Calcium-induced exposure of a hydrophobic surface on calmodulin

Abstract

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 bound to CaM, and these interactions were greatly enhanced by Ca2+. In the presence of Ca2+, the fluorescence intensity of 9-anthroylcholine (9AC) was increased 24-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, N-phenyl-1-naphthylamine. With all 3 fluorescent dyes, the fluorescence changes 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 4 to 6 molecules of 9AC or 2 to 3 molecules of Ans in a Ca2+-dependent manner. The effects of various amphiphilic compounds on the Ca2+-dependent complex formation between CaM and the [bovine heart] Ca2+-sensitive phosphodiesterase or troponin I were investigated. Trifluoperazine (TFP) and 9AC inhibited CaM stimulation of the Ca2+-sensitive phosphodiesterase to CaM-Sepharose was also inhibited by TFP, 9AC and Ans. 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. Binding of Ca2+ to CaM apparently results in exposure of a domain with considerable hydrophobic character, and binding of hydrophobic ligands to this domain antagonizes CaM-protein interactions. This hydrophobic domain may serve as the interface for the Ca2+-dependent binding of CaM to the phosphodiesterase or troponin I.