OPTICAL ROTATORY POWER OF BIOPOLYMERS

生物聚合物的旋光能力

• 批准号: 3268179
• 负责人: JEN T YANG
• 金额: $ 17.43万
• 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 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3268179
• 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; 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.

长期的目标是研究几个 蛋白质，并了解它们的结构-功能关系。 今后三年的具体目标有三个方面。 （一） 蛋白质构象的CD分析的进一步测试。 至少 目前有三种方法用于估计α-螺旋，β-螺旋， 折叠和β-转角。 几个问题应该 仍然可以解决：任意选择的数量和种类 蛋白质;缺乏鉴定次级 蛋白质结构的X射线数据;每个计数 构象片段的肽键数（基于CD 理论）与氨基酸残基;引入 CD分析中的两个约束（1>fi>0和sigma fi=1，其中fi是 第i个构象的分数）;和下限 可达到的CD波长。 (2)短的受阻折叠 多肽。 螺旋通过非极性、离子对和 电荷-螺旋偶极相互作用。 溶剂暴露的螺旋段 一组蛋白质将被调查，从他们的x射线衍射 数据和电荷螺旋偶极子的出现频率， 将比较离子对相互作用。 少了一两个 将选择多肽来测试螺旋偶极模型， 离子对形成。 它们在水溶液中的构象将 通过改变温度，pH值和离子强度的so- lution. 这些肽将通过有限的方法制备， 亲本蛋白质的蛋白水解或通过标准合成。 （三） 乙酰胆碱酯酶的构象研究。 的折叠 结构域和拓扑结构的活性中心，这 酶将是我们的主要关注点 活动中心由 一个酯化的，一个阴离子的和一个疏水的位点。 有限 蛋白水解将应用于这种酶;无论每个亚基 有一个以上的域，如果有，是否活动中心是 局限于一个领域，或者更可能涉及多个领域， 域将被确定。 有了这些信息片段， 酶将通过重组DNA技术合成， 将确定构象和可能的酶活性。 定点诱变可用于定位可能的突变位点。 季铵配体的结合位点和必需的 组氨酸在酯位点附近。 三人的作用 链内二硫键在酶的构象稳定性 将通过选择性地将半胱氨酸替换为 另一个氨基酸的突变。 的组合 有限蛋白水解和重组DNA技术将提供 关于这种突触酶的有用信息