Abstract
<p>The method of trace level radiochemical modification (an average of $<$1 group attached per protein) was used to survey calcium, peptide and enzyme binding by the ubiquitous, intracellular, regulatory protein calmodulin (CaM). Radiolabeling the 38 carboxylic acids of apo-CaM at increasing stoichiometries of calcium confirmed the first preference of calcium for the C-terminal pair of calcium binding loops. The reactivity changes at the lowest affinity loop 2 in the N-terminal domain correlated with those of the lysines in the CaM central helix. The largest calcium induced changes flanked the most hydrophobic sequence, helix D, located at the N-terminal end of the central helix. Radiolabeling the seven lysines of CaM when associated with the peptides Mastoparan X, Polistes mastoparan or M 5 resulted in the greatest protection at the CaM central helix. In comparison the protection patterns induced by lower affinity ligands or high affinity enzymes imply different modes of binding by CaM.The affinity based method of radiolabel selection successfully located the trypsin binding site on the basic pancreatic trypsin inhibitor protein. Its application to the lysines and carboxylic acids of calcium saturated CaM on binding the phosphatase calcineurin led to the definition of a plausible enzyme binding surface on the extended, crystallographic structure of CaM. The N terminal half of the CaM central helix exerted the greatest effect on enzyme binding. With the exception of glutamic acid 67, single site modification of most calcium binding residues had little effect on its affinity for the enzyme. These results guided the docking of M 13, the alpha helical CaM binding segment of skeletal muscle myosin light chain kinase, on a complementary, hydrophobic surface of CaM which included symmetrical contacts with both terminal domains and a prominent crossover at the central helix. The implications of this model agree with the results from other experiments on CaM and are complementary with studies on CaM target enzymes.</p>