Figure 3

Conformational changes of W67 KcsA channel probed by steady-state and time-resolved anisotropy measurements. Steady-state fluorescence anisotropy values of 6 µM W67 (A) and W67 E71A (B) KcsA in 5 mM DDM solutions under saturating concentrations of monovalent cations, either 200 mM of Cs⁺, Rb⁺, K⁺ or 5 mM TBA⁺. The Na⁺ concentration used was 200 mM for KcsA W67 and 1 M for W67 E71A KcsA at pH 7. Measurements of both channels were carried out at pH 7 (closed state) or 4 (open state) (C) Representative fluorescence anisotropy decays, r(t), of W67 KcsA at pH 7 in the presence of 5 mM TBA⁺ (SF average ion occupancy: (0), 200 mM Na⁺ (SF average ion occupancy: (1) and 200 mM K⁺ (SF average ion occupancy: (2) (λ_ex = 300 nm; λ_em = 345 nm). The solid lines are the best fit of Eq. 1 to r(t) with _g = 43 ns kept as a fixed parameter in the analyses. The 95% confidence intervals calculated for k₁ were [0.85,0.93]×10⁷ s^-1, [1.87,1.94]×10⁷ s^-1 and [3.60,3.83]×10⁷ s^-1 in the presence of 5 mM TBA⁺, 200 mM Na⁺ or K⁺, respectively, at pH 7 (D) Dependence of the reduced χ² on the energy transfer rate constant k₁. The relative χ² surfaces were obtained for the different ionic conditions by keeping r(0) as the only fitting parameter in Eq. 1. (E,F) represent the average lateral inter-W67 distances retrieved from fitting the anisotropy decays obtained for the detergent-solubilized W67 and W67 E71A KcsA channels, respectively, with the derived homo-FRET model. The data in panels A, B, E and F represent the mean ± SD of at least three independent experiments. Statistical significance was determined via two-way ANOVA with Bonferroni posttest ^*P < 0.001 for pH 7 relative to pH 4. P values greater than 0.05 were considered not significant (NS).