FIGURE 1.

Correlation between the conformational state of ϵ and the ATP hydrolysis activity of Bacillus PS3 F₁. Two introduced cysteines, ϵE31C and ϵ134C, were labeled by Cy3 and Cy5 randomly. F₁(ϵ^Cy3.5) was immobilized on a grass slide and excited at 532 nm. Fluorescent intensities of Cy3 and Cy5 of each molecule were used for calculation of FRET efficiency, E_FRET = I_Cy5/(I_Cy3 + I_Cy5). A, shown is a schematic illustration of ϵ subunit conformational change in F₁ and the distributions of FRET efficiency measured at different ATP concentrations (a–h; 100–110 molecules analyzed in each). F₁ was incubated with the indicated [ATP] for 1 h (30 min in the case of 1 mm ATP) at 25 °C before fixation to the glass slides. [ATP] in the solution for fluorescence observation infused after fixation was the same as in the incubation buffer except for h. In h the incubation was done in the presence of 1 mm ATP, whereas 0.1 μm ATP solution was infused after fixation; observation was started 10 min later. The ATP-regenerating system was present in all experiments. B, shown is the correlation between the fraction of high FRET and the fraction of active F₁. A FRET efficiency of 0.8–1.2 was defined as high FRET. Cyan bars represent the fractions of high FRET from the histograms in A, and ATP hydrolysis activities of F₁(ϵ), F₁(ϵ^Cy3), and F₁(ϵ^Cy3,5) divided by that of F₁(−ϵ) (taken from C) are plotted as green, red, and blue circles, respectively. The fractions of high FRET were fitted assuming a simple binding reaction after the fraction of the non-FRET pair was omitted (orange line). The apparent K_d from the fit was 34 μm. C, shown is ATP hydrolysis activity of F₁(−ϵ),F₁(ϵ), F₁(ϵ^Cy3), and F₁(ϵ^Cy3,5). ATP hydrolysis rate was measured in the presence of ATP-regenerating system at 25 °C. Velocities were taken at 3550–3600 s (1750–1800 s in the case of 1 mm ATP) after initiation of the reaction. Values were the average of two measurements.