GENETIC ANALYSIS OF BACTERIOPHAGE LYSOZYME STRUCTURE

噬菌体溶菌酶结构的遗传分析

• 批准号: 2062446
• 负责人: ANTHONY R. POTEETE
• 金额: $ 22.72万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1998-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2062446
• 关键词: bacteriophage T4; lysozyme; mutant; protein engineering; protein folding; protein sequence; protein structure; protein structure function; ultraviolet radiation

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.

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。 一个 提出了遗传学强化方法，重点关注噬菌体 T4 溶菌酶作为模型系统。 具体目标包括： 1.分离带有有害物质的 T4 溶菌酶突变体的回复体 单氨基酸取代，筛选二级位点回复体 其中，测序验证了第二位点回复体。 2. 表征所选突变体溶菌酶的稳定性 体内和体外、纯化和催化活性；合作中 与其他人一起确定他们的结构。 3. 研究溶菌酶分子的两个部分在 催化：先前涉及的残基AsP20，其性质 机械贡献现在受到质疑；和一个下部结构， 位于先前研究中确定的大域中， 提供关键催化残基 Glu11 的结构稳定性。 在这些位置带有取代的溶菌酶将被纯化，并且 它们的催化特性（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它们的性质决定的 三维结构，又由 它们的多肽亚基的序列。 了解规则 氨基酸序列折叠成独特的结构，将极大地帮助 在新蛋白质的设计以及现有蛋白质的修饰中， 用作精确特异性抗原或治疗剂。