Figure 2.

Na⁺ stimulation constant of the Trp→D²G FRET (K_0.5(Na+)) with MelB_St WT and mutants D55C and D59C. The FRET signals from Trp residues of MelB_St to the dansyl moiety of a fluorescent sugar (D²G) in response to increasing Na⁺ concentration were measured as described in Materials and methods. (a, c, and d) The purified WT MelB_St (a) or MelB_St single-site mutants D55C (c) or D59C (d) in a Na⁺-free buffer containing 20 mM Tris-HCl, pH 7.5, 50 mM ChCl, 0.035% UDM, and 10% glycerol were adjusted to a protein concentration of 1 µM. D²G in 20% DMSO was added at 1 min (red arrows) at a concentration of 10 µM (K_d value for D²G binding to MelB_St in the presence of Na⁺). NaCl solutions of increasing concentrations were consecutively added, up to a final concentration of ~50 mM for the WT MelB_St and ~200 mM for the mutants (black arrows). Finally, melibiose was added at an oversaturating concertation (green arrows). In control experiments, identical water volumes, instead of NaCl solution, was used (gray arrows) to control for sample dilution. Data collection and correction were as described in Materials and methods. (b) An increase in FRET intensity is expressed as _diffFRET (the difference before and after the addition of NaCl), and the value for K_0.5(Na+) was determined by fitting a hyperbolic function to the _diffFRET versus Na⁺ concentration. Error bars, standard error (SEM); the number of tests = 4.