CD_(126)功能域三维结构研究进展

ＣＤ１２６功能域三维结构的研究对研制具有不同生物学活性的新型ＩＬ－６Ｒ突变体和进行基于ＣＤ１２６三维结构的药物分子设计研究有重要的指导意义。本文概述ＣＤ１２６空间结构预测及同源模建的研究进展,并总结ＣＤ１２６与其配基ＩＬ－６形成高亲和力受体复合物时构－效关系分析的研究动态 。     

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。     

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对其搜索。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     

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。     

新型α-芋螺毒素Di1.1的克隆、合成及功能研究

目的：从中国南海长距芋螺中克隆出新的芋螺毒素序列并用固相方法合成该毒素,测定其折叠后的二硫键配对方式并初步研究其药理学特性。方法：根据芋螺毒素A超家族保守的信号肽序列设计引物,通过3＇-RACE扩增,从芋螺毒腺管中克隆出新的毒素基因;采用Fmoc-固相法合成线性多肽,通过空气氧化折叠获得含二硫键的折叠产物,用两步氧化折叠法测定多肽的二硫键连接方式;用双电极电压钳技术初步研究其药理学特性。结果：发现-种新的α-芋螺毒素Dil．1的cDNA序列,其成熟肽序列为CcVIESCHSNHIDECES;该肽二硫键连接方式以C1-C4、C2-C3为主,以C1-C3、C2-C4连接为辅,对烟碱型乙酰胆碱受体各亚型活性较弱。结论：Dil．1是-种新的α4／7型芋螺毒素,其折叠方式以C1-C4、C2-C3连接为主。     

朊病毒蛋白的三维结构

朊病毒病是一类引起人和动物的中枢神经组织退化的疾病,属于蛋白质构象病,涉及蛋白质分子肽链的错误折叠和空间结构的变化。这类疾病的临床表现为进行性共济运动失调、震颤、姿势不稳、痴呆或知觉过敏、行为反常等中枢神经系统症状,100％死亡。人们期望通过对朊病毒蛋白的三维结构的研究来了解其致病机制,以达到预防和治疗的目的。     

短肽蝎毒素的结构分类与功能特征

大量的资料已证实蝎毒中主要致死成分是一类由60～70个残基组成, 选择性地作用于电压门控Na⁺通道的长肽毒素.另一类由30～40个残基组成的短肽蝎毒素,由于其具有结构致密,易于合成改造的优点,特别是具有选择性地阻遏K⁺或Cl^－通道的特异药理功效,近年来倍受学术界的关注,并在结构与功能方面取得了很大的研究进展.     

用于研究植物质外体空间三维结构的树脂铸型技术

本文介绍了用于研究植物体质外体空间三维结构的树脂铸型技术。植物包含许多重要的质外体空间,如木质部管状分子的腔隙、与气孔相连的叶肉细胞间的通气系统、分泌腔等等。这种空间的三维结构可借助于扫描电子显微镜（ＳＥＭ）研究。但问题是,用ＳＥＭ直接观察不到一个组织或细胞切面内部的图像,因此,不能观察它们的全貌。通过运用树脂铸型技术,可以获得完整的组织或腔隙内部空间的铸型。反映管壁结构的各种形象被印在铸型的表面     

伊蚊浓核病毒三维结构的比较分析

细小病毒是目前已知的结构最小的病毒, 其宿主范围很广. 利用冷冻电镜和三维重构技术获得了伊蚊浓核病毒1.2 nm分辨率下的三维结构, 并利用计算机生物信息处理技术和氨基酸序列比对技术比较了该病毒和其他细小病毒的结构和序列的异同. 尽管均属于浓核病毒亚科, 但是无论从衣壳结构还是其蛋白质的氨基酸序列上, 伊蚊浓核病毒和其他昆虫的细小病毒都有很大差异. 相比之下, 伊蚊浓核病毒和人类B19细小病毒的衣壳蛋白相似性较大, 两者的结构蛋白和非结构蛋白一致性也高于伊蚊浓核病毒与其他昆虫的细小病毒的一致性. 此结果表明: 伊蚊浓核病毒和人类B19细小病毒有着密切关系, 前者可能来源于B19病毒的较近期变种.     

