Molecular basis for co-operativity in Ca2+ binding to calbindin D9k. 1H nuclear magnetic resonance studies of (Cd2+)1-bovine calbindin D9k.
The molecular basis for the co-operativity in binding of calcium ions by bovine calbindin D9k has been addressed by carrying out a comparative analysis of the solution conformation and dynamics of the apo, half saturated and fully saturated species using two-dimensional 1H nuclear magnetic resonance spectroscopy. Since the half saturated calcium form of the protein is not significantly populated under equilibrium conditions due to the co-operativity in binding of calcium ions, the half saturated cadmium form of the protein has been substituted for the calcium form. To verify that cadmium forms of calbindin D9k represent viable models for the calcium-bound species, the fully saturated cadmium form has been prepared and compared to the calcium-saturated protein. Virtually complete 1H resonance assignments have been obtained for both the (Cd2+)1 and the (Cd2+)2 states. Secondary structure elements and the global folding pattern were determined from nuclear Overhauser effects, backbone spin-spin coupling constants and slowly exchanging amide protons. Comparisons of the half saturated protein with the apo and calcium-saturated forms of calbindin D9k show that all three structures are highly similar. However, a change in the structural and dynamic properties of the protein does occur upon binding of the first ion; the half saturated form is found to be more similar to the calcium-saturated form than to the apo form. These results have important implications concerning the molecular basis for the co-operativity, and suggest that entropic effects associated with the protein dynamics play an important role.
Akke
M
M
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
Forsén
S
S
Chazin
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D006859:Hydrogen; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008961:Models, Structural; D008969:Molecular Sequence Data; D011487:Protein Conformation; D064030:S100 Calcium Binding Protein G / Q000737:chemistry / Q000378:metabolism*
D001059:Apoproteins; D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D006859:Hydrogen; D002118:Calcium