FIGURE 8.

Effects of azide on dissociation of F₁ from sensor-bound, wild-type ϵ. A, kinetics for F₁·ϵ dissociation measured by BLI. Prior to the F₁ dissociation phase shown, BLI sensors had been loaded with biotinylated wild-type ϵ and then incubated for 900 s in buffer with 20 nm F₁(−δϵ); the normalized BLI signal at time 0 represents bound F₁(−δϵ). Dissociation of F₁(−δϵ) from immobilized ϵ was done in hydrolysis conditions (1 mm ATP, 2 mm Mg²⁺) with increasing concentrations of azide, as indicated. Dissociation kinetics were biphasic, with “fast” (1–2.5 × 10⁻³ s⁻¹) and “slow” (0.8–1.6 × 10⁻⁴ s⁻¹) fractions (nonlinear regression fits not shown but essentially superimpose with data at scale shown; R² > 0.9995 for all fits). B, the azide dependence of F₁·ϵ dissociation. The fraction of complexes that dissociated fast versus slow shows hyperbolic dependence on azide concentration, with K½ = 14 ± 2.3 μm; R² = 0.9935.