用距离几何算法和同源建模法对水稻类金属硫蛋白(rgMT)的三维结构建模

水稻类金属硫蛋白(rgMT)的两端是高度保守的半胱氨酸富含区的结构域(CR区),中间是不含半胱氨酸的间隔区,呈典型的三段式结构.本研究分别采用距离几何算法和同源建模相结合的方法对水稻类金属硫蛋白进行三级结构建模.在排列出CR区的所有可能的半胱氨酸-金属硫络合的组合方式,并对每一种组合方式给出一定的限制条件后各生成20个随机构象.根据生成的随机构象是否能形成金属硫络合结构,从900个随机构象中最终选出6个构象(N端4种,C端2种组合)作为可能的结构模型.另一方面,采用GOR方法对间隔区进行了二级结构预测,随后用同源建模法对其建模.将上述建成的三部分模型连接起来后形成rgMT的整体三维构象.结果表明rgMT能像哺乳动物MT蛋白一样,可形成两个独立的、在结构和能量上均没有障碍的金属-硫络合结构.介于所有植物类金属硫蛋白都具有典型的三段式结构,其中的一部分还具有与rgMT相同的半胱氨酸排列方式,所以rgMT三维结构模型的建立对于其他植物类金属硫蛋白的结构研究具有重要的参考价值.     

植物类金属硫蛋白半胱氨酸富含区结构的建模

详细了解蛋白质的三级结构信息有助于理解其生物学功能.随着植物基因组研究的进展,已发现了50多个植物类金属硫蛋白(Metallothionein-Like, MT-L)基因.但至今只有少数几个MT-L蛋白得到了纯化,而其结构尚无报道,因此有必要建立分析这类蛋白结构特征的方法.本研究根据已知的哺乳动物MT的结构数据,分析得出了CXC、CXXC模式和金属-硫络合簇结构原子间的距离限制条件,并用距离几何算法计算得出预测蛋白可能的构象;然后通过统计分析筛选出目标函数值显著较小、构象能低的结构作为这些蛋白半胱氨酸富含区的预测结构,由此建成了适合于植物类金属硫蛋白半胱氨酸富含区的结构预测方法.从应用该方法正确地预测出了已知结构的蓝蟹MT的结构来看,该方法是可行的.并用该方法预测了油菜MT-L蛋白的半胱氨酸富含区的结构.     

植物类金属硫蛋白半胱氨酸富含区结构的建模

详细了解蛋白质的三级结构信息有助于理解其生物学功能。随着植物基因组研究的进展 ,已发现了 50多个植物类金属硫蛋白 (Metallothionein_Like ,MT_L)基因。但至今只有少数几个MT_L蛋白得到了纯化 ,而其结构尚无报道 ,因此有必要建立分析这类蛋白结构特征的方法。本研究根据已知的哺乳动物MT的结构数据 ,分析得出了CXC、CXXC模式和金属 硫络合簇结构原子间的距离限制条件 ,并用距离几何算法计算得出预测蛋白可能的构象 ;然后通过统计分析筛选出目标函数值显著较小、构象能低的结构作为这些蛋白半胱氨酸富含区的预测结构 ,由此建成了适合于植物类金属硫蛋白半胱氨酸富含区的结构预测方法。从应用该方法正确地预测出了已知结构的蓝蟹MT的结构来看 ,该方法是可行的。并用该方法预测了油菜MT_L蛋白的半胱氨酸富含区的结构。     

植物炭疽病真菌金属硫蛋白的三级结构预测

根据实验测定的Ⅰ类金属硫蛋白(metallothionein, MT)三级结构的实验数据,给出该类蛋白质的两种特征结构(CXC、CXXC一级结构,半胱氨酸-金属络合簇三级结构)的原子间距离约束条件,然后运用距离几何算法计算出一系列可能的构象.从这些构象中经统计分析筛选出目标函数值显著较小的结构作为所预测蛋白质的三级结构模型.用已知结构的蓝蟹MT对方法进行检验证实其可行性后,对植物炭疽病真菌金属硫蛋白CAP3进行了三级结构预测.     

