Tethered Domains As Regulatory elements

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是如何受到远离“活性部位”的事件的调节，以及蛋白质构象如何在信息传递中发挥作用。这个实验室的长期目标是确定构象调控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学和病理生理学联系起来，并最终足够好地理解蛋白质中的构象中介过程，以便能够将基本原理应用于更复杂的系统。这一广泛的目标将通过继续研究一个相对简单但极其有趣的变构系统来以特定的方式解决，该系统的基础是拴系结构域作为调节元件。主要研究人员的工作支持这样的假设，即效应器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，这两个过程可以通过特定的突变来分离或解偶联。本申请中描述的工作通过1)产生和分析包含不对称突变的杂交异位异构体来直接研究合作途径的方向性和位置的空间关系，2)通过热力学连锁分析研究被发现影响某些过程的突变体的耦合并探索连接螺旋在协同效应的转导中的作用，3)通过氢/氚交换分析确定结构域的相互关系和它们的对接接触的身份，4)对合理选择的突变体进行X射线结晶学分析。

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.