人白介素15及突变体的分子设计

以人白介素－２晶体结构为模板同源模型建人白介素１５及其两株突变体（Ｎ端缺失４个氨基酸、Ｃ端缺失３个氨基酸）的空间构象。在ＣＶＦＦ力场下,经过分子力学优化、常温分子动力学模拟获得稳定立体结构模型。借助空间构象残基的亲疏水性分析,利用Ｄｅｌｐｈｉ一性分析蛋白表达静电分布,进行从理论上预测人白介素１５及两株突变体生物学功能的性、差异性。结合分子生物实验,在ＰＢＶ２２０载体中克隆表达获得人白介素１５及两载     

蓖麻毒素A链突变体的设计、表达与活性研究

利用蛋白质结构同源模建并结合表观静电势分析,设计了拟具有生物学活性的蓖麻毒素A链的突变体.将PCR扩增的突变体基因,导入pKK223-3载体中,于大肠杆菌(E.coli)中获得高效、可溶性表达,而且,确证了表达产物具有预期的生物学活性.     

蓖麻毒素A链突变体的设计和结构模建

蓖麻毒素Ａ链（ＲＴＡ）有抑制蛋白质合成的功能,可用作“生物导弹”的弹头,但其免疫原性较强。我们根据国外所做的ＲＴＡ连续突变的实验结果,以及ＰＤＢ库中ＲＴＡ及其同源蛋白的结构信息,设计了一个ＲＴＡ突变体,缺失的５个片段与功能及结构保守性关系较小。然后,我们用同源模建的方法对设计出的ＲＴＡ突变体进行三维结构模建,初步验证表明模型基本合理     

蓖麻毒素A链突变体的设计和结构模建

蓖麻毒素Ａ链（ＲＴＡ）有抑制蛋白质合成的功能,可用作“生物导弹”的弹头,但其免疫原性较强。我们根据国外所做的ＲＴＡ连续突变的实验结果,以及ＰＤＢ库中ＲＴＡ及其同源蛋白的结构信息,设计了一个ＲＴＡ突变体,缺失的５个片段与功能及结构保守性关系较小。然后,我们用同源模建的方法对设计出的ＲＴＡ突变体进行三维结构模建,初步验证表明模型基本合理     

人肽抗生素hPAB—β分子的同源模建及其突变体设计

从人体基因组中克隆了一个编码肽抗生素样的基因。按基因编码产物的氨基酸序列 ,化学合成目的产物 ,经药物敏感测定法证实产物具有杀菌活性。旨在通过同源模建的方法 ,构建肽抗生素hPAB β的突变体分子 ,希望获得肽链更短 ,但生物活性不降低甚至更强的突变体。方法是在序列比较的基础上 ,利用同源分子对目的肽抗生素hPAB β进行同源模建 ,在此模型上 ,删除某些氨基酸后观察目的分子立体结构模型改变情况 ,从而确定突变体分子结构。同源模建结果表明hPAB β由一个α螺旋和 3个 β片层构成 ,二级结构保守 ;具有两亲性结构 ,推测与目的肽进…     

无色杆菌蛋白酶Ⅰ突变体的结构与稳定性研究

报道了用定位诱变技术,改变无色杆蛋白酶Ⅰ的酶原加工位点,使其向左或向右移动,导致突变体成熟酶的肽链从Ｎ端延长或缩短若干氨基酸残基。所获突变体显示了低于野生酶的活性。突变体的园二色谱和荧光光说研究表明,它们的结构发生了某些微小的改变。在不同的ＰＨ值的,温度和不同浓度的ＳＤＳ和盐酸胍条件下,也表现了低于天然酶的稳定性。     

卵清溶菌酶及其定点突变体热力学稳定性研究

测定了野生型卵清溶菌酶及其定点突变体的Ｅ、Ａ３１Ｖ、Ｉ５５Ｌ、Ｓ９１Ａ、Ｄ１０１Ａ、ＳＩＳ、ＳＶＳ、ＴＩＴ、ＴＶＳ（后者三个字母为该酶序列中第４０、５５、９１位的氨基酸残基,野生型为ＴＩＳ）在不同浓度脲溶液中的变性热力学参数和酶活性,稳定性有变化 １６．８６－２８．６７ｋＪ／ｍｏｌ,变性中点脲浓度为４．８９－６．２４ｍｏｌ／Ｌ。并对这个过程的可能机理,定点突变引起的过渡热,和学参数的变化,及个别氨     

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。     

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。     

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。     

The quaternary structure of carbonmonoxy hemoglobin ypsilanti

We present a geometric analysis of the allosteric interface in the new Y state quaternary structure observed in liganded mutant hemoglobin Ypsilanti (β99 Asp → Tyr) by Smith, F.R., Lattman, E.E., Carter, C.W., Jr. (Proteins 10:81–91, 1991). The classical T to R quaternary structure change being a rotation of αβ dimers about an axis which is approximately parallel to the dimer axis of pseudosym-metry, the new quaternary structure is obtained by applying to R an additional rotation about an axis orthogonal to the first. This suggests that Y is a modified R state rather than an intermediate on the T to R pathway. Computer docking experiments designed to simulate the quaternary structure change support this suggestion. © 1993 Wiley-Liss, Inc.     

