Table 1.

Stern–Volmer quenching constants for I⁻ with MIANS-labeled permease mutants

Mutant	KSV, M−1
V331C	0.06
E126A/V331C	0.62
R144A/V331C	0.58
R144K/V331C	0.24

MIANS labeling of each single-Cys permease mutant was performed with the protein bound to avidin-sepharose. Reactions were terminated with DTT, and excess reagent was removed by extensive washing before elution of the labeled protein from the affinity column. Fluorescence emission maxima (see Fig. 5) then were recorded (excitation, 330 nm) in the presence of concentrations of KI ranging from 0.025 M to 0.4 M, and the spectra were corrected for identical measurements carried out in the presence of the same concentrations of KCl. The data were fit to the Stern–Volmer equation by using a linear regression program as described (13).