脱氮硫杆菌硫化合物载体SoxYZ蛋白的同源建模和结构分析

脱氮硫杆菌(Thiobacillus denitrificans)中的Sox蛋白在硫代谢过程中起着至关重要的作用,硫化合物需先与硫氧化基因族(sox)编码的蛋白质Sox YZ二聚体共价连接后才能与其他酶发生相互作用。利用同源建模法构建硫化合物载体Sox YZ蛋白的二聚体结构并验证了其合理性。二聚体相互作用分析发现Sox YZ蛋白的溶剂可及表面积(solvent accessible surface,SAS)为10 922.92,疏水率为50.85%;亚基Sox Y和Sox Z界面处共含有12个氢键和1个Π键来维持其三维结构的稳定性;二聚体表面呈现明显的正负电势互补,两亚基界面处氨基酸残基的VDW作用能和静电作用能分别为-80.925 13kcal/mol和-323.856 57kcal/mol,这说明静电作用是二聚体形成的主要驱动力;Sox Z亚基的残基Thr28、Arg31、Lys32、Ser64、Gly65、Val66、Ser67对Sox Y亚基活性位点构象的稳定有重要作用。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21(Saccharomyces cerevisiae Cu-21)及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE－52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半脱氨酸和金属元素,具有典型的巯基吸收特性。     

岸蟹（Carcinus maenas）金属硫蛋白cDNA及其基因的克隆

利用已知的Ｃ．ｍａｅｎａｓ金属硫蛋白氨基酸序列资料,用全简并的ＰＣＲ引物,从鳃组织总ＲＮＡ中扩增出两种金属硫蛋白ｃＤＮＡ片断,并将其克隆到ｐＧＥＭ－Ｔ载体中,序列测定表明,其中一种ｃＤＮＡ片断核苷酸序列和推知的Ｃ．ｍａｅｎａｓ金属硫蛋白核苷酸序列完全吻合;另一种ｃＤＮＡ片断则在３’端有较大变异。根据前者ｃＤＮＡ片段序列设计特异性引物,扩增并克隆了其编码区全长ｃＤＮＡ和其编码基因,测序结果表明,岸蟹     

锌胁迫对小球藻抗氧化酶和类金属硫蛋白的影响

通过对抗氧化酶活性和类金属硫蛋白的测定,考察在0、5、10、20、50和100 μmol/L Zn²⁺(氯化锌)胁迫下锌对普通海洋小球藻的生物学影响。结果表明:不同浓度Zn²⁺均能抑制小球藻的生长,当Zn²⁺浓度大于10 μmol/L时,小球藻生物量随培养时间延长而迅速下降;过氧化物歧化酶 (SOD)活性随Zn²⁺胁迫浓度的增加而增加,当Zn²⁺浓度为50 μmol/L时SOD活性达到最大,但继续增加Zn²⁺胁迫浓度反而导致SOD活性下降;而过氧化物酶 (POD)活性则随着Zn²⁺胁迫浓度的增加而降低。同时,实验发现藻细胞内有两种主要的锌结合形态,其中Zn结合类金属硫蛋白(Zn-MT-like)与兔肝金属硫蛋白(MT)的分子量相近,且随着Zn²⁺胁迫浓度的增加而出现规律性地增多。因此,藻细胞内Zn-MT-like蛋白的诱导量可作为小球藻受Zn²⁺胁迫的响应指标。     

酵母菌类金属硫蛋白的分离、纯化及性质鉴定

经过对酵母菌的金属抗性试验和培养条件试验,确定了高产类金属硫蛋白的酿酒酵母菌株Cu-21（Saccharomyces cerevisiae Cu-21）及其最佳培养条件。对该菌株的菌体蛋白进行SephadexG-100、DEAE-52结合的两次柱分离纯化,获得了类金属硫蛋白的三个亚型,蛋白性质鉴定结果表明含三个亚型、分子量小、富含半胱氨酸和金属元素,具有典型的巯基吸收特性。     

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.     

Homology modeling by the ICM method

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the noncon-served portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed. © 1995 Wiley-Liss, Inc.     