Heme-based sensors: theoretical modeling of heme-ligand–protein interactions

Heme proteins are uniquely adapted to bind the important diatomic molecules O(2), NO and CO. An increasing number of heme proteins are being discovered that sense these molecules and thereby regulate a variety of biochemical responses. The interactions of diatomic molecules with heme, and with the surrounding protein, are therefore of great interest. Recent theoretical modeling, using density functional theory, captures many features of these interactions, as exemplified by the well-characterized heme protein myoglobin. Important details, however, especially the mutual influence of the bound diatomic molecule and the proximal ligand, remain to be clarified.     

NMR investigation of the dynamics of tryptophan side-chains in hemoglobins

NMR relaxation measurements of 15N spin-lattice relaxation rate (R(1)), spin-spin relaxation rate (R(2)), and heteronuclear nuclear Overhauser effect (NOE) have been carried out at 11.7T and 14.1T as a function of temperature for the side-chains of the tryptophan residues of 15N-labeled and/or (2H,15N)-labeled recombinant human normal adult hemoglobin (Hb A) and three recombinant mutant hemoglobins, rHb Kempsey (betaD99N), rHb (alphaY42D/betaD99N), and rHb (alphaV96W), in the carbonmonoxy and the deoxy forms as well as in the presence and in the absence of an allosteric effector, inositol hexaphosphate (IHP). There are three Trp residues (alpha14, beta15, and beta37) in Hb A for each alphabeta dimer. These Trp residues are located in important regions of the Hb molecule, i.e. alpha14Trp and beta15Trp are located in the alpha(1)beta(1) subunit interface and beta37Trp is located in the alpha(1)beta(2) subunit interface. The relaxation experiments show that amino acid substitutions in the alpha(1)beta(2) subunit interface can alter the dynamics of beta37Trp. The transverse relaxation rate (R(2)) for beta37Trp can serve as a marker for the dynamics of the alpha(1)beta(2) subunit interface. The relaxation parameters of deoxy-rHb Kemspey (betaD99N), which is a naturally occurring abnormal human hemoglobin with high oxygen affinity and very low cooperativity, are quite different from those of deoxy-Hb A, even in the presence of IHP. The relaxation parameters for rHb (alphaY42D/betaD99N), which is a compensatory mutant of rHb Kempsey, are more similar to those of Hb A. In addition, TROSY-CPMG experiments have been used to investigate conformational exchange in the Trp residues of Hb A and the three mutant rHbs. Experimental results indicate that the side-chain of beta37Trp is involved in a relatively slow conformational exchange on the micro- to millisecond time-scale under certain experimental conditions. The present results provide new dynamic insights into the structure-function relationship in hemoglobin.     

Computerized scheme for the reaction of hemoglobin with ligands

A scheme for the reaction of hemoglobin with ligands is described, which postulates the functional heterogeneity of the chains, considers all possible combinations of the distribution of the ligand on the four chains of hemoglobin, and does not require simplifying assumptions about the hemoglobin reactivity. Ten tetrameric species are considered, together with 16 reactions between these species, each with an on and an off rate constant. The dissociation of hemoglobin tetramers into dimers is also considered, with four on and four off rate constants for the reactions between dimers, and ten equilibrium constants for the reactions between tetramers and dimers. Moreover, some side reactions, such as the trapping of ligands by a hemoglobin competitor, are included. A FORTRAN program, suitable for microcomputers, is described for handling this scheme, with some examples showing its advantages.     

