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了解蛋白质如何受到远离“活性位点”发生的事件的调节，以及蛋白质构象如何在信息转导中发挥作用。该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学和病理生理学联系起来，并最终理解蛋白质中构象介导的过程，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一个具体的在本申请中，通过继续研究相对简单但非常有趣的变构系统，该变构系统具有作为其基础的拴系结构域作为调节元件。主要研究者的工作支持了效应物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。他的研究小组已经表明，这些结构域在四聚体系统中的相互作用产生了两个不同的合作过程，它们可以通过特定的突变分离或解偶联。此外，他的小组正在成功地精确定位蛋白质的结构区域，这些结构区域参与合作过程并控制蛋白质结构域的空间相互关系。本申请中描述的工作通过以下方式扩展了这项工作：1）生产和分析含有不对称突变的杂合异源四聚体，以直接研究合作途径的方向性和位点的空间关系，2）通过热力学连接分析研究发现影响某些过程的突变体的偶联，并探索连接螺旋在协同效应转导中的作用，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，以及4）通过合理选择的突变体的X射线晶体学。

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.