Tethered Domains As Regulatory elements

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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.