人脑和人血清胆碱酯酶三维结构的计算机模拟研究

本文以同源的电鳐胆碱酯酶（Ｔ．ＡＣｈＥ）的三维结构为模板,模拟预测了人脑和血清胆碱酯酶（Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ）的三维结构和活力中心的组成。指Ｔ．ＡＣｈＥ,Ｈ．ＡＣｈＥ和Ｈ．ＢｕＣｈＥ宁间结构差异,并讨论了ＡＣｈ和Ｈ。ＡＣｈＥ的对接（ｄｏｃｋｉｎｇ）。Ｈ。ＡＣｈＥ和Ｈ．ＢｕＣｈＥ三维结构的确定将为进一步深入研究它的中毒机理和合理药物设计提供靶子。     

重组乳酸乳球菌X-脯氨酰二-肽酰基-氨基肽酶的纯化及动力学特性(英文)

重组的乳酸乳球菌X 脯氨酰 二肽酰基 氨基肽酶是一个工具酶 ,它对基因构建的神经肽或活性多肽的转活具有重要意义 .通过细菌细胞的破碎 ,洗涤 ,冷冻离心 ,透析 ,DEAE 纤维素 5 2柱层析等工艺过程达到电泳纯 .该酶比活为 11.92 6U mg ,纯化倍数为 14.37倍和总活性收率为5 5 .5 6% .通过SDS PAGE和凝胶柱层析法 ,测得该二肽酶有单肽链组成 ,分子量 89KD .在该酶的动力学研究中 ,针对特异性底物L 甘氨酰 L 脯氨酰 对 硝基苯胺 ,求得该酶的米氏方程式 1 V =0 0 4 8 [S]+ 0 2 5 66(r =0 994 ) .它的Km 值为 0 1871mmol L ,最大反应速度Vmax为 3 897μmol·L-1·min-1.该酶可被苯甲酰基磺酰氟 ,胰蛋白酶抑制剂和Mn2 +,Ba2 +,Cu2 +andZn2 +等金属离子抑制 ,但可被Mg2 +激活 .进一步试验显示 ,当Cu2 +和Zn2 +浓度增加到 3 72 6mmol L ,抑制作用明显增强 .低浓度的EDTA Na2 (≤ 0 62 12mmol L)不影响酶的活性 .因此 ,该X 脯氨酰 二肽酰基 氨基肽酶是一个金属离子非依赖性的丝氨酸蛋白酶     

人心脏低维动力学模型的计算机模拟

心脏窦房结与异位心室搏动非线性相互作用,导致调制并行收缩反常节律。本文从新的低维动力学心脏模型（双参量圆周映射）出发,研究由周期性到不规则、混沌动力学过渡的性质。模型的数值实验揭示出在双参量空间不同区域中,某些特征性步入混沌的道路和总体标度关系。一个明显特征是,在双参量空间宽广范围内观察到无穷层次的嵌套结构,即嵌在混沌带中的周期递加序列。而且,出现周期性的位置或者周期窗口的宽度,均存在普适的标度关系。另一方面,在参量空间一部分,包含相互交叠的不同周期性稳定轨道,有极复杂图象。本文结果显示,运用数值方法计算李雅普诺夫指数谱,可以基本上确定人心脏圆周映射的二维分岔结构和标度性质。     

植物Rubisco活性中心的模拟分析

通过对与不同配基结合的植物Rubisco复合物结构的重叠比较分析 ,发现Rubisco的活性差异是由其中一段Loop6环序列所造成的 ;金属离子与活性中心的结合会造成活性中心巨大的构象变化 .进一步用SwissPDBViewer软件模拟不同配基的植物Rubisco活性中心与此Loop环的氢键相互作用 .结果表明 ,有 3个Lys残基Lys2 0 1、Lys334、Lys175与Rubisco是否处于活性状态密切相关 ,这些残基的结构变化对分子设计可能有重要的参考价值     

TNF-α拮抗肽拮抗机制的计算机模拟研究

为了了解从肽库中筛选到的TNF-α拮抗肽的作用机制,用分子模拟程序模建了4条肽序列(TBP1、2、3、6)的结构,然后利用分子对接程序建立了它们与TNF-α作用的理论模型。在得到的模型中,拮抗肽TBP2、3与TNF-α相互作用的区域位于TNF-α分子中形成三聚体的区域,提示这些肽是通过影响TNF-α三聚体的形成而发挥作用的。     

生物大分子体系及其相互作用的计算机模拟

生物大分子体系及其相互作用的计算机模拟陈凯先（中国科学院上海药物研究所２０００３１）随着分子生物学的发展和计算机技术的进步,运用各种数学、物理学的理论方法,以计算机作为工具进行生物学的基本理论研究,特别是进行生物大分子体系及其相互作用的计算机分子图形...     

