Calcium-induced exposure of a hydrophobic surface on calmodulin

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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.

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