Key Determinants for Calcium Binding Affinity of EF-Hand Proteins

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0092486YangThe specific goals of this project are to understand site-specific calcium binding properties of EF-hand loops and identify the key factors contributing to calcium affinity of EF-hand motifs in proteins. Calcium binding is essential for the biological functions of EF-hand proteins in controlling cellular calcium levels and in regulating many cellular processes through calcium-induced conformational change. Site-specific information about calcium binding properties of individual EF-hand motifs is essential for defining the sequence for the calcium binding process and the contribution of each EF-hand motif to the conformational change of the intact protein. In this research, the PI will investigate the intrinsic calcium binding affinity of isolated EF-hand motifs by engineering a single EF-calcium-binding site into a scaffold protein. This approach focuses specifically on the intrinsic binding ability of an individual EF-hand motif without the complexities encountered in cooperative, multi-site systems of natural EF-hand proteins. To obtain site-specific information about calcium-binding properties of EF-hand loops, the building blocks of EF-hand motifs will be systematically introduced into the host scaffold. To identify contributions of charged ligands to calcium affinity of EF-loops, the number and geometric arrangement of negatively charged ligands in isolated EF-loops will be also varied.