计算机模拟酶解制备驴乳清蛋白抗氧化肽的研究

本文选用驴乳清蛋白为原材料,以DPPH自由基清除率为指标,利用计算机模拟酶解驴乳清蛋白,筛选出能够产生抗氧化活性肽的最适蛋白水解酶,以pH、酶解温度、酶底比(质量比)为自变量,采用Design-Expert V8.0.6设计响应面试验,确定以α-胰凝乳蛋白酶酶解驴乳清蛋白制备抗氧化肽的最佳工艺条件。结果表明在底物浓度4%,酶解时间4 h的条件下,当温度达到39℃,pH 8,酶底比4%时得到的酶解肽抗氧化活性最强,10 mg/mL驴乳清蛋白酶解肽的DPPH自由基清除率最高可达46.23%。     

肠炎沙门菌肽脯氨酰顺反异构酶SlyD的基因敲除、表达及酶学特征

【目的】以肠炎沙门菌肽脯氨酰顺反异构酶SlyD为对象,构建基因缺失株及表达纯化该蛋白,为研究其在肠炎沙门菌致病性与应激等方面的作用奠定基础。【方法】参考Gen Bank登录的肠炎沙门菌基因组序列设计用于slyD基因敲除及原核表达的特异引物,运用自杀质粒介导的同源重组技术对肠炎沙门菌C50041 slyD基因进行敲除,构建C50041ΔslyD缺失株;原核表达SlyD蛋白,通过α-糜蛋白酶耦联法对其PPIase活性进行测定;利用生物信息学相关软件,分析SlyD蛋白的氨基酸序列及功能域。【结果】PCR鉴定与测序结果证明成功构建了肠炎沙门菌C50041ΔslyD缺失株,其生长特性与野生株基本一致;SDS-PAGE及PPIase活性分析表明,获得了具有生物活性的可溶性SlyD蛋白;生物信息学分析显示SlyD蛋白由FKBP样肽脯氨酰顺反异构酶结构域、分子伴侣功能域和金属结合区域3个功能区域组成。【结论】成功获得了肠炎沙门菌C50041ΔslyD缺失株和具有PPIase活性的重组SlyD蛋白。     

RasGAP作用下ras P21水解GTP的计算机模拟分析

利用PDB(brookhaven protein data bank)文库提供的晶体结构数据,从大分子的计算机三维模拟分析入手,分析了rαs P21在RasGAP(GTPase-aetivating protein of RAS)作用下水解GTP的具体机制。对rαs P21核苷酸结合口袋的微环境和rαs P21在GTP水解过程中的结构变化进行了讨论。在分析rαs P21空间三维结构的基础上,分析了催化水解过程中RasGAP的变化。讨论了对于Rsa GAP和rαs P21的结合定位以及对水解过程中rαs P21的Switchl的构象变化起重要作用的Lys949手指结构,并给出了rαs P21在RasGAP催化下水解GTP的一个模型,其结果是进一步的变异分析的基础。     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

Summary The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[d-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[d-Dab-Gly-Trp-d-Leu] (II) (DNS=dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a β-turn at residues 2 and 3.     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[D-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[D-Dab-Gly-Trp-D-Leu] (II) (DNS = dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a -turn at residues 2 and 3.     

Modeling the paramyxovirus hemagglutinin-neuraminidase protein

The paramyxovirus hemagglutinin-neuraminidase (HN) protein exhibits neuraminidase activity and has an active site functionally similar to that in influenza neuraminidases. Earlier work identified conserved amino acids among HN sequences and proposed similarity between HN and influenza neuraminidase sequences. In this work we identify the three-dimensional fold and develop a more detailed model for the HN protein, in the process we examine a variety of protein structure prediction methods. We use the known structures of viral and bacterial neuraminidases as controls in testing the success of protein structure prediction and modeling methods, including knowledge-based threading, discrete three-dimensional environmental profiles, hidden Markov models, neural network secondary structure prediction, pattern matching, and hydropathy plots. The results from threading show that the HN protein sequence has a 6 β-sheet propellor fold and enable us to assign the locations of the individual β-strands. The three-dimensional environmental profile and hidden Markov model methods were not successful in this work. The model developed in this work helps to understand better the biological function of the HN protein and design inhibitors of the enzyme and serves as an assessment of some protein structure prediction methods, especially after the x-ray crystallographic solution of its structure. Proteins 29:264–281, 1997. © 1997 Wiley-Liss, Inc.     

