Calcium binding domains of calmodulin. Sequence of fill as determined with terbium luminescence.
Terbium, a trivalent lanthanide, effectively substituted for Ca2+ in calmodulin as judged by several criteria: intrinsic fluorescence spectra, altered mobilities on polyacrylamide gel electrophoresis, formation of a stable complex with troponin I or calcineurin, and stimulation of phosphodiesterase. Calmodulin harbors four Ca2+ binding domains; domains I and II contain no tyrosine, whereas domains III and IV each have one tyrosine. The binding of Tb3+ to calmodulin was followed by the increase of Tb3+ fluorescence at 545 nm upon binding to calmodulin. This fluorescence was elicited either by exciting Tb3+ directly at 222 nm or by exciting the calmodulin tyrosine at 280 nm with resulting energy transfer from tyrosine to Tb3+. Fluorescence generated by direct excitation measures binding of Tb3+ to any of the Ca2+ binding domains, whereas energy transfer through indirect excitation is effective only when Tb3+ is within 5 A of tyrosine, indicating that Tb3+ necessarily occupies a Ca2+ binding domain that contains tyrosine. A judicious use of the direct and indirect excitation could reveal the sequence of fill of the binding domains. Our results suggest these domains are filled in the following sequence: 1) domain I or II; 2) domains III and IV; and 3) domain II or I that has not been filled initially.
D000818:Animals; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D007700:Kinetics; D008163:Luminescent Measurements; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013725:Terbium; D014336:Troponin / Q000378:metabolism; D019210:Troponin I
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D014336:Troponin; D019210:Troponin I; D013725:Terbium; D010727:Phosphoric Diester Hydrolases; D002118:Calcium