Akihiko Ishijima.
Yanagida Biomotron Project, ERATO, JRDC, Mino, Osaka, Japan.
We have refined total internal reflection fluorescence microscopy (TIRFM) to visualize single fluorescent dye molecules in aqueous solution at real time (Funatsu, et al., Nature,374,555 '95). We extended this method to measurements of individual ATP turnovers. Individual ATP turnover events by single myosin molecules were detected by directly observing association-(hydrolysis)-dissociation of fluorescent ATP analogue, in which Cy3 was attached to ribose. Elementary mechanical events of single myosin molecules were measured by optical trapping nanometry (Finer, et al. Nature 94). For simultaneous measurements of single motor mechanics and the ATP turnover, we combined the optics for single molecule imaging with optical trapping nanometry for single motor mechanics. An actin filament was brought into contact with a single one-or two-headed myosin molecule in a myosin-rod cofilament bound to a chemically etched cover slip in the presence of 50 nM Cy3-ATP. Both individual displacement steps and ATP turnovers could be simultaneously measured. Steps of force generation and detachment of the myosin head from the actin were roughly associated with dissociation and association of Cy3-ATP with the myosin head, respectively. Thus, we could get correlation between the elementary steps of chemical and mechanical reaction of a single myosin molecule.