Differential native state ruggedness of the two Ca2+-binding domains in a Ca2+ sensor protein

Characterization of near-native excited states of a protein provides insights into various biological functions such as co-operativity, protein-ligand, and protein-protein interactions. In the present study, we investigated the ruggedness of the native state of EhCaBP using nonlinear temperature dependence of backbone amide-proton chemical shifts. EhCaBP is a two-domain EF-hand calcium sensor protein consisting of two EF-hands in each domain and binds four Ca2+ ions. It has been observed that approximately 30% of the residues in the protein access alternative conformations. Theoretical modeling suggested that these low-energy excited states are within 2-3 kcal/mol from the native state. Further, it is interesting to note that the residues accessing alternative conformations are more dominated in the C-terminal domain compared with its N-terminal counterpart suggesting that the former is more rugged in its native state. These distinct characteristics of N- and C-terminal domains of a calcium sensor protein belonging to the super family of calmodulin would have implications for domain dependent Ca2+ signaling pathways.