STRUCTURE & FUNCTION OF HELIX DESTABILIZING PROTEIN

本研究的目的是研究噬菌体的结构 T4基因32产物，一种螺旋不稳定蛋白，并研究其在 DNA复制、重组和修复。 其具体目的是 目的是阐明32个蛋白质与自身及与其它蛋白质的相互作用 与其他蛋白质的相互作用，并了解这种相互作用如何控制和 协调单个酶的活动以及整体 复制-重组复合物的结构和活性。 在过去的拨款期间，我们开始了一系列的研究， 32种蛋白质的有限蛋白水解产物， 蛋白质的功能。 结果表明，32种蛋白质具有 几个离散域和一些域似乎是专门 参与蛋白质相互作用。 我们计划继续 研究范围扩大到包括突变和化学 通过强化结构研究进行改性蛋白质研究。 拟议的实验包括制备各种32蛋白质片段 和突变或化学修饰的蛋白质;化学和物理 研究，以检查其结构和其大分子 复合物;广泛使用32种蛋白质亲和层析来检查 蛋白质-蛋白质和蛋白质-DNA相互作用以及鉴定 32-结合蛋白;接触面积的测绘和 通过差异化学修饰的构象变化;和使用32 体外复制反应中的蛋白质片段和修饰的蛋白质 将物理相互作用与功能相互作用联系起来。

The objective of this research is to study the structure of bacteriophage T4 gene 32 product, a helix destabilizing protein, and examine its roles in DNA replication recombination and repair. The specific aim of this proposal is to elucidate interactions of 32 protein with itself and those with other proteins and understand how such interactions control and coordinate the activities of individual enzymes as well as the overall structure and activities of the replication-recombination complex. During the past granting periods, we started a line of research using limited proteolysis products of 32 protein to correlate the structure and functions of the protein. The results indicated that 32 protein has several descrete domains and some of the domains seemed to be specifically involved in protein-protein interactions. We plan to continue the same line of research with expansion to include mutationally and chemically modified protein research with intensified structure studies. Proposed experiments consist of preparation of various 32 protein fragments and mutationally or chemically modified proteins; chemical and physical studies to examine their structure and that of their macromolecular complexes; extensive use of 32 protein affinity chromatography to examine protein-protein and protein-DNA interactions as well as identification of 32-binding proteins; mapping of the contact area and detection of conformational changes by differential chemical modification; and use of 32 protein fragments and modified proteins in in vitro replication reactions to correlate the physical interactions with the functional ones.