OPTICAL ROTATORY POWER OF BIOPOLYMERS

长期目标是研究一些的构象 蛋白质并了解其结构与功能的关系。 未来三年的具体目标有三个。 (1) 蛋白质构象CD分析的进一步测试。 至少 目前使用三种方法来估计α-螺旋、β-螺旋 蛋白质分子中的折叠和β-转角。 几个问题应该 仍需解决：数量和种类的任意选择 蛋白质；缺乏统一的二级标准 来自 X 射线数据的蛋白质结构；每个的计数 按肽键数量划分的构象片段（基于 CD 理论）与氨基酸残基；引入的问题 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 CD 可达到的波长。 (2) 短螺旋折叠 多肽。 螺旋通过非极性、离子对和 电荷螺旋偶极子相互作用。 暴露于溶剂的螺旋段 将通过 X 射线衍射来调查一组蛋白质的 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。 一两短 将选择多肽来测试螺旋偶极子模型和 离子对的形成。 它们在水溶液中的构象将 通过改变温度、pH 值和离子强度来研究 解决方案。 这些肽将通过有限的 母体蛋白的蛋白水解或通过标准合成。 (3) 乙酰胆碱酯酶的构象研究。 的折叠 结构域和活性中心的拓扑结构 酶将是我们主要关注的问题。 活性中心由 酯位点、阴离子位点和疏水位点。 有限公司 该酶将进行蛋白水解；是否每个亚基 有多个域，如果有，活性中心是否是 仅限于一个领域，或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，其 将确定构象和可能的酶活性。 定点诱变可用于定位可能的 季铵配体的结合位点和必需的 酯化位点附近的组氨酸。 三人的角色 链内二硫键对酶构象稳定性的影响 将通过选择性地替换半胱氨酸来研究 另一种氨基酸再次通过诱变。 的组合 有限的蛋白水解和重组 DNA 技术将提供 有关这种突触酶的有用信息。

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.