Kinetics of structural changes of reconstituted skeletal muscle thin filaments observed by fluorescence resonance energy transfer.
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.
Miki
M
M
Department of Applied Chemistry and BioTechnology, Fukui University, Japan.
Iio
T
T
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003545:Cysteine / Q000378:metabolism; D004735:Energy Transfer; D005453:Fluorescence; D005454:Fluorescence Polarization; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014335:Tropomyosin / Q000737:chemistry / Q000378:metabolism; D014336:Troponin / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D014335:Tropomyosin; D014336:Troponin; D008239:Lysine; D003545:Cysteine; D002118:Calcium