残基突变对冷休克蛋白热稳定性影响的计算分析研究(英文)

残基突变是提高蛋白质热稳定性最直接有效的方式。在本文中,我们选取一对冷休克蛋白质作为研究对象,其中一个来自嗜温的Bacillus subtilis(Bs-CspB),另一个来自嗜热的Bacillus caldolyticus(Bc-Csp),这两个蛋白质在序列和结构上具有高度的相似性,但两者的耐热能力却相差很大。我们利用全原子模型计算残基突变前后蛋白质的自由能和氨基酸之间相互作用能的变化,分析残基突变对冷休克蛋白热稳定性的影响。通过对比两个蛋白质对应位置上残基的能量,我们成功鉴别出对Bc-Csp的高热稳定性有突出贡献的残基。我们计算了这些残基突变前后,该残基的静电相互作用和范德华相互作用的变化,以分析该残基对Bc-Csp高热稳定性的主要贡献。同时,我们分析了离子键对蛋白质热稳定性的贡献。我们的计算结果和实验结果吻合得很好,关键在于利用该方法可以详细地说明残基突变影响蛋白质热稳定性的根本原因。本文为研究残基突变对蛋白质热稳定性的影响提供了一种计算思路和方法,并有助于设计具有高耐热能力的蛋白质。     

计算方法研究HIV-1蛋白酶及其变异与小分子GRL-0519的相互作用

艾滋病病毒在世界范围内的传播,严重地威胁到人们的身心健康.HIV-1蛋白酶的残基变异严重地削弱了药物的治疗效果.为了研究残基变异D30N、I54M和V82A对蛋白酶结合抑制剂GRL-0519的影响,本研究进行了4个30 ns的分子动力学(MD)模拟,并采用溶解相互自由能(SIE)方法计算了蛋白酶和抑制剂的结合能.计算结果表明,极性相互作用不利于变异的蛋白酶结合抑制剂,而对于野生型的蛋白酶(WT),极性相互作用有微弱的贡献,极性相互作用是残基变异抗药性的主要原因,计算得到的总结合能与实验的数据一致.为了说明每个残基在抗药性中的贡献,采用分子力场的方法计算了每一个残基与小分子作用的范德华作用能,并分析了抑制剂与蛋白酶形成的氢键.范德华作用分析表明,V82A残基变异对结合模式的影响较小,相对于WT,D30N有5个残基的范德华贡献差异大于0.4 kcal/mol,I54M残基变异的蛋白酶有6个残基.氢键的分析说明,D30N和I54M变异丢失了几个氢键;范德华作用和氢键的分析结果与SIE的计算结果一致.研究结果为设计新的更有效的抗HIV-1蛋白酶变异的抑制剂提供了理论指导.     

蛋白质相互作用热点残基的预测北大核心CSCD

氨基酸突变扫描实验揭示了在蛋白质相互作用的结合过程中大部分的结合自由能是由极少数热点残基贡献的,通常定义结合自由能变化△△G≥2.0 kcal/mol的蛋白质残基为热点残基。热点残基对蛋白质相互作用具有重要意义。因此,如何有效进行热点残基的预测,仍然是一个研究课题。综合蛋白质氨基酸理化属性的加权疏水性、加权残基接触数、结构属性溶剂可接近面积和残基突出指数等特征,提出利用机器学习支持向量机算法来预测热点残基的方法。所提方法在丙氨酸热力学数据库数据和结合界面数据库选定的数据集上有很好的效果。在一定程度上对以后的研究发展有所帮助。     

残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

残基相互作用网络特征与木聚糖酶耐热性的关系

残基相互作用网络是体现蛋白质中残基与残基之间协同和制约关系的重要形式。残基相互作用网络的拓扑性质以及社团结构与蛋白质的功能和性质有密切的关系。本文在构建一系列耐热木聚糖酶和常温木聚糖酶的残基相互作用网络后,通过计算网络的度、聚类系数、连接强度、特征路径长度、接近中心性、介数中心性等拓扑参数来确定网络拓扑结构与木聚糖酶耐热性的关系。识别残基相互作用网络的hub点,分析hub点的亲疏水性、带电性以及各种氨基酸在hub点中所占的比例。进一步使用GA-Net算法对网络进行社团划分,并计算社团的规模、直径和密度。网络的高平均度、高连接强度、以及更短的最短路径等表明耐热木聚糖酶残基相互作用网络的结构更加紧密;耐热木聚糖酶网络中的hub节点比常温木聚糖酶网络hub节点具有更多的疏水性残基,hub点中Phe、Ile、Val的占比更高。社团检测后发现,耐热木聚糖酶网络拥有更大的社团规模、较小的社团直径和较大的社团密度。社团规模越大表明耐热木聚糖酶的氨基酸残基更倾向于形成大的社团,而较小的社团直径和较大的社团密度则表明社团内部氨基酸残基的相互作用比常温木聚糖酶更强。     

