OPTICAL ROTATORY POWER OF BIOPOLYMERS

生物聚合物的旋光能力

• 批准号: 3268180
• 负责人: JEN T YANG
• 金额: $ 18.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-05-01 至 1992-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268180
• 关键词: acetylcholinesterase; acidity /alkalinity; circular dichroism; conformation; disulfide bond; enzyme structure; genetic manipulation; ionic strengths; optical rotation; peptide structure; protein engineering; protein folding; protein signal sequence; protein structure function; proteins; recombinant DNA; site directed mutagenesis; synthetic peptide; thermodynamics

The long-range objective is to study the conformation of a few proteins and to understand their structure-function relationship. The specific aims for the next three years are threefold. (1) Further tests of CD analysis of protein conformation. At least three methods are currently used for estimating alpha-helix, beta- sheet and beta-turn in a protein molecule. Several problems should still be addressed: the arbitrary choice of the number and kind of proteins; lack of uniform criteria for identifying secondary structures of proteins from x-ray data; the counting of each conformation segment by the number of peptide bonds (based on CD theory) vs. amino acid residues; the question of introducing the two constraints in CD analysis (1>fi>0 and sigma fi=1, where fi is the fraction of the i-th conformation); and the lower limit of attainable wavelength for CD. (2) Helix folding of short polypeptides. Helices are stabilized by nonpolar, ion-pair and charge-helix dipole interactions. Solvent-exposed helical segments of a set of proteins will be surveyed from their x-ray diffraction data and the frequencies of occurrence of charge-helix dipole and ion-pair interactions will be compared. One or two short polypeptides will be chosen to test the helix dipole model and the ion-pair formation. Their conformation in aqueous solution will be studied by varying temperature, pH and ionic strength of the so- lution. These peptides will be prepared either by limited proteolysis of the parent protein or by standard synthesis. (3) Conformation studies of acetylcholinesterase. The folding of structural domains and the topology of the active center of this enzyme will be our major concern. The active center is composed of an esteratic, an anionic and a hydrophobic site. Limited proteolysis will be applied to this enzyme; whether each subunit has more than one domain and, if so, whether the active center is confined within one domain or, more likely, involves more than one domain will be determined. With this information fragments of the enzyme will be synthesized by recombinant DNA technique and their conformation and possible enzymatic activity will be determined. Site-directed mutagenesis can be used to locate the possible binding site for quaternary ammonium ligands and the essential histidine near the esteratic site. The role of the three intrachain disulfides in the conformation stability of the enzyme will be studied by selectively replacing the half cystines with another amino acid again through mutagenesis. A combination of limited proteolysis and recombinant DNA technique will provide useful information about this synaptic enzyme.

长期目标是研究几个化合物的构象 并了解它们的结构-功能关系。 未来三年的具体目标有三个方面。(1) 蛋白质构象的CD分析的进一步测试。至少 目前用于估计α-螺旋、β-螺旋的方法有三种。 蛋白质分子中的片状和β-转角。应该注意的几个问题 仍然需要解决的问题：对数字和种类的任意选择 指蛋白质；缺乏识别次级蛋白质的统一标准 来自x射线数据的蛋白质结构.每种蛋白质的计数 多肽键数目的构象片段(以CD为基础 理论)与氨基酸残基；引入 CD分析中的两个约束(1&gt；fi&gt；0和sigma fi=1，其中fi是 第i个构象的分数)；以及 CD的可获得波长。(2)短的螺旋折叠 多肽。螺旋由非极性、离子对和 电荷-螺旋偶极相互作用。暴露在溶剂中的螺旋段 一组蛋白质的X-射线衍射图 电荷螺旋偶极子和电荷螺旋偶极子的数据和出现频率 我们将对离子对相互作用进行比较。一到两个短 将选择多肽来检验螺旋偶极模型和 离子对的形成。它们在水溶液中的构象将 通过改变温度、pH和离子强度来研究SO- 迷幻药。这些多肽将由限量制备 蛋白质降解亲本蛋白质或通过标准合成。(3) 乙酰胆碱酯酶的构象研究。折叠式的 结构域及其活动中心的拓扑结构 酶将是我们的主要关注点。活动中心被组成 一个酯基、一个阴离子和一个疏水基位。有限 蛋白水解酶将被应用于该酶；无论每个亚基 有多个域，如果有，活动中心是否 局限于一个域，或者更有可能涉及多个域 域名将被确定。有了这个信息， 将利用重组DNA技术合成酶及其相互作用 构象和可能的酶活性将被确定。 定点突变可以用来定位可能的 季铵盐类配体的结合部位及其必需的 酯化部位附近的组氨酸。三者的作用 链内二硫键对酶构象稳定性的影响 将通过选择性地用半胱氨酸取代半胱氨酸来研究 另一种氨基酸再次通过诱变。一种组合 有限的蛋白质分解和重组DNA技术将提供 关于这种突触酶的有用信息。