Distance measurements between metal-binding sites of calmodulin and from these sites to Cys-133 of troponin I in the binary complex.
Distances between the four Ca2+-binding sites of calmodulin (CaM) have been measured by fluorescence energy transfer techniques using Eu3+ and Tb3+ as energy donors and a number of other lanthanide ions (Ln3+) as acceptors. It was shown previously that lanthanide ions preferentially bind to sites I and II of CaM with an affinity higher than that for sites III and IV (Kilhoffer, M.-C., Demaille, J. G., and Gerald, D. (1980) FEBS Lett. 116, 269-272; Wang, C.-L. A., Aquaron, R. R., Leavis, P. C., and Gergely, J. (1982) Eur. J. Biochem. 124, 7-12). Thus upon direct excitation with a laser the luminescence lifetimes of Eu1Ln1CaM and Tb1Ln1CaM provide information on the distance between sites I and II. On the other hand, since Tb3+ ions bound to sites III and IV are sensitizable through tyrosine residues, lifetime measurements of Tb2Ln2CaM excited by UV light yield the distance between sites III and IV. Both pairs of sites were found to be separated by a distance of 1.05 +/- 0.07 nm. Binding of Ca2+ to sites III and IV does not alter the distance between sites I and II. We have also attached a chromophoric label, dimethylaminophenylazobenzene, to Cys-133 of skeletal troponin I and carried out distance measurements on its complex with CaM by both direct and indirect excitation. The averaged distances from sites I and II in the N-terminal half and from sites III and IV in the C-terminal half of the CaM molecule to the label on troponin I are 2.7 and 2.5 nm, respectively.
D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003553:Cystine / Q000378:metabolism*; D005063:Europium / Q000378:metabolism; D007811:Lanthanum / Q000378:metabolism; D007834:Lasers; D008163:Luminescent Measurements; D046911:Macromolecular Substances; D008433:Mathematics; D008670:Metals / Q000378:metabolism*; D013725:Terbium / Q000378:metabolism; D014336:Troponin / Q000378:metabolism*; D019210:Troponin I
D002147:Calmodulin; D046911:Macromolecular Substances; D008670:Metals; D014336:Troponin; D019210:Troponin I; D013725:Terbium; D005063:Europium; D003553:Cystine; D007811:Lanthanum