Investigation of the mechanism of calcium binding to calmodulin. Use of an isofunctional mutant with a tryptophan introduced by site-directed mutagenesis.

A mutant calmodulin, in which phenylalanine 99 of calcium binding site III was changed to a tryptophan by using cassette-based, site-directed mutagenesis, has been used to analyze the mechanism of calcium binding. The combined study of direct calcium binding, modification of tryptophan fluorescence properties upon calcium binding, and terbium titration allows some discrimination among proposed mechanisms of cation binding to calmodulin. Calmodulin appears to have six cation binding sites, four of which are selective for calcium, that seem to be coupled.