GENETIC ANALYSIS OF BACTERIOPHAGE LYSOZYME STRUCTURE

拟议研究的广泛、长期目标是了解 蛋白质结构和功能的序列决定因素。 一 提出了一种遗传密集的方法，重点是噬菌体T4 溶菌酶作为模型系统。 具体目标包括： 1 .一、分离具有有害生物的T4溶菌酶突变体的回复突变体 单个氨基酸取代，筛选二级位点回复突变体 其中，测序验证了第二位点回复突变体。 2.表征选定的突变体溶菌酶的稳定性， 体内和体外，纯化和催化活性;合作 和其他人一起决定他们的结构 3.研究溶菌酶分子的两个部分参与 催化：先前涉及的残基AsP 20，其性质 机械贡献现在受到质疑;以及子结构， 位于大域，在以前的研究中确定， 以提供关键催化残基Glu 11的结构稳定性。 将纯化在这些位置上具有取代的溶菌酶， 它们的催化性能（对底物的亲和力、催化效率） 将在动力学实验中确定。 蛋白质的功能特性是由其 三维结构，这反过来又取决于 它们的多肽亚基的序列。 关于规则的知识， 氨基酸序列折叠成独特的结构，将大大有助于 设计新的蛋白质，以及修改现有的蛋白质， 作为特异性抗原或治疗剂。

The broad, long-term objective of the proposed research is understanding of the sequence determinants of protein structure and function. A genetics-intensive approach is proposed, focused on bacteriophage T4 lysozyme as a model system. Specific aims include: 1 . Isolating revertants of T4 lysozyme mutants bearing deleterious single amino acid substitutions, screening for secondary site revertants among them, and sequencing verified second-site revertants. 2. Characterizing selected mutant lysozymes with regard to stability in vivo and in vitro, purification, and catalytic activity; in collaboration with others, determining their structures. 3. Studying the involvement of two parts of the lysozyme molecule in catalysis: the previously implicated residue AsP20, the nature of whose mechanistic contribution is now called into question; and a substructure, located in the large domain, identified in previous studies, that appears to provide structural stabilization of the key catalytic residue Glu11. Lysozymes bearing substitutions in these positions will be purified, and their catalytic properties (affinity for substrate, catalytic efficiency) will be determined in kinetic experiments. The functional properties of proteins are determined by their three-dimensional structures, which in turn are determined by the sequences of their polypeptide subunits. Knowledge of the rules by which sequences of amino acids fold into unique structures, would greatly aid in the design of new proteins, as well as modification of existing ones, to serve as exquisitely specific antigens or therapeutic agents.