Thermodynamics and kinetics of calmodulin/roponic chimeras (969.1)

Abstract

Calmodulin (CaM) and Troponic C (TnC) belong to an EF‐hand protein family which contains 4 EF‐hands and share very similar primary sequences and three dimension structures. However, their specificities to target proteins are quite different. For example, enzymes activated or recognized by CaM are generally not activated by TnC. In the past, chimera proteins containing EF‐hands from CaM and TnC have been used to understand the function of individual EF‐hands of CaM, assuming that the sequence difference between CaM and TnC plays the major role in target recognition. However, it is unclear whether such EF‐hand switching affects the structure and function. Here we generated four chimera proteins (1TnC, 2TnC, 3TnC, and 4TnC), in which the particular EF‐hand of CaM is replaced with the corresponding EF‐hand of TnC, and investigated their structural change upon Ca2+ binding using isothermal titration calorimetry, fluorescence, different scanning calorimetry, and circular dichroism. Furthermore, the interaction of these chimera proteins to the peptides corresponding to the CaM binding sequence of CaM dependent kinase II, Nitric Oxide Synthase, Orai, and Fas receptor, was performed in a similar manner and the resulting data were used to compare to structures built from molecular modeling.Grant Funding Source: This work is supported in part by National Science Foundation.