): The conserved C2 domain has been recognized in over 500 proteins, where it plays a central role in targetting proteins to new cellular locations during Ca2+ signals. The most common type of targetting driven by this ubiquitous motif is Ca2+-triggered membrane docking, which initiates critical signaling processes including neurotransmitter and hormone release, activation or inactivation of phosphorylation and G protein signaling cascades, inflammation, and cell cycle control. The present new research proposal aims to develop a molecular picture of C2 domain function, mechanism and structure. The five broad goals of the research are as follows. (i) Distinct classes of C2 domains, differing in their Ca2+ activation parameters and even their mechanisms, will be resolved by comparative equilibrium and kinetic studies of isolated C2 domains from functionally diverse proteins. (ii) The mechanisms by which these different C2 domain classes dock to membranes will be elucidated, and residues essential for membrane docking will be identified. (iii) A medium resolution structure of the protein-membrane interface will be determined via a novel strategy. (iv) The mechanism by which Ca2+ triggers membrane docking will be investigated. Finally, (v) activation parameters and mechanistic models developed by studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. To achieve these goals, a range of methods will be employed. Equilibrium dialysis, fluorescence titrations and stopped flow kinetics will be used to quantitate the equilibrium and kinetic features of selected C2 domains. Scanning cysteine mutagenesis and careful solution measurements will identify critical residues and forces that drive membrane docking. A novel combination of EPR distance measurements and constraint-based modelling will reveal the structure of the protein-membrane interface, and will probe the Ca2+ triggering mechanism. Finally, hypotheses arising from in vitro studies of isolated C2 domains will be tested in multi-domain proteins and in living cells. Overall, this research will provide the first detailed molecular portrait of one of the most prevalent signaling motifs in nature, and will develop new methods to probe the challenging protein-membrane interface. Furthermore, comparative studies of C2 domains will provide information crucial to genomic analyses of many signaling pathways.
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