FIGURE 2.

pK_a determination using two different iodoacetamide-based compounds and single cysteine mutants of AhpC provide pK_a values for C46 and C165 close to 8.5. (a) Reduced C165S (—●—) or C46S (- -○- -) AhpC (24 μM) in various pH buffers was incubated with 180 μM 5-iodoacetamidofluorescein for various amounts of time, quenched with excess 2-mercaptoethanol, then analyzed on a 12% SDS-polyacrylamide gel to determine the % fluorescence in the protein fraction; the data were fit to a single exponential equation to determine k_obs at each pH. Data plotted as k_obs versus pH were fit to equation 1 as described in Methods. (b) Reduced wild-type AhpC (40 μM) in various pH buffers was incubated with 400 μM d₀-IAAn over a time course, and aliquots were quenched with excess 2-mercaptoethanol. A standard amount of AhpC labeled with d₅-IAAn was added to each sample, and the protein was digested with trypsin overnight as described in Experimental Procedures. The extent of alkylation of C46 (—●—) and C165 (- -○- -) was determined by measuring the ratio of the peak intensities at 3836/3841 or 1745/1750 Da, respectively. The ratios were fit to a single exponential equation to determine the k_obs and used to calculate the pK_a of each residue as in panel (a).