用于蛋白质同源模建及三维结构预测的结构比较方法

继前面的工作把测试蛋白从三族扩大到十一族,寻求联配参数的普适“缺省值“;比较不同的主链曲率和挠率的计算方法,进一步确认主链的折红红分几何刻划方法的有效性;寻找有效的可变缺失突变惩罚函数的形式。结果表明,编制的蛋白质多重联配软件系统是满意的,可用于蛋白质三维结构预测。     

嗜热厌氧乙醇杆菌α-葡萄糖苷酶基因的表达及酶学性质的研究

用PCR方法从嗜热厌氧乙醇杆菌(Thermoanaerobacter ethanolicus)JW200中扩增出编码a-葡萄糖苷酶的基因,将其克隆到大肠杆菌(Escherichia coli)表达载体pTrc99A上并获得表达a-葡萄糖苷酶的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测出蛋白相对分子量约89kDa,经阴离子交换层析和凝胶层析纯化后的a-葡萄糖苷酶最适反应温度为70℃,最适反应pH为5～5.5,且在pH 5.5～6.5之间有较高的稳定性。重组a-葡萄糖苷酶在70℃下105 min后酶活仍达到80%。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

嗜热淀粉芽孢杆菌来源β-葡萄糖苷酶的重组表达与酶学性质

【背景】β-葡萄糖苷酶(EC 3.2.1.21,β-glucosidase),是纤维素分解酶系中的重要组成部分,目前工业上应用的β-葡萄糖苷酶多数来源于植物和真菌,来源于细菌的较少,且应用中还存在酶活力偏低、热稳定性差、反应条件适用范围窄、酶活力易受产物反馈抑制等问题,增加了经济成本。嗜热微生物具有特殊的遗传信息资源,极有可能从中挖掘到酶学性质优良的新型β-葡萄糖苷酶,从而解决工业难题。【目的】从嗜热淀粉芽孢杆菌(Bacillus thermoamylovorans)基因组中挖掘新型β-葡萄糖苷酶基因,通过基因重组、异源表达和蛋白纯化技术制备新型β-葡萄糖苷酶,并探究其酶学性质,为新型β-葡萄糖苷酶在纤维素水解等领域的应用奠定基础。【方法】人工合成新型β-葡萄糖苷酶基因bgl52,构建重组表达质粒pET22b-bgl52,并用电脉冲法转化到大肠杆菌BL21(DE3)中实现可溶性表达,利用Ni-NTA亲和层析纯化得到高纯度的β-葡萄糖苷酶Bgl52。【结果】实现重组表达质粒pET22b-bgl52在大肠杆菌BL21(DE3)中的可溶性表达,并获得β-葡萄糖苷酶Bgl52纯蛋白,蛋白分子量...     

用于蛋白质分子设计的环区模建方法

利用距离约束的数据库搜索方法和接触势能分析技术，提出了一种用来模建蛋白质结构环区的分子模建方法。通过对珠蛋白、丝氨酸蛋白酶、钙结合蛋白和溶菌酶中的５０个环区的模建，证明上述方法是可行的。对总区５０个环区的模建表明，８６％的环区可以模建，只有１４％的环区不能模建。研究结果表明这种方法非常适用于蛋白质工程中的环区模建。     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

嗜热栖热菌HB 8耐热α-葡萄糖苷酶的提纯和性质

嗜热栖热菌HB 8(Thermus thermophilus<.I> H8 8 )的耐热α-葡萄糖苷酶(α-glucosidaseEC．.2.1.20)经硫酸铵分步沉淀、DEAE-纤维素柱层析和垂直板制备凝胶电泳提纯,经盘状凝胶电泳鉴定为单一区带,比活提高17倍。酶作用最适温度80℃,最适pH5．8,分子量67000,等电点Pl为4.5。该酶能够水解对硝基酚α--D-葡萄糖苷(PNPG)、蔗糖和麦芽糖,但不水解纤维二糖、蜜二糖、可溶性淀粉。酶作用于PNPG的米氏常数(Km)为0.4mmol／L,最大反应速度Vmax为0.29umol·nmin-1·mg-1。金属离子Mg2+、Mn2+、Ca2+和Ba2+对酶有激活怍用,Hg2+和Cu2+有强烈抑制作用。酶表现出极好的热稳定性,在90℃保温10小时后,仍保留90％的原始酶活力,在95℃的失活半寿期(t1/2)为108分钟．经蛋白质侧链化学修饰研究表明,羧基和组氨酸残基为其表现话力所必需。     

