G protein-coupled signal transduction controls many cellular processes, including vision, olfaction and hundreds of others that involve signaling triggered via physical stimuli and small molecule and peptide ligands. Proteins of the signaling pathways are key pharmacological targets, and naturally occurring mutations in these proteins account for a host of human diseases. Despite this high level of biological and medical importance, there is only a fragmentary level of understanding of the molecular mechanisms involved. A fundamental difficulty in sorting out the mechanisms is that the proteins apparently rely on a high degree of molecular flexibility involving both intrinsically disordered domains and global conformational equilibria. Crystallization of flexible proteins generally requires engineering and the resulting structures at best provide a view of a single member of a conformational ensemble. Moreover, recent evidence suggests that the rate of exchange between conformational substates may be functionally important for G-protein coupled receptors (GPCRs). To make progress, experimental tools are needed to identify dynamically disordered domains, to resolve conformational substates and determine their global fold, and to measure exchange rates between them. Recently developed strategies in site directed spin labeling (SDSL) that employ static high-pressure, pressure- jump and time-domain EPR promise to meet these needs and will be further elaborated. The research proposed here will represent the first application of these technologies to proteins of signal transduction with a primary focus on the visual system, but including the 2adrenergic receptor to identify common features. The overall goals are to reveal conformational equilibria of the receptors, the cognate G protein and arrestin to test the pre-equilibrium model of protein-protein interactions, and to measure the kinetics of exchange rates. Success in reaching these goals will provide new insight into the dynamic mechanism of signal transduction, and provide a foundation for understanding biased agonism and allosteric activation of the receptors.
The project aims to elucidate molecular mechanisms of G-protein coupled signal transduction, the proteins of which are key pharmacological targets and for which mutations account for a host of human diseases. Success in the proposed research will likely provide a new view of the role of dynamics in the process, and novel targets for drug design may be revealed.
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