Prolidase activity in fibroblasts is regulated by interaction of extracellular matrix with cell surface integrin receptors

Prolidase (EC 3.4.13.9) is a ubiquitously distributed imidodipeptidase that catalyzes the hydrolysis of C-terminal proline or hydroxyproline containing dipeptides. The enzyme plays an important role in the recycling of proline for collagen synthesis and cell growth. An increase in enzyme activity is correlated with increased rates of collagen turnover indicative of extracellular matrix (ECM) remodeling, but the mechanism linking prolidase activity and ECM is poorly understood. Thus, the effect of ECM-cell interaction on intracellular prolidase activity is of special interest. In cultured human skin fibroblasts, the interaction with ECM and, more specifically, type I collagen mediated by the β₁ integrin receptor regulates cellular prolidase activity. Supporting evidence comes from the following observations: 1) in sparse cells with a low amount of ECM collagen or in confluent cells in which ECM collagen was removed by collagenase (but not by trypsin or elastase) treatment, prolidase activity was decreased; 2) this effect was reversed by the addition of type I collagen or β₁ integrin antibody (agonist for β₁ integrin receptor); 3) sparse cells (with typically low prolidase activity) showed increased prolidase activity when grown on plates coated with type I collagen or on type IV collagen and laminin, constituents of basement membrane; 4) the relative differences in prolidase activity due to collagenase treatment and subsequent recovery of the activity by β₁ integrin antibody or type I collagen treatment were accompanied by parallel differences in the amount of the enzyme protein recovered from these cells, as shown by Western immunoblot analysis. Thus, we conclude that prolidase activity responded to ECM metabolism (tissue remodeling) through signals mediated by the integrin receptor. J. Cell. Biochem. 67:166–175, 1997. Published 1997 Wiley-Liss, Inc.     

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.     

Discovery of Fragment Molecules That Bind the Human Peroxiredoxin 5 Active Site

The search for protein ligands is a crucial step in the inhibitor design process. Fragment screening represents an interesting method to rapidly find lead molecules, as it enables the exploration of a larger portion of the chemical space with a smaller number of compounds as compared to screening based on drug-sized molecules. Moreover, fragment screening usually leads to hit molecules that form few but optimal interactions with the target, thus displaying high ligand efficiencies. Here we report the screening of a homemade library composed of 200 highly diverse fragments against the human Peroxiredoxin 5 protein. Peroxiredoxins compose a family of peroxidases that share the ability to reduce peroxides through a conserved cysteine. The three-dimensional structures of these enzymes ubiquitously found throughout evolution have been extensively studied, however, their biological functions are still not well understood and to date few inhibitors have been discovered against these enzymes. Six fragments from the library were shown to bind to the Peroxiredoxin 5 active site and ligand-induced chemical shift changes were used to drive the docking of these small molecules into the protein structure. The orientation of the fragments in the binding pocket was confirmed by the study of fragment homologues, highlighting the role of hydroxyl functions that hang the ligands to the Peroxiredoxin 5 protein. Among the hit fragments, the small catechol molecule was shown to significantly inhibit Peroxiredoxin 5 activity in a thioredoxin peroxidase assay. This study reports novel data about the ligand-Peroxiredoxin interactions that will help considerably the development of potential Peroxiredoxin inhibitors.     

Crystal Structure of the Ectoine Hydroxylase,a Snapshot of the Active Site

Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.     

