Tethered Domains As Regulatory elements

受限域作为监管要素

• 批准号: 6754377
• 负责人: GREGORY A GRANT
• 金额: $ 33.92万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6754377
• 关键词: X ray crystallography; alcohol dehydrogenase; allosteric site; chemical binding; conformation; enzyme mechanism; enzyme structure; phosphoglycerate; protein engineering; protein structure function; site directed mutagenesis; structural biology; thermodynamics

DESCRIPTION (provided by applicant): The long term objective of this research is to understand, at the molecular level, how proteins are regulated by events that occur away from the "active sites" and how protein conformation functions in the transduction of information. The long term goals of this laboratory are to determine the relationship between structure and function in conformationally regulated control mechanisms, to eventually relate that to the physiology and pathophysiology mediated by these enzymes and by analogous systems, and to eventually understand conformational mediated processes in proteins well enough to be able to apply basic principles to more complex systems.This broad objective will be addressed in a specific manner in this application through the continuation of the study of a relatively simple, but extremely interesting, allosteric system that has as its basis tethered domains functioning as regulatory elements. The principal investigator's work supports the hypothesis that effector binding exerts its inhibitory effect by modulating the spatial arrangements of protein domains. His group has shown that the interplay of these domains within the context of a tetrameric system produces two distinct cooperative processes, which can be separated or uncoupled by specific mutations. Moreover, his group is succeeding in pinpointing the structural regions of the protein that participate in the cooperative processes and govern the spatial inter-relationships of the protein domains.The work described in this application extends this work by 1) producing and analyzing hybrid heterotetramers containing asymmetric mutations to directly study the directionality of the cooperative pathways and the spatial relationship of the sites, 2) studying the coupling of mutants found to affect certain processes by thermodynamic linkage analysis and to explore the role of connecting helices in transduction of the cooperative effects, 3) determining the inter-relationship of domains and the identity of their docking contacts by hydrogen/deuterium exchange analysis, and 4) by X-ray crystallography of rationally selected mutants.

描述（由申请人提供）：这项研究的长期目标是在分子水平上了解蛋白质如何受到远离“活跃位点”的事件的调节，以及蛋白质构象如何在信息转导中起作用。该实验室的长期目标是确定在构象调节的控制机制中的结构与功能之间的关系，最终将这些酶与生理学和病理生理学联系起来，这些酶介导的生理学和病理生理学通过类似的系统和类似的系统介导的生理学和病理生理学，并最终了解蛋白质中的构象介导的过程，以便通过更复杂的系统将基本原理应用于该蛋白质的良好的应用程序。但是非常有趣，变构的系统作为其基础束缚域，作为调节元素。主要研究者的工作支持这样的假设，即效应子结合通过调节蛋白质结构域的空间排列来发挥其抑制作用。他的小组表明，在四聚体系统的背景下，这些域的相互作用产生了两个不同的合作过程，这些过程可以通过特定突变将其分开或取消耦合。此外，他的小组成功地指出了参与合作过程的蛋白质的结构区域，并控制了蛋白质域的空间相互关系。本应用中所描述的工作通过1）进行了1），通过1）生产和分析混合异位术语，从突变体的耦合通过热力学链接分析影响某些过程，并探索连接螺旋在合作效应转导中的作用，3）确定域的相互关系以及通过氢/氘交换分析的对接接触的相互关系，以及4）通过X射线晶体素描术的X射线晶体术的X射线晶体术。