Studying the interaction between three synthesized heterocyclic sulfonamide compounds with hemoglobin by spectroscopy and molecular modeling techniques

The interaction between synthesized heterocyclic benzene sulfonamide compounds, N-(7-benzyl-56-biphenyl-2m-tolyl-7H-pyrrolo[23-d]pyrimidine–4–yl)-benzene sulfonamide (HBS₁), N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d] pyrimidine-4-yl)-4-methyl- benzene sulfonamide (HBS₂), and N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d]pyrimidine-4-yl)-4-chloro-benzene sulfonamide (HBS₃) with Hb was studied by fluorescence quenching, zeta potentional, circular dichroism, and molecular modeling techniques. The fluorescence spectroscopy experiments were performed in order to study the conformational changes, possibly due to a discrete reorganization of Trp residues during binding between HBS derivatives and Hb. The variation of the K_SV value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The K_SV1 ans K_SV2 values of HBS derivatives with Hb are .6 × 10¹³ and 3 × 10¹³ M^?1 for Hb–HBS₁, 1 × 10¹³ and 4 × 10¹³ M^?1 for Hb–HBS₂, .9 × 10¹³, and 6 × 10¹³ M^?1 for Hb–HBS₃, respectively. The molecular distances between Hb and HBS derivatives in binary and ternary systems were estimated according to Förster’s theory of dipole–dipole non-radiation energy transfer. The quantitative analysis data of circular dichroism spectra demonstrated that the binding of the three HBS derivatives to Hb induced conformational changes in Hb. Changes in the zeta potential of the Hb–HBS derivatives complexes demonstrated a hydrophobic adsorption of the anionic ligand onto the surface of Hb as well as both electrostatic and hydrophobic adsorption in the case of the complex. The modeling data thus confirmed the experimental results. This study is expected to provide important insight into the interaction of Hb with three HBS derivatives to use in various toxicological and therapeutic processes.     

Regulation of oxygen affinity by quaternary enhancement: Does hemoglobin ypsilanti represent an allosteric intermediate?

Recent crystallographic studies on the mutant human hemoglobin Ypsilanti (beta 99 Asp-->Tyr) have revealed a previously unknown quaternary structure called "quaternary Y" and suggested that the new structure may represent an important intermediate in the cooperative oxygenation pathway of normal hemoglobin. Here we measure the oxygenation and subunit assembly properties of hemoglobin Ypsilanti and five additional beta 99 mutants (Asp beta 99-->Val, Gly, Asn, Ala, His) to test for consistency between their energetics and those of the intermediate species of normal hemoglobin. Overall regulation of oxygen affinity in hemoglobin Ypsilanti is found to originate entirely from 2.6 kcal of quaternary enhancement, such that the tetramer oxygenation affinity is 85-fold higher than for binding to the dissociated dimers. Equal partitioning of this regulatory energy among the four tetrameric binding steps (0.65 kcal per oxygen) leads to a noncooperative isotherm with extremely high affinity (pmedian = .14 torr). Temperature and pH studies of dimer-tetramer assembly and sulfhydryl reaction kinetics suggest that oxygenation-dependent structural changes in hemoglobin Ypsilanti are small. These properties are quite different from the recently characterized allosteric intermediate, which has two ligands bound on the same side of the alpha 1 beta 2 interface (see ref. 1 for review). The combined results do, however, support the view that quaternary Y may represent the intermediate cooperativity state of normal hemoglobin that binds the last oxygen.     

膜联蛋白AnxB1序列缺失突变体的结构模建及活性分析

为获得低分子量、低免疫原性的膜联蛋白AnxB1的序列缺失突变体 ,以AnxB1C端的 4个内部同源结构域为基础 ,模拟构建了 4个突变体群 .利用同源模建的方法对各突变体进行结构模建和分子优化 ,最后选择 4个结构最为合理的突变体在大肠杆菌GST融合表达系统中表达 .结果显示 :GST M3和GST M4均表达出较强的抗凝血活性 ,且免疫原性也降低为原来的 1 2 ,为进一步构建兼具抗凝、溶栓双重功能的靶向性溶栓药物奠定了基础 .     

The mutation beta 99 Asp-Tyr stabilizes Y--a new, composite quaternary state of human hemoglobin

Carbonmonoxy hemoglobin Ypsilanti (beta 99 Asp-Tyr) exhibits a quaternary form distinctly different from any structures previously observed for human hemoglobins. The relative orientation of alpha beta dimers in the new quaternary form lies well outside the range of values observed for normal unliganded and liganded tetramers (Baldwin, J., Chothia, C., J. Mol. Biol. 129:175-220, 1979). Despite this large quaternary structural difference between carbonmonoxy hemoglobin Ypsilanti and the two canonical structures, the new quaternary structure's hydrogen bonding interactions in the "switch" region, and packing interactions in the "flexible joint" region, show noncovalent interactions characteristic of the alpha 1 beta 2 contacts of both unliganded and liganded normal hemoglobins. In contrast to both canonical structures, the beta 97 histidine residue in carbonmonoxy hemoglobin Ypsilanti is disengaged from quaternary packing interactions that are generally believed to enforce two-state behavior in ligand binding. These features of the new quaternary structure, denoted Y, may therefore be representative of quaternary states that occur transiently along pathways between the normal unliganded, T, and liganded, R, hemoglobin structures.