Y. Ishii, T. Funatsu, T. Wazawa, T. Yoshida, M. Nishishita, H. Yokota and T. Yanagida
Yanagida Biomotron Project, ERATO, JST, Mino, Osaka 562, Japan
Conformational state of individual protein molecules could be directly visualized by combining fluorescence resonance energy transfer (FRET) technique and single molecule imaging technique (Funatsu et al. Nature 374, 555 (1995)). Both images and spectra of single fluorescence molecules were successfully obtained in aqueous solution. Troponin and actin were specifically labeled with two dyes. Cys-98 of troponin-C was labeled with tetramethylrhodamine (TMR) as a donor and Cys-133 of troponin-I was labeled with Cy5 as an acceptor. Individual troponin molecules had different FRET efficiency, indicating that the conformation of subunits complex varies from molecule to molecule. In addition, the conformation of single troponin molecules changed with time in the time range of seconds. The FRET efficiency and the distance between the donor and acceptor were determined for individual troponin molecules. The slow conformational fluctuation was greater in the absence of Ca2+. Similar experiments were done with actin. Gln-41 and Cys-374 were specifically labeled with TMR and Cy5, respectively. Dynamic changes in the FRET efficiency were observed for single monomer molecules in F-actin. Thus, the FRET technique is proved to be a powerful tool to explore the properties of proteins at the single molecular level.