The central goal of this proposal is to contribute to our understanding, on a detailed molecular level, of the means whereby calmodulin (CaM) mediates a wide range of cellular reponses to changes in intracellular calcium concentration. In prior work, complexes of melittin with CaM have been studied by proton nuclear magnetic resonance (NMR) as a model system for eventual studies concerning the structural basis for Ca2+ -CaM- dependent enzyme regulation. We now propose to apply similar and additional strategies to peptides derived from the CaM- binding domains of skeletal and smooth muscle myosin light chain kinase (MLCK), an enzyme central to the process of muscle contraction. Newly introduced spectroscopic (isotopic manipulation) and assignment (main chain directed) strategies will be employed. In particular, perdeuterated calmodulin will be used to simplify the spectrum of the complex in tandem with 15 N directed nuclear Overhauser effects (NOE) from 15N enriched peptide to avoid ambiguity in the origin of amide NH NOEs. Structural constraints required to construct a refined three dimensional model of the peptides while bound to calmodulin will be obtained by analysis of two dimensional nuclear Overhauser effect spectroscopy. The distance geometry transform will then be employed to generate a detailed molecular model of the bound peptide. Upon definition of the peptide's structure, the effects of its binding on calmodulin will be examined. This will require an extensive set of 1H resonance assignments of calmodulin which will be obtained by use of 70% randomly deuterated calmodulin (which has significant technical advantages) and will employ data obtained at 600 MHz. These studies will lead directly to experiments designed to delineate the structural requirements for tight binding of peptide to calmodulin. Overall, the results of this proposal are expected to provide insights into the detailed molecular basis for Ca2+-triggered calmodulin-dependent enzyme regulation as well as the nature of protein-protein interactions in general.
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