title: 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 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 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-phyenyl-1-naphthylamine. With all three 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 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.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002417:Cattle; D004533:Egtazic Acid; D005456:Fluorescent Dyes; D009206:Myocardium / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014336:Troponin
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D014336:Troponin; D004533:Egtazic Acid; D010727:Phosphoric Diester Hydrolases; D002118:Calcium