MAPK/ERK kinase 2(MEK2)的全长三维结构

作为一种枢纽性的信号通路, Ras/Raf/MEK/ERK级联可被众多细胞外刺激激活, 进而将不同刺激信号传递到不同的底物分子. 其中MEK分子只有MEK1和MEK2两种, 它们如何介导众多信号, 一直是人们感兴趣的问题. 但由于技术局限, 一直难以得到MEK分子的完整三维结构, 限制了对此复杂机理分子水平上的研究. 利用同源模建与分子动力学模拟相结合, 构建了MEK2分子的完整结构, 并研究了其分子动力学特性. 结果显示, MEK2的N端部分具有非常高的柔性, 富脯氨酸环区和C端也具有相当的柔性. 这些结构特性提示, 对于不同的上游信号, MEK2有可能以相应的不同构象与ERK和/或其他上下游蛋白作用, 从而导致相应不同的下游效应.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

二聚体蝎钠通道毒素的1.4(A)分辨率晶体结构:反常非脯氨酸顺式肽键及其内在结构因素

报道了以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构．蛋白质中的肽键是局部双键,不可旋转,因此具有顺式（cis）和反式（trans）两种构型,它们不能通过旋转操作相互转换．非脯氨酸顺式肽键是指形成该肽键的氨基是由脯氨酸以外的氨基酸提供的（Xaa-nonPro）,这类肽键的顺式构型的自由能远比反式高,因此极少出现在天然蛋白质结构中．事实上,在长时间中,多肽链的“反式肽键连接”被视为蛋白质结构的一条基本规则,把顺式肽键视为不可能．随着高分辨率精确蛋白质结构数量的增加,近年来有详细的统计分析揭示,非脯氨酸顺式肽键（Xaa-nPro）在蛋白质结构中出现的几率为0．03％～0．05％,而且大多存在于功能敏感的结构区域,可能具有重要意义．但由于所用的基本结构数据都来自晶体结构,对这种反常肽键是否由结晶环境影响而形成,存在疑问．此前曾在以单体形式存在的蝎神经毒素mono-BmK M1的高分辨率结构中发现其中肽键Pr09-His10是非脯氨酸顺式肽键,并详细分析了其结构．功能意义．以二聚体存在的dimo-BmK M1的1．4A分辨率晶体结构表明,它与mono．BmK M1有不同的空间群、不同的分子堆积方式,不同的晶体环境．结构模型被高度精化,Rcryst达到0．109．dimo-BmK M1结构显示,在不对称单位中的两个M1分子在同一位置（残基9．10之间）都清晰地存在顺式肽键．立体化学分析显示,这一肽键的几何参数和局部结构与mono．BmK M1中的（9．10）顺式肽键基本相同．这一结果表明,非脯氨酸顺式肽键9．10的存在与结晶环境无关,是BmK M1分子的固有结构特征．在此基础上,综合分析了与顺式、反式肽键相关的结构元素,发现与残基（8．19）序列模体-KPXNC-（X为任意氨基酸）所决定的特征回折结构可能是分子内在的主要结构因素,其中第8位残基是Lys或Asp对决定肽键是顺式还是反式有关键作用．近来的突变实验及其晶体结构测定已证实,Lys8／Asp8是（9-10）肽键顺式／反式异构的结构开关,它们对该类分子与不同种属钠通道作用的专一选择性具有重要作用．通过BLAST搜索,发现在其他18个蛋白质中也存在相同的序列模体．KPXNC-,推测在这些蛋白质的相应肽键位置也可能存在反常的脯氨酸顺式肽键。     

Characterization of the venom of the vermivorous cone snail Conus fulgetrum

Over 200 components with molecular mass ranging mainly from 400 to 4000 Da were characterized from the venom of the vermivorous cone snail Conus fulgetrum that inhabit Egyptian Red Sea. One major component having a molecular mass of 2946 Da was purified by HPLC, and its primary structure was determined by a combination of Edman degradation and MS/MS analysis.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Three-Dimensional Structure of Selenocosmia huwena Lectin-I (SHL-I) from the Venom of the Spider Selenocosmia huwena by 2D-NMR

The three-dimensional structure of native SHL-I, a lectin from the venom of the Chinese bird spider Selenocosmia huwena, has been determined from two-dimensional ¹H NMR spectroscopy recorded at 500 and 600 MHz. The best 10 structures have NOE violation <0.3 Å, dihedral violation <2 deg, and average root-mean-square differences of 0.85 + 0.06 Å over backbone atoms. The structure consists of a three-stranded antiparallel -sheet and three turns. The three disulfide bridges and three-stranded antiparallel -sheet form a inhibitor cystine knot motif which is adopted by several other small proteins, such as huwentoxin-I, -conotoxin, and gurmarin. The C-terminal fragment from Leu28 to Trp32 adopts two sets of conformations corresponding to the cis and trans conformations of Pro31. The structure of SHL-I also has high similarity with that of the N-terminus of hevein, a lectin from rubber-tree latex.     

