Binding of trifluoperazine and fluorene-containing compounds to calmodulin and adducts

Calmodulin can be specifically acylated with a fluorene-containing hydrophobic spin-labeling reagent at just Lys 75 or at Lys 75 and Lys 148. The binding of trifluoperazine to calmodulin and the two adducts was determined using a Hummel-Dreyer procedure, and binding of the phenothiazine was found to be characterized by apparent positive cooperativity and an apparent limiting stoichiometry of about seven binding sites per protein molecule. Two non-reactive fluorene-containing compounds were synthesized, and both reagents exhibited far less binding to calmodulin than did trifluoperazine. One of these was also assayed for binding to the monolabeled adduct, and this binding was about half that observed with calmodulin and was non-cooperative. Thus, the qualitative and quantitative binding parameters of hydrophobic groups to calmodulin can be quite different.