Fig. 2.

Fast aspartate binding after slow processes associated with the binding of two Na⁺. (A) The tryptophan fluorescence transients upon the rapid addition of 100 µM l-aspartate to L130W Glt_Ph preequilibrated with 100 mM NaCl (upper curve) or mixing 100 µM l-aspartate and 250 mM NaCl with empty L130W Glt_Ph (lower curve). NaCl (500 mM) was equimolarly substituted with choline chloride. The black lines represent experimental data, and the fits to the sums of exponential functions as well as a linear function to account for photobleaching are in gray. (B) Fluorescence transients and corresponding exponential fits after mixing 100 µM l-aspartate with L130W Glt_Ph preincubated with three different [Na⁺] (the fit results of all experiments are in Table S1). (C) The relative amplitudes of the highest rates obtained from the fits (k_obs,fast) as a function of [Na⁺]. The continuous line represents the fit to a Hill function with n_Hill = 1.7 ± 0.3 and K_D = 22 ± 3 mM. For comparison, the result of a fit with n_Hill fixed to one is shown as a broken line. (D) The Na⁺ titration curve of WT Glt_Ph emission at 308 nm after excitation at 289 nm in 500 mM choline chloride fitted with a Hill function (n_Hill = 1.9 ± 0.2, K_D = 25 ± 2 mM) is shown as a continuous line. The fraction of bound protein is calculated by dividing the relative fluorescence change caused by each addition by the maximum change. All error bars represent ±SEM of three experiments.