肽基载体蛋白结构功能的研究进展

肽基载体蛋白(peptidyl carrier protein,PCP)是非核糖体肽合成酶(non-ribosomal peptide synthetase,NRPS)的核心结构域。根据NRPS的装配机制,每个模块都至少包含一个PCP,PCP对于非核糖体肽合成中氨基酸残基及多肽在不同催化结构域中的传递起着重要作用,并为氨基酸残基和多肽向模块内其他修饰酶的转移提供一个平台。本文主要对PCP的结构功能、与其他催化结构域的相互作用及重组模块活性降低的问题等方面进行了综述,期望为重组NRPS模块的构建提供理论依据。     

抑制剂Amprenavir与HIV-2蛋白酶作用机制的分子动力学研究

HIV-2蛋白酶已成为治疗艾滋病的一个重要药物靶标。作者采用分子动力学模拟并结合自由能计算,研究了抑制剂Amprenavir(AMP)与HIV-2蛋白酶的作用机制。研究结果表明范德华作用是AMP与HIV-2蛋白酶结合的主要力量。采用基于残基的自由能分解方法计算抑制剂-残基相互作用,结果表明疏水性的CH-π和CH-O相互作用驱动了抑制剂AMP与HIV-2蛋白酶的结合。作者期望这一研究结果能为抗艾滋病双重抑制剂的研发提供一定的理论指导。     

基于密度聚类和社区探测的蛋白质相互作用热区的预测方法

在蛋白质相互作用基础上,热点残基有聚集在一起形成模块的倾向,这样的模块被定义为热区。热区对揭示生物体的生命活动起着重要的作用,因此,如何有效而精确地对热区进行预测,是一个重要的研究方向。作者结合热区空间密度普遍大于非热区空间密度的特性,先采用聚类算法得到簇集,而后利用相关计算模型得到的结合自由能值过滤掉簇中非热点残基及某些不符合条件的簇。最后,使用社区探测技术处理过滤后的簇集,从而得到最终的预测热区。实验结果显示该热区预测方法具有较好的预测效果。     

肺炎链球菌双组份系统中的组氨酸激酶(YycG)的同源模建与分析

对肺炎链球菌双组份系统中的组氨酸激酶YycG进行同源模建, 并分析其与底物ADP的相互作用, 为寻找特异性的激酶抑制剂提供了理论依据。采用同源模建的方法构建YycG蛋白的三维结构, 并用ProCheck、Profile_3D软件对此结构模型的合理性进行验证; 用Autodock4.0软件将结构模型与ADP进行自动对接, 分析二者之间的相互作用。序列比对结果显示肺炎链球菌YycG蛋白与Thermotoga maritima X-ray晶体结构序列的同一性达33%; YycG模建后的结构与模板能很好的叠合; 在活性口袋处的保守的氨基酸残基Asn145、Asn149、Lys152以及口袋内部的疏水残基在结合、水解底物ADP的过程中发挥重要作用。组氨酸激酶YycG的模建合理, 该结构模型可作为设计抗菌药的研究起点。     

小鼠肝质膜蛋白质的生物信息学研究

随着高通量蛋白质组研究技术的发展,使用生物信息学方法对鉴定出的蛋白质进行批量的物理化学性质和功能属性的研究显得越来越重要．对2-DE分离的小鼠肝质膜中鉴定的209个蛋白质运用生物信息学方法进行了一系列的功能属性分析,包括统计分析ProtParam软件计算出的209个蛋白质的理论相对分子质量、等电点以及疏水值的分布情况,使用TMHMM预测蛋白质的跨膜区数目,运用系统发生谱方法预测蛋白质相互作用网络,根据其相互作用网络预测部分未知蛋白质的功能．     

Theoretical probes of conformational fluctuations in S-peptide and RNase A/3′–UMP enzyme product complex

