a significant functional activity in the parasite. The model obtained will help in the design of mutagenesis experiments aimed to elucidate the mechanism of gene expression in trypanosomatids and serve as a starting point for its exploration as a potential source of targets for a rational chemotherapy.     

Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.     

Structural relationships of homologous proteins as a fundamental principle in homology modeling

Protein structure prediction is based mainly on the modeling of proteins by homology to known structures; this knowledgebased approach is the most promising method to date. Although it is used in the whole area of protein research, no general rules concerning the quality and applicability of concepts and procedures used in homology modeling have been put forward yet. Therefore, the main goal of the present work is to provide tools for the assessment of accuracy of modeling at a given level of sequence homology. A large set of known structures from different conformational and functional classes, but various degrees of homology was selected. Pairwise structure superpositions were performed. Starting with the definition of the structurally conserved regions and determination of topologically correct sequence alignments, we correlated geometrical properties with sequence homology (defined by the 250 PAM Dayhoff Matrix) and identity. It is shown that both the topological differences of the protein backbones and the relative positions of corresponding side chains diverge with decreasing sequence identity. Below 50% identity, the deviation in regions that are structurally not conserved continually increases, thus implying that with decreasing sequence identity modeling has to take into account more and more structurally diverging loop regions that are difficult to predict. © 1993 Wiley-Liss, Inc.     

Application of molecular modeling to analysis of inhibition of kinesin motor proteins of the BimC subfamily by monastrol and related compounds

Application of molecular modeling approaches has potential to contribute to rational drug design. These approaches may be especially useful when attempting to elucidate the structural features associated with novel drug targets. In this study, molecular docking and molecular dynamics were applied to studies of inhibition of the human motor protein denoted HsEg5 and other homologues in the BimC subfamily. These proteins are essential for mitosis, so compounds that inhibit their activity may have potential as anticancer therapeutics. The discovery of a small-molecule cell-permeable inhibitor, monastrol, has stimulated research in this area. Interestingly, monastrol is reported to inhibit the human and Xenopus forms of Eg5, but not those from Drosophila and Aspergillus. In this study, homology modeling was used to generate models of the Xenopus, Drosophila, and Aspergillus homologues, using the crystal structure of the human protein in complex with monastrol as a template. A series of known inhibitors was docked into each of the homologues, and the differences in binding energies were consistent with reported experimental data. Molecular dynamics revealed significant changes in the structure of the Aspergillus homologue that may contribute to its relative insensitivity to monastrol and related compounds.     

Matrix representation of cell structure,functional processes and evolution: Part V of a general theory

The purpose of this paper is to formulate a compact analytical representation of cell structure, functional processes and evolution. In this formulation, the individual molecular structures are represented by their force-field surfaces. Complementary active sites on these surfaces permit molecular interactions. In cells, these interactions are further regulated by barrier systems in time, space, specificity, and energy. In terms of these parameters, evolution can be represented (modeled) as a random walk in a multi-dimensional space, subject to constraints. In this paper, the various parameters are integrated into a single compact matrix (stack) representation (a three index array). Cell life cycle and functional processes can be represented as a sequence of quantized, time-dependent changes in the representation matrix, subject to specified constraints. Cell evolution can be modeled by generating allowed matrix combinations. This theoretical approach has applications in: (1) ordering and interpreting experimental findings into the matrix representation. Missing matrix elements can be predicted, to be confirmed experimentally; (2) theoretical analysis and prediction of cell regulatory processes and the possible pathological failures; (3) theoretical derivation of the possible biological structures and functional processes, modeling possible pathways of cell and molecular evolution in terms of the matrix representation.