Conformational changes induced by binding of divalent cations to calregulin.
Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037282:Calreticulin; D002413:Cations, Divalent; D002417:Cattle; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012261:Ribonucleoproteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D002413:Cations, Divalent; D012261:Ribonucleoproteins; D015032:Zinc; D002118:Calcium