The dynamic properties of the RNase A/3′–UMP enzyme/product complex and the S-peptide of RNase A have been investigated by molecular dynamics simulations using suitable generalization of ideas introduced to probe the energy landscape in structural glasses. We introduce two measures, namely, the kinetic energy fluctuation metric and the force metric, both of which are used to calculate the time needed for sampling the conformation space of the molecules. The calculation of the fluctuation metric requires a single trajectory whereas the force metric is computed using two independent trajectories. The vacuum MD simulations show that for both systems the time required for kinetic energy equipartitioning is surprisingly long even at high temperatures. We show that the force metric is a powerful means of probing the nature and relative importance of conformational substates which determine the dynamics at low temperatures. In particular the time dependence of the non-bonded force metric is used to demonstrate that at low temperatures the system is predominantly localized hi a single cluster of conformational substates. The force metric is used to show that relaxation of long range (in sequence space) interactions must be mediated by a sequence of local dihedral angle transitions. We also argue that the time needed for compact structure formation is intimately related to the time needed for the relaxation of the dihedral angle degrees of freedom. The tame for non-bonded interactions, which drive protein molecules to fold under appropriate conditions, to relax becomes extremely long as the temperature is lowered suggesting that the formation of maximally compact structure hi proteins must be a very slow process. © 1993 Wiley-Liss, Inc.     

Comparison of energy-minimized crystal structures of 2,3-dilauroyl-D-glycerol, 3-palmitoyl-DL-glycerol-1-phosphorylethanolamine and 1,2-dilauroyl-DL-phosphatidylethanolamine:acetic acid

Computational procedures have been developed by which the total energy of a lipid multibilayer can be calculated and minimized. The energy is expressed as a sum of non-bonded, electrostatic, hydrogen bonded and torsional energy terms and includes intramolecular and intermolecular components. Calculations were carried out on three lipid crystals for which structural data are available from X-ray diffraction analysis. For each crystal, the energy was minimized as a function of all bond rotations, molecular rotations and translations and the lattice constants. The minimized structures differed by only small amounts from the experimental structures, which confirms the validity of the current set of energy functions and parameters for use with lipids. The intermolecular energy of each crystal is analyzed in terms of lateral interactions, interactions between the two monolayers of the same bilayer and interactions between bilayers. The intermolecular non-bonded energy per CH2 or CH3 group in the acyl chains is also given.     

On the conformation of the disulphide group in L-cystine and its derivatives.

The terminal groups in cystine are asymmetric and hence the dissulphide group with right and left chirality may not have equal energy. An attempt is made to resolve this problem through energy calculation using semi-empirical methods involving non-bonded and electrostatic interactions and simple Pauling type torsional potential for the disulphide group. The results are discussed in the light of experimental data.     

The non-bonded interactions in D-glucose and β-maltose: an ab initio study of conformations produced by empirical force-field calculations

Minimal basis set SCF-MO computations on conformations of α-D-gluco-pyranose, β-D-glucopyranose, and β-maltose resulting from empirical energy minimisation reproduce known trends in relative energy. Analysis of electron population and molecular orbital valency-state energy leads to separation of atoms into classes, two for oxygen, three for carbon, and two for hydrogen, the classes being correlated with the chemical environments of the atoms. Partitioning of the total energy into two-centre terms gives a quantification of non-bonded interactions, leading to potential energy curves for interactions of all types of atom present. Peculiar details of the electronic structure at and around the anomeric carbon atoms are noted: C(1s) chemical shift is insensitive to configuration, but depends on conformation.     

Alteration of the DNA double helix conformation upon incorporation of mispairs as revealed by energy computations and pathways of point mutations.

To explain biochemical and genetic data on spontaneous nucleotide replacements in nucleic acid biosynthesis all the 8 mispairs in normal tautomeric forms have been considered. Possible B-conformations of DNA fragments containing each of such mispairs incorporated between Watson-Crick pairs have been found using computations of the energy of non-bonded interactions via classical potential functions. These conformations have no reduced interatomic contacts. The values of each dihedral angle of the sugar-phosphate backbone fall within the limits of those of double-helical fragments of B-DNA in crystals. These values differ from those of the corresponding angles for the low-energy polynucleotide conformations consisting of canonical pairs by no more than 30 degrees (except for the fragment with the U:U pair for which the C4'-C3'-O-P angle differs by about 50 degrees). The difference in experimentally observed frequencies of various nucleotide replacements in DNA biosynthesis correlates with the difference in the energy of non-bonded interactions and with the extent of the sugar-phosphate backbone distortion for the fragments containing the mispairs which serve as intermediates for the replacements.     

A novel algorithm for non-bonded-list updating in molecular simulations.

Simulations of molecular systems typically handle interactions within non-bonded pairs. Generating and updating a list of these pairs can be the most time-consuming part of energy calculations for large systems. Thus, efficient non-bonded list processing can speed up the energy calculations significantly. While the asymptotic complexity of current algorithms (namely O(N), where N is the number of particles) is probably the lowest possible, a wide space for optimization is still left. This article offers a heuristic extension to the previously suggested grid based algorithms. We show that, when the average particle movements are slow, simulation time can be reduced considerably. The proposed algorithm has been implemented in the DistanceMatrix class of the molecular modeling package MESHI. MESHI is freely available at .     

