GENETIC ANALYSIS OF BACTERIOPHAGE LYSOZYME STRUCTURE

拟议研究的广泛、长期目标是理解 蛋白质结构和功能的序列决定因素。一个 提出了以T4噬菌体为研究对象的遗传密集型方法 溶菌酶作为一种模型系统。具体目标包括： 1.T4溶菌酶致病突变株的筛选 单一氨基酸替换，筛选二级位点反转体 其中，和测序证实了第二个位点的回复。 2.筛选出的突变型溶菌酶的稳定性 体内和体外，纯化和催化活性；合作 与其他人一起，决定他们的结构。 3.研究溶菌酶分子的两部分参与 催化：先前涉及的残基AsP20，其性质 机械论的贡献现在受到了质疑；而一个子结构， 位于先前研究中确定的大范围内，似乎 以提供关键催化残基Glu11的结构稳定性。 在这些位置含有取代的溶菌酶将被提纯，以及 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它们的 三维结构，而这些结构又由 它们的多肽亚基的序列。对规则的了解 氨基酸序列折叠成独特的结构，将极大地帮助 在设计新的蛋白质以及对现有蛋白质进行修饰时， 作为精致的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.