Cloning, Expression, and Homology Modeling of GroEL Protein from Leptospira interrogans Serovar Autumnalis Strain N2

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and se- quenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indi- cated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and vali- dated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a pro- tein. The superposition of the Cα traces of cpn60 structure and predicted structure of leptospiral GroEL indi- cates good agreement of secondary structure elements with an RMSD value of 1.5 . Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component.     

Protein structure modeling in the proteomics era

Structural proteomics aims to understand the structural basis of protein interactions and functions. A prerequisite for this is the availability of 3D protein structures that mediate the biochemical interactions. The explosion in the number of available gene sequences set the stage for the next step in genome-scale projects – to obtain 3D structures for each protein. To achieve this ambitious goal, the slow and costly structure determination experiments are supplemented with theoretical approaches. The current state and recent advances in structure modeling approaches are reviewed here, with special emphasis on comparative protein structure modeling techniques.     

胰岛素受体底物PH结构域相互作用蛋白结构和功能研究进展

PHIP是一种与胰腺β细胞中胰岛素受体底物（IRS）的PH结构域相互作用的蛋白。根据小鼠PHIP（mPHIP）mRNA翻译的不同起始位点,除全长的PHIP1外,mPHIP基因还编码其他3种不同变异体。在胰岛素诱导的信号途径中,主要分布于细胞核的PHIP1和IRS-1的PH结构域相互作用,介导IRS蛋白酪氨酸的磷酸化。IRS-2和PHIP1的共表达能诱导IRS在细胞膜上的定位,促进葡萄糖转运蛋白4（GLUT4）向细胞质膜的转移。PHIP1的表达能提高β-细胞内细胞周期蛋白D2的表达,促进β细胞的生长。PHIP1的表达活化蛋白激酶B（PKB）,活化的PKB能明显抑制β细胞的凋亡。PHIP与胰岛素信号传导途径中其他信号分子的相互作用机制尚不明确。     

基于SwissPdbViewer和MATLAB对东北虎Sox蛋白三级结构建模及分析

SOX蛋白具有一个与DNA特异结合的高保守HMG-box结构域。为研究东北虎SOX蛋白三级结构的分子机理,利用MATLAB的Bioinformatics工具从GenBank中下载东北虎SOX蛋白序列信息,以三级结构已知的SOX2为模板,联合SwissPdbViewer与MATLAB,采用同源建模方法对SOX蛋白HMG-box进行建模、预测;利用MATLAB的Visualization Tool分析预测结果的三维结构。结果显示PtSox蛋白的HMG-box由3个α-螺旋和2个loop区构成;热稳定性分析表明PtSox蛋白loop区的热力学结构不稳定;表面静电分布显示出PtSox蛋白C-端的中间有一个可能与其它小分子或蛋白质的相互作用位点的N/C腔,上述空间结构可能与其活性与功能的调控有关。     

Protein structure prediction constrained by solution X-ray scattering data and structural homology identification

Here we perform a systematic exploration of the use of distance constraints derived from small angle X-ray scattering (SAXS) measurements to filter candidate protein structures for the purpose of protein structure prediction. This is an intrinsically more complex task than that of applying distance constraints derived from NMR data where the identity of the pair of amino acid residues subject to a given distance constraint is known. SAXS, on the other hand, yields a histogram of pair distances (pair distribution function), but the identities of the pairs contributing to a given bin of the histogram are not known. Our study is based on an extension of the Levitt-Hinds coarse grained approach to ab initio protein structure prediction to generate a candidate set of C(alpha) backbones. In spite of the lack of specific residue information inherent in the SAXS data, our study shows that the implementation of a SAXS filter is capable of effectively purifying the set of native structure candidates and thus provides a substantial improvement in the reliability of protein structure prediction. We test the quality of our predicted C(alpha) backbones by doing structural homology searches against the Dali domain library, and find that the results are very encouraging. In spite of the lack of local structural details and limited modeling accuracy at the C(alpha) backbone level, we find that useful information about fold classification can be extracted from this procedure. This approach thus provides a way to use a SAXS data based structure prediction algorithm to generate potential structural homologies in cases where lack of sequence homology prevents identification of candidate folds for a given protein. Thus our approach has the potential to help in determination of the biological function of a protein based on structural homology instead of sequence homology.