Mechanisms of Calcium-Calmodulin Mediated Ion Channel Gating

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Project Summary As a central molecular hub in calcium signaling, calmodulin (CaM) is a key regulator of hundreds of target proteins including a wide range of ion channels. Essential for understanding the diversity of calmodulin mediated cellular processes is a thorough understanding of the physical relationships and interactions between calcium ions, the four EF- hand binding sites of calmodulin and the individual target proteins. We have developed new methods that allow the characterization of the four binding sites individually, including their binding affinities and cooperative interactions. Our previous work has shown, by site-specific binding measurements and evolutionary informatics that CaM's four EF-hand binding sites have different and distinct binding properties that have undergone strong selective pressures to remain different from each other. The overall goal of this proposal is to employ new state of the art experimental and analytic methods to understand the energetics and molecular mechanisms of calcium binding at each binding sites and how they are altered by occupancy at neighboring sites (cooperativity) and by binding to targets (transduction). We bring to these efforts powerful new experimental and theoretical approaches that we have developed that promise to lead to an unprecedented understanding of the CaM signal transduction mechanism that will have implications for ion channel regulation, calcium signaling and allosteric mechanisms in general. Our aims are to: (1) Determine by lanthanide luminescence spectroscopy the site-specific affinity and cooperativity of Ca2+ and Ln3+ binding to each of the four EF hands of free CaM in solution; (2) Determine how each of the four EF-hand ligand binding affinities are changed upon binding to specific target proteins or appropriate peptide fragments and how calmodulin peptide affinity and stoichiometry are modulated by calcium binding; (3) Determine the amino attributes that determine the unique binding properties of the four EF hand Ca2+ binding sites in CaM using lanthanide luminescence spectroscopy on calmodulin binding site chimeras.