This proposal is part of a long term research program in which theoretical chemistry and computer simulation are used to investigate the relationships between the primary secondary, and tertiary structures of calcium-binding proteins and their biological functions. The present goal is to use the structures and molecular properties of calcium-specific calcium recognition and binding, the dependence of these mechanisms on dynamic structural details of the proteins, and the structural consequences of calcium binding. The dynamic properties of calcium-binding proteins in a family including calmodulin, troponin C, and calbindin-D will be stimulated by molecular dynamics calculations in order to learn the relation between Ca2+ binding and specific structural components. Protein regions and molecular mechanisms through which interactions with external agents (e.g., inhibitors, drugs, other proteins) modulate the biological functions of calcium binding proteins will be identified from structural perturbations induced by calcium, by exploring the dynamical evolution of the protein in the presence and absence of Ca2+. To understand the basis for CA2+ selectivity, structures and properties of high and low affinity binding sites will be compared, and the structures will be perturbed by mutations. Factors dependent on the electronic structure of the calcium ion will be identified by calculating ion affinities and by exploring ion substitutions (e.g., with Na+, Mg2+, Cd2+, etc). Results will be compared with experimentally determined rank order of ion affinities. The role of structure in the experimentally determined cooperativity between calcium-binding sites will be explored by dynamics simulation to identify mechanisms of interaction between sites, and by probing the structural effects of binding at one site on the other sites and on the intervening structural effects of binding at one site on the other sites and on the intervening structural elements such as liner segments. Comparisons of results from simulated mutations and from the single and double inferences from the calculations. The role that tightly bound solvent molecules and ions identified in the crystal structures of the proteins have in maintaining structures capable of binding calcium and in propagating signals from on structural unit to another will be characterized. Long term plans call for inclusion of bulk solvent in all simulations to identify the nature of this effect and the magnitude of its contribution to the phenomena we study.