Characterization of the Endoribonuclease Active Site of Human Apurinic/Apyrimidinic Endonuclease 1

Apurinic/apyrimidinic endonuclease 1 (APE1) is the major mammalian enzyme in DNA base excision repair that cleaves the DNA phosphodiester backbone immediately 5′ to abasic sites. Recently, we identified APE1 as an endoribonuclease that cleaves a specific coding region of c-myc mRNA in vitro, regulating c-myc mRNA level and half-life in cells. Here, we further characterized the endoribonuclease activity of APE1, focusing on the active-site center of the enzyme previously defined for DNA nuclease activities. We found that most site-directed APE1 mutant proteins (N68A, D70A, Y171F, D210N, F266A, D308A, and H309S), which target amino acid residues constituting the abasic DNA endonuclease active-site pocket, showed significant decreases in endoribonuclease activity. Intriguingly, the D283N APE1 mutant protein retained endoribonuclease and abasic single-stranded RNA cleavage activities, with concurrent loss of apurinic/apyrimidinic (AP) site cleavage activities on double-stranded DNA and single-stranded DNA (ssDNA). The mutant proteins bound c-myc RNA equally well as wild-type (WT) APE1, with the exception of H309N, suggesting that most of these residues contributed primarily to RNA catalysis and not to RNA binding. Interestingly, both the endoribonuclease and the ssRNA AP site cleavage activities of WT APE1 were present in the absence of Mg²⁺, while ssDNA AP site cleavage required Mg²⁺ (optimally at 0.5-2.0 mM). We also found that a 2′-OH on the sugar moiety was absolutely required for RNA cleavage by WT APE1, consistent with APE1 leaving a 3′-PO₄²⁻ group following cleavage of RNA. Altogether, our data support the notion that a common active site is shared for the endoribonuclease and other nuclease activities of APE1; however, we provide evidence that the mechanisms for cleaving RNA, abasic single-stranded RNA, and abasic DNA by APE1 are not identical, an observation that has implications for unraveling the endoribonuclease function of APE1 in vivo.     

Evidence for an Elevated Aspartate pKa in the Active Site of Human Aromatase

Aromatase (CYP19A1), the enzyme that converts androgens to estrogens, is of significant mechanistic and therapeutic interest. Crystal structures and computational studies of this enzyme shed light on the critical role of Asp³⁰⁹ in substrate binding and catalysis. These studies predicted an elevated pK_a for Asp³⁰⁹ and proposed that protonation of this residue was required for function. In this study, UV-visible absorption, circular dichroism, resonance Raman spectroscopy, and enzyme kinetics were used to study the impact of pH on aromatase structure and androstenedione binding. Spectroscopic studies demonstrate that androstenedione binding is pH-dependent, whereas, in contrast, the D309N mutant retains its ability to bind to androstenedione across the entire pH range studied. Neither pH nor mutation perturbed the secondary structure or heme environment. The origin of the observed pH dependence was further narrowed to the protonation equilibria of Asp³⁰⁹ with a parallel set of spectroscopic studies using exemestane and anastrozole. Because exemestane interacts with Asp³⁰⁹ based on its co-crystal structure with the enzyme, its binding is pH-dependent. Aromatase binding to anastrozole is pH-independent, consistent with the hypothesis that this ligand exploits a distinct set of interactions in the active site. In summary, we assign the apparent pK_a of 8.2 observed for androstenedione binding to the side chain of Asp³⁰⁹. To our knowledge, this work represents the first experimental assignment of a pK_a value to a residue in a cytochrome P450. This value is in agreement with theoretical calculations (7.7–8.1) despite the reliance of the computational methods on the conformational snapshots provided by crystal structures.     

胡椒碱分子键合人血清白蛋白位点的表征

利用荧光光谱法、紫外光谱法并结合计算机模拟技术在分子水平上研究了胡椒碱与人血清白蛋白(human serum albumin HSA)的键合作用.同步荧光及紫外光谱图表明,胡椒碱对HSA微环境有影响.位点竞争试验证明,胡椒碱分子键合在HSA的位点Ⅱ区.通过荧光光谱滴定数据求得不同温度下(300K 310K和318 K)药物与蛋白相互作用的结合常数及结合位点数.分子模拟的结果显示了胡椒碱与HSA的键合区域和键合模式,表明药物与蛋白有较强的键合作用;维持药物与蛋白质的相互作用力主要是疏水用,兼有氢键(位于氨基酸残基Arg 257,Arg 222及Arg218位).通过实验数据计算得到的热力学参数(ΔH0与ΔS0的值分别为原33.11 kJ·mol-1和原18.90 J·mol原1·K-1)确定了胡椒碱与HSA分子的相互作用力类型主要为氢键兼范德华力.