Calcium-Induced Exposure of a Hydrophobic Surface on Calmodulin

Interactions between calmodulin (CaM) and several hydrophobic fluorescent probes were characterized in order to determine if CaM expresses hydrophobic binding sites in the presence of Ca²⁺. Several classes of fluorescent probes capable of sensing exposure of hydrophobic binding sites on proteins were found to bind to CaM, and these interactions were greatly enhanced by Ca²⁺. In the presence of Ca²⁺, the fluorescence intensity of 9-anthroylcholine (9AC) was increased24-fold by CaM, with a shift in the fluorescence emission maximum from 514 to 486 nm. The fluorescence intensity of 8-anilino-1-naphthalenesulfonate (Ans) was enhanced 27-fold with an emission maximum shift from 540 to 488 nm in the presence of CaM and Ca²⁺. Similar results were obtained with the uncharged fluorescent ligand, TV-phenyl-1- naphthylamine. With all three fluorescent dyes, the fluorescencechanges caused by CaM in the absence of Ca²⁺ were minor compared to those observed with CaM and Ca²⁺. Direct binding studies using equilibrium dialysis demonstrated that CaM can bind four to six molecules of 9AC or two to three molecules of Ans in a calcium-dependent manner. The effects of various amphiphilic compounds on the Ca²⁺-dependent complex formation between CaM and the Ca²⁺-sensitive phosphodiesterase or troponin I were investigated. Trifluoperazine (TFP) and 9AC inhibited CaM stimulation of the Ca²⁺-sensitive phosphodiesterase. The Ca²⁺-dependent binding of the phosphodiesterase to CaM-Sepharose was also inhibited by TFP, 9AC, and Ans. Furthermore, binding of CaM to troponin I-Sepharose was inhibited by these ligands. Consistent with these data was the observation that troponin I antagonized binding of 9AC to CaM. These data indicate that binding of Ca²⁺ to CaM results in exposure of a domain with considerable hydrophobic character, and binding of hydrophobic ligands to this domain antagonizes CaM-protein interactions. It is proposed that this hydrophobic domain may serve as the interface for the Ca²⁺-dependent binding of CaM to the phosphodiesterase or troponin I.