Figure 3.

Conformational changes during the ATP hydrolysis cycle of NT Pgp in NDSCs at 37 °C. A, emission spectra from NDSCs containing NT Pgp labeled with donor only (Tb³⁺ chelate) or donor and acceptor (Tb³⁺ chelate and Bodipy FL). Traces were normalized to the Tb³⁺ emission at 546 nm. B, emission spectra of the catalytically-inactive mutant NT/AA Pgp in NDSCs. C, time course of the changes in Bodipy FL-sensitized emission, recorded at 520 nm, in response to sequential additions of NaATP, MgSO₄, and V_i. The gaps during the recording correspond to time periods where additions and manual mixing were performed. The intensity was normalized to the maximum obtained after addition of V_i. D, Bodipy FL sensitized emission decays, recorded at 520 nm, from NT Pgp in NDSCs. The inset displays the same curves in a semi-log scale. R_i represents the weighted residuals of the multiexponential fits (gray lines in the main graph and inset). Decays were normalized to the emission of the ATP-bound protein at 200 μs. All the traces in the figures are representative of at least seven independent experiments and were obtained at 37 °C. Emission was recorded after a 200-μs delay from the 337-nm excitation pulse. Apo, nucleotide- and drug-free buffer with 1 mm EDTA; ATP-bound, + 5 mm NaATP; MgATP, +10 mm MgSO₄.