白色念珠菌羊毛甾醇14α—去甲基化酶三维结构分子？…

基于四种原核细胞色素Ｐ４５０晶体蛋白Ｐ４５０ＢＭ３、Ｐ４５０ｃａｍ、Ｐ４５０ｔｅｒｐ、４５０ｅｒｙＦ模建白色念珠菌羊毛甾醇１４α－去甲基化酶三维结构。序列匹配采用四种晶体结构比较结果基础之上提出的细胞色素Ｐ４５０超家族蛋白基于结构知识的序列匹配方法。以Ｐ４５０ＢＭ３晶体结构坐标模建目标蛋白结构保守区主链结构,结构保守区侧链构象来源于四种晶体蛋白与模建蛋白对应残基同源性得分最高残基构象。建模结果用分     

用于蛋白质分子设计的三维模体搜索

三维结构模体,常是蛋白质中离散残基片断组成的功能性子结构,传统的序列依赖结构比较方法无法地其搜索,本文采用序列无关的结构比较算法,实现了搜索程序,经多例测试,证明本程序是快速和良好的识别三维结构模式的工具,在小分子设计和多肽模拟中有重要的应用。     

Myostatin三维结构模建及分子进化分析

Myostatin(MST)为肌肉生长负调节因子,其功能受抑制可导致肌肉量增加.对MST核酸序列进行序列比对,构建进化树;采用同源模建方法首次模建MST成熟肽生物活性二聚体的四级结构,并预测MST与其受体ActRIIB的相互作用模式.进化树将肌肉生长抑制素基因(MSTN)分成4个亚家族:哺乳动物MSTN,鸟类MSTN以及鱼类MSTN 1和2.MST受纯化选择作用,在不同物种的直系同源基因具有较高的刚源性,其中哺乳动物、鸟类MST C端活性肽氨基酸序列高度保守.表明哺乳动物、鸟类MST的结构、功能类似,且信号传导路径可能一致;而鱼类MST的调控机制可能存在较大差异.MST结构及其表面静电势和疏水氨基酸分布表明静电力和疏水相互作用在MST与其受体结合过程中可能起到十分重要的作用.     

Three Dimensional Structure of Haemoglobin Rainier

The difference electron density map of deoxyhaemoglobin Rainier (α₂β₂^145Tyr→Cys) shows that the cysteine introduced by the mutation forms a disulphide bridge with the reactive cysteine, F9(93)β, of the same β-chain. The resulting structural distortions affect the C-terminus of the β-chain, thereby inhibiting part of the alkaline Bohr effect and haem–haem interaction.     

猪SLA-DR二级结构预测及同源模建

应用EXPASY服务器（http：／／www．expasy．oh／tools）上的SOPMA法对SLA—DR的α链和口链进行二级结构的预测,并与人的HLA—DR相应的α链和β链的氨基酸和二级结构成分比较,在此基础上同源模建SLA—DRα链、β链及复合体SLA—DR的三级结构。结果显示,SLA—DR的α链各二级结构成分α螺旋、β折叠、转角和无规则卷曲的数目分别为36、61、4和69,β链中分别为42、56、16和74。α链和β链各二级结构成分与复合体SLA—DR具有高度的符合率,分别达到98．7％（α螺旋）、99．1％（β折叠）、83．3％（转角）和97．2％（无规则卷曲）。各功能区分析,SLA—DR的α1和β1区为结合抗原的高变化区。同源模建和三级结构分析表明SLA—DR的α链和口链具有独立的三级结构,并可以组成复合体SLA—DR。     

