Stabilization, characterization, and crystallization of the NhaA mutant. (A) The NhaA double mutant (A109T and Q277G) and NhaA triple mutant (A109T, Q277G, L296M) are more stable in detergent as shown by the longer unfolding half-life (_t1/2) in LDAO at 40°C. (B) The ATP synthase and NhaA wild-type and mutants were co-reconstituted in liposomes. ATP-driven proton pumping establishes a ΔpH (acidic inside) as monitored by the quenching of 9-amino-6-chloro-2-fluorescence (ACMA). Proton efflux is initiated by the addition of increasing concentrations of NaCl/LiCl, and apparent ion-binding affinities for NhaA wild type (closed circle) and mutant (open circle) at pH 8.5 were calculated: K_MNa⁺ wild type (mean ± SD): 1.8 ± 0.2; K_MNa⁺ mutant: 1.6 ± 0.1; K_MLi⁺ wild type: 4.1 ± 0.5; K_MLi⁺ mutant: 4.1 ± 0.3. (C) pH dependence of NhaA Na⁺(Li⁺)–H⁺ antiporter activity for wild type (closed circle) and mutant (open circle) were measured in proteoliposomes by the level of ACMA dequenching as in B at the indicated pH values after the addition of saturating NaCl/LiCl at pH 8.5; all experiments were repeated in triplicate and representative traces are shown.