Theoretical conformational analysis of 2-phosphopropionic acid, 2-phospho,3-olo-propionic acid and D-2,3-diphosphoglycerate.

Partition energy method (PEM) has been used to calculate the molecular conformation of three organic phosphates of biological interest, namely the 2-phosphopropionic acid, the 2-phospho-3-olo-propionic acid and the d-2,3 diphosphoglycerate (DPG). The analysis shows that the results for the symmetrically independent conformations of these phosphates indicate a strong dependence upon the electrostatic contributions. This contribution is critical in the case of DPG because of the presence of two phosphate groups. Considering only van der Waals non-bonded and torsional energy contributions, seven conformations are allowed. Including the electrostatic energy contributions an extended conformation is preferred. The derived data are discussed in relation to experimental results.     

Computer analysis of protein-protein interaction

An automatic procedure which generates possible modes of protein-protein association is developed and applied to the bovine pancreatic trypsin inhibitor-trypsin complex as a test case. Using a simplified model in which each residue is replaced by one interaction center, all possible modes of interaction between the inhibitor and the active center of the enzyme are generated systematically. The non-bonded interactions between the molecules and the protein surface area buried in the generated interfaces are evaluated and used as criteria for selecting stable complexes. We show that satisfactory estimates of accessible and buried surface areas can be made using the simplified model.The procedure leads to about nine structures having non-bonded interactions and buried surface areas similar to those of the native complex. This suggests that the major contributions to the free energy of dissociation are taken into account by our selection procedure, though complementarity and specificity are not properly represented in the simplified model. However, it makes it possible to scan a much larger number of configurations than would otherwise be feasible, chiefly through elimination of side-chain detail.     

Conformational analysis of xyloglucans

Xyloglucan isolated from the elongating regions of pea stems was examined using X-ray diffraction and energy calculations. The X-ray fibre pattern suggested that the backbone (1----4)-beta-D-glucan takes an extended two-fold helix similar to common cellulose. In order to study side chains (xylosyl or fucosyl-galactosyl-xylosyl residues) of the polysaccharide, energetically preferable conformations were searched by calculation of interactions between non-bonded atom pairs. A stepwise calculation for the conformation of fucosyl-galactosyl-xylosyl residue gave 10 allowed area (phi-psi) maps which are useful to deduce xyloglucan conformations of both monocotyledons and dicotyledons in the walls of growing plant cells.     

Ribosomal subunit orientations determined in the monomeric ribosome by single and by double-labeling immune electron microscopy

The energies of two and three-chain antiparallel and parallel β-sheets have been minimized. The chains were considered to be equivalent. In each case, chains consisting of four and of eight l-alanine residues, respectively, with CH₃CO- and -NHCH₃ end groups were examined. Computations were carried out both for chains constrained to have a regular structure (i.e. the same φ and ψ dihedral angles for each residue) and for chains in which the regularity constraint was relaxed. All computed minimum-energy β-sheets were found to have a right-handed twist, as observed in proteins. As in the case of right-handed α-helices, it is the intrastrand non-bonded interaction energy that plays the key role in forcing β-sheets of l-amino acid residues to adopt a right-handed twist. The non-bonded energy contribution favoring the right-handed twist is the result of many small pairwise interatomic interactions involving the C^βH₃ groups. Polyglycine β-sheets, lacking the C^βH₃ side-chains, are not twisted. The twist of the poly-l-alanine sheet diminishes as the number of residues per chain increases, in agreement with observations. The twist of the four-residue chain increases somewhat (because of interstrand non-bonded interactions, also involving the C^βH₃ groups) in going from a single chain to a two-chain antiparallel structure, but then decreases slightly in going from a two-chain to a three-chain structure. β-Sheets in observed protein structures sometimes have a larger twist than those in the structures computed here. This may be due to irregularities in amino acid sequence and in hydrogenbonding patterns in the observed sheets, or to long-range interactions in proteins. The minimized energies of parallel β-sheets are considerably higher than those of the corresponding antiparallel β-sheets, indicating that parallel β-sheets are intrinsically less stable. This finding about the two kinds of β-sheets agrees with suggestions based on analyses of β-sheets observed in proteins. The energy difference between antiparallel and parallel β-sheets is due to closer packing of the chains and a more favorable alignment of the peptide dipoles in the antiparallel structures. The hydrogen-bond geometry in the computed antiparallel structures is very close to that proposed by Arnott et al. (1967) for the β-form of poly-l-alanine.