Towards a molecular and atomic anatomy of calmodulin and calmodulin-binding proteins.

The molecular mechanisms by which calcium regulates cellular processes such as metabolic and mechanochemical events probably involve interactions with a variety of molecules. A large body of evidence suggests that the targets of calcium's regulatory effects inside the cell are calcium-binding proteins. Our work attempts to correlate calcium-binding protein structure with activities. In this chapter we have presented some of our recent studies on these functional domains in calmodulin and calmodulin-binding proteins. Selected chemical modifications of known amino acid sequence positions have demonstrated the presence of multiple functional domains on calmodulin, have allowed the dissociation of calmodulin functions, and have provided the necessary tools for further investigations of the molecular basis of calmodulin action. One of these modifications, iodination of tyrosine-99, has allowed us to develop procedures to reproducibly detect calmodulin-binding proteins by a binding technique. This method is technically simple. It allows us to detect and study calmodulin-binding proteins (e.g., myosin heavy chain and membrane gap junction proteins) that would be difficult to study with immobilized calmodulin. We have developed a library of antibodies to calmodulin and related proteins such as troponin C and S100 beta. Some of these antisera appear to be site-specific gamma globulins. We have demonstrated that reactivity can be contained in an amino acid sequence as short as seven residues. We have demonstrated the feasibility of using an immunochemical mapping approach to study calmodulin and calmodulin-binding proteins. Comparative sequence analyses combined with functional analyses have allowed correlation of function and structure and have suggested logical candidates for functional domains on calmodulin and calmodulin-binding proteins. Although not discussed in detail in this chapter, these calmodulin-binding proteins appear to contain amino acid sequence homologies. This suggests, analogous to the approach used for calmodulin and related proteins, a logical starting point for domain analyses of calmodulin-binding proteins. Interestingly, structural homologies among calmodulin-binding proteins are reminiscent of the different phosphorylation sites found in many of the physiological substrates for protein kinases. Since a number of calmodulin-binding proteins are themselves substrates for protein kinases, these results suggest another possible point of interrelationships between calcium and cyclic nucleotide regulation.