Akke M, Forsén S, Chazin W J
Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, CA 92037.
J Mol Biol. 1991 Jul 5;220(1):173-89. doi: 10.1016/0022-2836(91)90389-n.
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.
通过使用二维¹H核磁共振光谱对脱辅基、半饱和和完全饱和状态的溶液构象和动力学进行比较分析，研究了牛钙结合蛋白D9k结合钙离子时协同作用的分子基础。由于钙离子结合的协同作用，蛋白质的半饱和钙形式在平衡条件下含量不显著，因此用蛋白质的半饱和镉形式替代钙形式。为了验证钙结合蛋白D9k的镉形式代表钙结合状态的可行模型，制备了完全饱和的镉形式并与钙饱和蛋白进行比较。已获得（Cd²⁺）₁和（Cd²⁺）₂状态的几乎完整的¹H共振归属。通过核Overhauser效应、主链自旋-自旋耦合常数和缓慢交换的酰胺质子确定二级结构元件和整体折叠模式。半饱和蛋白与钙结合蛋白D9k的脱辅基和钙饱和形式的比较表明，所有三种结构高度相似。然而，在结合第一个离子后，蛋白质的结构和动力学性质确实发生了变化；发现半饱和形式与钙饱和形式比与脱辅基形式更相似。这些结果对协同作用的分子基础具有重要意义，并表明与蛋白质动力学相关的熵效应起着重要作用。
