Mutant versions of the calmodulin of Drosophila melanogaster have been prepared for use in the study of Ca2+ binding and Ca2(+)-induced conformational changes. In each mutant, a conserved glutamic acid residue indicated to play a critical role in Ca2+ binding has been mutated to glutamine in one of the Ca2(+)-binding sites. Thus a series of four proteins, each with an analogous mutation in one of the four binding sites, has been generated. Here the Ca2(+)-induced conformational changes in these proteins have been examined by use of the fluorescent hydrophobic reporter molecule, 9-anthroyl choline. These studies confirm earlier work which indicates that the carboxyl-terminal pair of Ca2(+)-binding sites shows cooperative Ca2+ binding to produce a major conformational change in the protein. However, these studies provide evidence that the sites of the amino-terminal pair are more independent in their Ca2+ binding properties and contribute individually to the conformational changes associated with Ca2+ binding in the amino-terminal half of the protein. This work also indicates that mutation of either of the amino-terminal Ca2(+)-binding sites can influence the conformational change produced by Ca2+ binding to the carboxyl-terminal sites.
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