Key Determinants for Calcium Binding Affinity of EF-Hand Proteins

该项目的具体目标是了解EF-hand环的位点特异性钙结合特性，并确定影响蛋白质中EF-hand基元钙亲和力的关键因素。钙结合对于EF-hand蛋白在控制细胞钙水平和通过钙诱导的构象变化调节许多细胞过程中的生物学功能至关重要。关于单个EF-hand基序的钙结合特性的位点特异性信息对于确定钙结合过程的序列以及每个EF-hand基序对完整蛋白构象变化的贡献是必不可少的。在这项研究中，PI将通过将单个ef -钙结合位点设计成支架蛋白来研究分离的EF-hand基序的内在钙结合亲和力。这种方法特别关注单个EF-hand基序的内在结合能力，而不像天然EF-hand蛋白的合作多位点系统那样复杂。为了获得关于EF-hand环钙结合特性的位点特异性信息，将系统地将EF-hand基元的构建块引入宿主支架中。为了确定带电配体对ef -环的钙亲和力的贡献，在孤立的ef -环中，带负电荷的配体的数量和几何排列也会发生变化。

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.