M. Miki*, T. Kobayashi#, A. Hagiwara*, H. Kimura*, K. Sano#, and Y. Maeda#
*Department of Applied Chemistry & BioTechnology, Fukui University, Fukui-shi, 910 Japan and # International Institute for Advanced Research, Matsushita Electric Industrial, 3-4 Hikari-dai, Seika, Kyoto, 619-02 Japan.
Fluorescence resonance energy transfer has been measured to detect a conformational change of the troponin-tropomyosin-actin complex in response to a change in the Ca2+ concentration. (1) The single cysteine-9 of a mutant TnI (Ala-9-Cys, Cys-48-Ala, Cys-64-Ala, Cys-133-Ser, Trp-160-Phe) was labeled with IAEDANS and Troponin was reconstituted with TnC, TnI and the labeled TnI. Cys-190 of tropomyosin was labeled with DABMI. The resonance energy transfer between these probes on the reconstituted thin filament was measured under various conditions. A significant change in the efficiency of resonance energy transfer was observed between the presence (0.18) and absence (0.27) of Ca2+, which corresponds to the change in the distance of 0.5 nm. TnI moves on tropomyosin as a molecular switch. (2) Cys-87 of mutant Tm (Ser-87-Cys, Cys-190-Ile) was labeled with 4-acetoamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS). Lys-61 on actin was labeled with FITC. The resonance energy transfer between these probes on the reconstituted thin filament was measured. The transfer efficiency did not change whether Ca2+ is present or not.