This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.A range of biochemical and structural studies suggest that a unique structural element, the central linker, plays a major role in conferring the calmodulin (CaM) low binding specificity. The central linker region serves as a molecular hinge, allowing CaM to assume large conformational changes throughout the protein, in order to present a structurally dynamic surface of hydrophobic and acidic residues to recognize, bind, and ultimately transmit the calcium signal to its numerous target proteins. Attempts to decipher what interactions are responsible for the CaM low binding specificity have recently focused on trying to improve CaM binding affinity towards a single target molecule. In contrast to these previous studies, which attempted to increase binding specificity by redesigning particular binding interactions a priori, a different approach is to explore binding affinity/specificity through the generation of combinatorial protein libraries. The proposed research would involve using the binary patterning approach towards the creation of high-quality, productive combinatorial libraries of the CaM central linker to test the hypothesis that CaM binding affinity and specificity are a result of a combination of amino acid contacts and structural plasticity provided by this unique region. Biochemical characterization of selected proteins would not only contribute to the understanding of the native CaM's function, but will also validate the application of this de novo protein design approach towards a new protein scaffold. In addition, these libraries would serve as a starting point for obtaining proteins with altered ligand specificities using high throughput screens and selections.
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