Tethered Domains As Regulatory elements

描述（由申请人提供）：本研究的长期目标是在分子水平上理解蛋白质是如何被远离“活性位点”的事件调节的，以及蛋白质构象如何在信息转导中起作用。本实验室的长期目标是确定构象调节控制机制中结构和功能之间的关系，最终将其与这些酶和类似系统介导的生理和病理生理联系起来，并最终充分理解蛋白质中的构象介导过程，以便能够将基本原理应用于更复杂的系统。这个广泛的目标将在本应用中以一种特定的方式解决，通过对一个相对简单但非常有趣的变构系统的继续研究，该系统的基础是作为调控元件的栓系结构域。首席研究员的工作支持效应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的假设。他的团队已经证明，在四聚体系统的背景下，这些结构域的相互作用产生了两个不同的合作过程，可以通过特定的突变分离或解耦。此外，他的团队成功地确定了参与合作过程的蛋白质结构区域，并控制了蛋白质区域的空间相互关系。本应用程序中描述的工作扩展了该工作：1)产生和分析含有不对称突变的杂化异四聚体，以直接研究合作途径的方向性和位点的空间关系；2)通过热力学链接分析研究影响某些过程的突变体的耦合，并探索连接螺旋在合作效应转导中的作用。3)通过氢/氘交换分析确定结构域的相互关系及其对接接触的身份；4)通过x射线晶体学合理选择突变体。

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.