Miki M, Iio T
Department of Applied Chemistry and BioTechnology, Fukui University, Japan.
J Biol Chem. 1993 Apr 5;268(10):7101-6.
Fluorescence resonance energy transfer was measured between probes attached to Troponin-I and actin in the reconstituted skeletal muscle thin filament. Cys-133 on TnI was labeled with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid, and Lys-61 on actin was labeled with fluorescein 5-isothiocyanate. A large difference in the efficiency of resonance energy transfer was observed between the presence (0.55) and absence (0.76) of Ca2+, which corresponds to the change in the distance of 0.53 nm and is in good accordance with a previous report (Miki, M. (1990) Eur. J. Biochem. 187, 155-162). The fluorescence titration curve of this reconstituted thin filament was measured versus free Ca2+ concentration under physiological conditions in the presence of 8 mM MgCl2. The transfer efficiency changed sharply between pCa 6.9 and 5.75, with a midpoint at pCa 6.25, suggesting that a conformational change of thin filaments occurs with a highly cooperative mode. The time course of this conformational change was followed by measuring the fluorescence intensity change (energy transfer efficiency change) of the labeled reconstituted thin filament after rapid decrease or increase of free Ca2+ concentration with a stopped-flow fluorometer. When the free Ca2+ concentration was increased in the presence of 5 mM MgCl2, the fluorescence intensity increased (the transfer efficiency decreased) with a rate constant of 530 +/- 170 s-1 at 20 degrees C. On the other hand, when the free Ca2+ concentration was decreased, the fluorescence intensity decreased with a rate constant of 43 +/- 5 s-1. The results indicate that the conformational change observed by fluorescence resonance energy transfer corresponds well to the regulation mechanism by troponin-tropomyosin on the reconstituted skeletal muscle thin filament.
在重构的骨骼肌细肌丝中，测量了附着于肌钙蛋白I和肌动蛋白上的探针之间的荧光共振能量转移。肌钙蛋白I上的Cys-133用4-乙酰氨基-4'-马来酰亚胺基芪-2,2'-二磺酸标记，肌动蛋白上的Lys-61用异硫氰酸荧光素5标记。在有Ca2+（0.55）和无Ca2+（0.76）存在时，观察到共振能量转移效率有很大差异，这对应于0.53 nm的距离变化，与之前的报道（Miki, M. (1990) Eur. J. Biochem. 187, 155 - 162）非常吻合。在8 mM MgCl2存在的生理条件下，测量了这种重构细肌丝的荧光滴定曲线与游离Ca2+浓度的关系。转移效率在pCa 6.9和5.75之间急剧变化，中点在pCa 6.25，表明细肌丝的构象变化以高度协同的方式发生。通过用停流荧光计快速降低或升高游离Ca2+浓度后，测量标记的重构细肌丝的荧光强度变化（能量转移效率变化），跟踪这种构象变化的时间进程。当在5 mM MgCl2存在下增加游离Ca2+浓度时，荧光强度增加（转移效率降低），在20℃时速率常数为530±170 s-1。另一方面，当降低游离Ca2+浓度时，荧光强度以43±5 s-1的速率常数降低。结果表明，通过荧光共振能量转移观察到的构象变化与肌钙蛋白-原肌球蛋白对重构骨骼肌细肌丝的调节机制非常吻合。