蝎氯毒素结构、功能与应用研究

蝎氯毒素是一类能特异阻断神经胶质瘤氯电流的短链蝎毒素。它们具有高度的同源性、保守的基因序列与相似的3-D结构。根据其结构与功能的关系,推测它们可能有相似的药理功能。其中,Chlorotoxin(Cltx)能与神经胶质瘤细胞特异相互作用,并抑制其侵袭与转移。     

Antimicrobial and Antibiofilm Activity of Mauriporin,a Multifunctional Scorpion Venom Peptide

Many pathogenic free living and biofilm forming bacterial organisms can cause serious infections to humans that could consequently have devastating effects on human health. A significant number of these microbial organisms are resistant to almost all known conventional antibiotics and the ability of some these strains to form sessile communities of biofilms increases the resistance ability of bacteria to antibiotic treatment. Global research is currently focused on finding novel therapies to counteract the threat of bacterial and biofilm infections rather than using conventional antibiotics. Mauriporin, a novel cationic α-helical peptide identified from the venom derived cDNA library of the scorpion Androctonus mauritanicus was reported to display selective cytotoxic and anti-proliferative activity against prostate cancer cell lines. In the present study, we investigated the antimicrobial and antibiofilm activities of Mauriporin. Our results show that Mauriporin displays potent antimicrobial activities against a range of Gram-positive and Gram-negative planktonic bacteria with MIC values in the range 5 µM to 10 µM. Mauriporin was also able to prevent Pseudomonas aeruginosa biofilm formation while showing weak hemolytic activity towards human erythrocytes. Studies on the mechanism of action of Mauriporin revealed that the peptide is probably inducing bacterial cell death through membrane permeabilization determined by the release of β-galactosidase enzyme from peptide treated Escherichia coli cells. Moreover, DNA binding studies found that Mauriporin can cause potent binding to intracellular DNA. All these results indicate that Mauriporin has a considerable potential for therapeutic application as a novel drug candidate for eradicating bacterial infections.     

Molecular Evolution and Diversity of Conus Peptide Toxins,as Revealed by Gene Structure and Intron Sequence Analyses

Cone snails, which are predatory marine gastropods, produce a cocktail of venoms used for predation, defense and competition. The major venom component, conotoxin, has received significant attention because it is useful in neuroscience research, drug development and molecular diversity studies. In this study, we report the genomic characterization of nine conotoxin gene superfamilies from 18 Conus species and investigate the relationships among conotoxin gene structure, molecular evolution and diversity. The I1, I2, M, O2, O3, P, S, and T superfamily precursors all contain three exons and two introns, while A superfamily members contain two exons and one intron. The introns are conserved within a certain gene superfamily, and also conserved across different Conus species, but divergent among different superfamilies. The intronic sequences contain many simple repeat sequences and regulatory elements that may influence conotoxin gene expression. Furthermore, due to the unique gene structure of conotoxins, the base substitution rates and the number of positively selected sites vary greatly among exons. Many more point mutations and trinucleotide indels were observed in the mature peptide exon than in the other exons. In addition, the first example of alternative splicing in conotoxin genes was found. These results suggest that the diversity of conotoxin genes has been shaped by point mutations and indels, as well as rare gene recombination or alternative splicing events, and that the unique gene structures could have made a contribution to the evolution of conotoxin genes.     

Structure and Mechanism of Metallocarboxypeptidases

Metallocarboxpeptidases cleave C-terminal residues from peptide substrates and participate in a wide range of physiological processes, but they also contribute to human pathology. On the basis of structural information, we can distinguish between two groups of such metallopeptidases: cowrins and funnelins. Cowrins comprise protozoan, prokaryotic, and mammalian enzymes related to both neurolysin and angiotensin-converting enzyme and their catalytic domains contain 500–700 residues. They are ellipsoidal and traversed horizontally by a long, deep, narrow active-site cleft, in which the C-terminal residues are cut from oligopeptides and unstructured protein tails. The consensus cowrin structure contains a common core of 17 helices and a three-stranded β-sheet, which participates in substrate binding. This protease family is characterized by a set of spatially conserved amino acids involved in catalysis, HEXXH+EXXS/G+H+Y/R+Y. Funnelins comprise structural relatives of the archetypal bovine carboxypeptidase A1 and feature mammalian, insect and bacterial proteins with strict carboxypeptidase activity. Their ～ 300-residue catalytic domains evince a consensus central eight-stranded β-sheet flanked on either side by a total of eight helices. They also contain a characteristic set of conserved residues, HXXE+R+NR+H+Y+E, and their active-site clefts are rather shallow and lie at the bottom of a funnel-like cavity. Therefore, these enzymes act on a large variety of well-folded proteins. In both cowrins and funnelins, substrate hydrolysis follows a common general base/acid mechanism. A metal-bound solvent molecule ultimately performs the attack on the scissile peptide bond with the assistance of a strictly conserved glutamate residue.     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.