Fig. 3. Effect of pH on the binding and release of TPP⁺ to EmrE–His wild type and the E14 replacement mutants. (A) EmrE–beads were incubated with 25 nM [³H]TPP⁺ for 1 h in solutions over a range of pH values. Values are graphed as a percentage of maximal TPP⁺ binding. Purified wild-type EmrE–His (•) and E14D EmrE–His mutant (□) or E14C EmrE–His membranes (▴) were assayed for TPP⁺ binding over a range of pH values. The inset graph shows TPP⁺ binding at pH 5 and 8. For this experiment, TPP⁺ bound to EmrE–His was separated from free TPP⁺ using size exclusion chromatography over a Sephadex G–50 column. After the TPP⁺ binding to EmrE–His at the desired pH, the binding reaction was run over the column and the bound [³H]TPP⁺ collected in scintillation vials by centrifugation and then counted. The black bars represent the data from wild-type EmrE–His and the open bars represent data from the E14D EmrE–His mutant construct. (B) EmrE–beads were incubated with 25 nM TPP⁺ for 30 min at 4°C. EmrE–beads bound to TPP⁺ at equilibrium levels were diluted 1:75 in 60 mM buffered solutions at the desired pH and incubated at 4°C for 2 h. This time was determined to be sufficient for binding reactions to reach equilibrium in experiments not shown here. Values are graphed as a percentage of maximal TPP⁺ binding. Wild-type EmrE–His (•) and the EmrE–His E14D mutant (□) were assayed. For (A) and (B), each point represents the average of triplicate binding reactions. The error bars represent the average deviation of the triplicate measurements.