Figure 6: Substrate-induced proton release.

MdfA was solubilized and purified to homogeneity and dialysed to remove buffering agents. The protein was diluted to 2 μM and the solution acidity was monitored in a time-dependent manner by measuring the pH-sensitive fluorescence of the probe fluorescein. Before the assay, the protein was titrated by NaOH or HCl to a fluorescent signal of ~550 (pH~6.5). Notably, the absence of a buffering agent generates spontaneous slow drifts in the acidity, creating a background signal. The substrate-induced changes are distinguished from the background by their much higher rate of changes. The arrows denote additions of HCl (2 μM), TPP (150 μM), Pent (300 μM) or Dq (50 μM). HCl was added in an MdfA-equimolar amount to reveal the signal expected by a stoichiometric release of a single proton per MdfA molecule. This signal is nearly identical to that of substrates (TPP/Dq/Pent), suggesting that all substrates release a single proton from MdfA. Substrates were added at saturating concentrations. As a control, substrates were added twice to examine the saturability of binding and the related proton release. In addition, substrates did not appreciably affect fluorescence when added to a solution without MdfA (lower panel). The experiments were repeated at least three times and the results shown are representative.