Figure 3

Functional characterization of E26T/D34M/A150E. (a) Fluorescence measurement of a solution containing 2 µM E26T/D34M/A150E (red), revealing its ability to release one proton per protein molecule upon chloramphenicol (Cm) binding. As a comparison, the addition of Cm to a solution containing 2 µM E26T/D34M (blue), failed to trigger the release of H⁺. (b) Cm/H⁺ antiport observed in the everted membrane vesicles expressing E26T/D34M/A150E (red). H⁺ movement was monitored by measurement of acridine orange fluorescence, which is shown in arbitrary units (a.u.). By contrast, Cm was unable to elicit H⁺ movement in the everted membrane vesicles harboring E26T/D34M (blue) or vector (black). The traces are representative of experiments performed in duplicate using two different preparations of everted membrane vesicles.