Prediction of Three Dimensional Structure of Calmodulin

Calmodulin (CaM) is an important human protein, which has multiple structures. Numerous researchers studied the CaM structures in the past, and about 50 different structures in complex with fragments derived from CaM-regulated proteins have been discovered. Discovery and analysis of existing and new CaM structures is difficult due to the inherent complexity, i.e. flexibility of 6 loops and a central linker that constitute part of the CaM structure. The extensive interest in CaM structure analysis and discovery calls for a comprehensive study, which based on the accumulated expertise would design a method for prediction and analysis of future and existing CaM structures. It is also important to find the mechanisms by which the protein adjusts its structure with respect to various factors. To this end, this paper analyzes the known CaM structures and finds four factors that influence CaM structure, which include existence of Ca²⁺ binding, different binding segments, measuring surroundings, and sequence mutation. The degree of influence of specific factors on different structural regions is also investigated. Based on the analysis of the relation between the four factors and the corresponding CaM structure a novel method for prediction of the CaM structure in complex with novel segments, given that the surroundings of the complex, is developed. The developed prediction method is tested on a set aside, newest CaM structure. The prediction results provide useful and accurate information about the structure verifying high quality of the proposed prediction method and performed structural analysis.     

Predicting the Structure of the Flavodoxin from Eschericia coli by Homology Modeling,Distance Geometry and Molecular Dynamics

Abstract

As part of our on-going development of a method, based upon distance geometry calculations, for predicting the structures of proteins from the known structures of their homologues, we have predicted the structure of the 176 residue Flavodoxin from Escherichia coli. This prediction was based upon the crystal structures of the homologous Flavodoxins from Anacystis nidulans, Chondrus crispus, Desulfovibrio vulgaris and Clostridium beijerinckii, whose sequence identities with Escherichia coli were 44%, 33%, 23% and 16%, respectively. A total of 13,043 distance constraints among the alpha-carbons of the Escherichia coli structure were derived from the sequence alignments with the known structures, together with 8,893 distance constraints among backbone and sidechain atoms of adjacent residues, 978 between the alpha-carbons and selected atoms of the flavin mononucleotide cofactor, 116 constraints to enforce conserved hydrogen bonds, and 452 constraints on the torsion angles in conserved residues. An ensemble of ten random Escherichia coli structures was computed from these constraints, with an average root mean square coordinate deviation (RMSD) among the alpha carbons of 0.85 Ångstroms (excluding the first 1 and last 6 residues, which have no corresponding residues in any of the homologues and hence were unconstrained); the corresponding average heavy-atom RMSD was 1.60 Å.

Since the distance geometry calculations were performed without hydrogen atoms, protons were added to the resulting structures and these structures embedded in a 50 × 50 × 40 Å solvent box with periodic boundary conditions. They were then subjected to a 20 picosecond dynamical simulated annealing procedure, starting at 300 K and gradually reduced to 10K, in which all the distance and torsion angle constraints were maintained by means of harmonic restraint functions. This was followed up by 1000 iterations of unrestrained conjugate gradients minimization. The goal of this energy refinement procedure was not to drastically modify the structures in an attempt at a priori prediction, but merely to improve upon the predictions obtained from the geometric constraints, particularly with regard to their local backbone and sidechain conformations and their hydrogen bonds. The resulting structures differed from the respective starting structures by an average of 1.52 Å in their heavy atom RMSD's, while the average RMSD among the heavy atoms of residues 2-170 increased slightly to 1.66 Å. We hope these structures will be good enough to enable the phase problem to be solved for the crystallographic data that is now being collected on this protein.     

