Wang C L
J Biol Chem. 1986 Aug 25;261(24):11106-9.
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.
利用铕（Eu3+）和铽（Tb3+）作为能量供体，以及其他一些镧系离子（Ln3+）作为受体，通过荧光能量转移技术测量了钙调蛋白（CaM）四个钙离子结合位点之间的距离。先前的研究表明，镧系离子优先结合到CaM的位点I和位点II，其亲和力高于位点III和位点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）。因此，在用激光直接激发时，Eu1Ln1CaM和Tb1Ln1CaM的发光寿命提供了关于位点I和位点II之间距离的信息。另一方面，由于结合在位点III和位点IV的Tb3+离子可通过酪氨酸残基被敏化，因此对紫外光激发的Tb2Ln2CaM进行寿命测量可得出位点III和位点IV之间的距离。发现这两对位点之间的距离均为1.05±0.07纳米。钙离子结合到位点III和位点IV不会改变位点I和位点II之间的距离。我们还将发色团标记物二甲基氨基苯基偶氮苯连接到骨骼肌肌钙蛋白I的Cys - 133上，并通过直接和间接激发对其与CaM的复合物进行了距离测量。从CaM分子N端一半的位点I和位点II以及C端一半的位点III和位点IV到肌钙蛋白I上标记物的平均距离分别为2.7纳米和2.5纳米。