Molecular Modeling of Immunoglobulin Superfamily Proteins: Predicting the Three Dimensional Structure of the Extracellular Domain of CTLA-4 (CD152)

The interactions between CD28/CTLA-4 (CD152) on T cells and their ligands CD80/CD86 on antigen presenting cells provide costimulatory signals critical for T cell activation. CD28/CTLA-4 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). CD28 and CTLA-4 both contain a single extracellular immunoglobulin (Ig) domain which binds CD80/CD86. Here we report modeling studies on the three-dimensional (3D) structure of the CTLA-4 binding domain. Since CTLA-4 displays only very weak sequence homology to proteins with known 3D structure, conventional modeling techniques were difficult to apply. Structure-oriented sequence comparison, consensus residue analysis, conformational searching, and inverse folding calculations were employed to aid in the generation of a comparative CTLA-4 model. Regions of high and low prediction confidence were identified, and the sequence-structure compatibility of the model was determined. Characteristics of the modeled structure, which resembles an Ig V domain, were analyzed, and the model was used to map N-linked glycosylation sites and residues critical for CTLA-4 function. The modeling approach described here can be applied to predict 3D structures of other IgSF proteins.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s008940050025     

Molecular Modeling of Immunoglobulin Superfamily Proteins: Predicting the Three Dimensional Structure of the Extracellular Domain of CTLA-4 (CD152)

The interactions between CD28/CTLA-4 (CD152) on T cells and their ligands CD80/CD86 on antigen presenting cells provide costimulatory signals critical for T cell activation. CD28/CTLA-4 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). CD28 and CTLA-4 both contain a single extracellular immunoglobulin (Ig) domain which binds CD80/CD86. Here we report modeling studies on the three-dimensional (3D) structure of the CTLA-4 binding domain. Since CTLA-4 displays only very weak sequence homology to proteins with known 3D structure, conventional modeling techniques were difficult to apply. Structure-oriented sequence comparison, consensus residue analysis, conformational searching, and inverse folding calculations were employed to aid in the generation of a comparative CTLA-4 model. Regions of high and low prediction confidence were identified, and the sequence-structure compatibility of the model was determined. Characteristics of the modeled structure, which resembles an Ig V domain, were analyzed, and the model was used to map N-linked glycosylation sites and residues critical for CTLA-4 function. The modeling approach described here can be applied to predict 3D structures of other IgSF proteins.     

决明查尔酮合成酶的同源模建和分子模拟对接

查尔酮合成酶(chalcone synthase,CHS)是植物中类黄酮生物合成途径的关键酶,其催化对-香豆酰辅酶A和丙二酸单酰辅酶A发生缩合反应.本研究以苜蓿CHS的晶体为模板,利用同源建模构建决明CHS的三维模型.经过动力学优化后,决明CHS的三维模型与苜蓿CHS的结构极为相似,主要由α-螺旋和β-折叠构成,其中有13个α螺旋,占32.82％,15个β折叠,占19.23％,无规则卷曲占47.95％.模型验证结果表明决明CHS的三维模型具有合理的立体化学性质与氨基酸相容性.决明CHS含有两个重要的结构域:对-香豆酰辅酶A结合域与丙二酸单酰辅酶A结合域.决明CHS与对-香豆酰辅酶A、丙二酸单酰辅酶A的结合主要通过氢键与范德华力.决明CHS中Cys164、His303与活性中心的H2O能够形成电子传递体系,参与对-香豆酰辅酶A形成CHS-对-香豆酰基中间产物.本研究结果为利用此类CHS三维模型研究其催化机理和分子工程改造奠定基础.     

大肠杆菌P4 FliC蛋白结构分析及同源建模

为了进一步认识大肠杆菌鞭毛蛋白(Escherichia coliFliC)的结构及功能,深入研究细菌鞭毛的生物学特性,采用生物信息学方法,对组成Escherichia coliP4 FliC蛋白质的理化性质、二级结构及三级结构等进行生物信息学分析,并在三级结构的基础上进行同源建模。理化性质分析结果表明,E.coliP4 FliC蛋白为酸性结构蛋白,与沙门氏菌鞭毛蛋白性质较为接近,二级结构以α-螺旋和随机卷曲为主要构件,其空间结构与Salmonella typhimurium的3a5xA蛋白相似性较高,以此为模板成功构建了可靠的三维结构分子模型。FliC蛋白为多种细菌的鞭毛蛋白,与细菌的运动功能关系密切,本研究为深入研究细菌的运动机制及致病机理奠定基础。