Figure 7: Affinity and stoichiometry of Dq binding by purified MdfA.
From: Export of a single drug molecule in two transport cycles by a multidrug efflux pump
(a) Quenching of 0.2 μM Dq fluorescence by 2 μM wild-type MdfA or MdfA(R112M). Effect of adding 1 mM TPP to the mixture is also shown. Error bars indicate s.d. of triplicate measurements (*P<10−4, two-tailed t-test). (b) Quenching of 0.2 μM Dq fluorescence by increasing MdfA concentrations. The binding of Dq to MdfA was analysed by nonlinear regression fitting (line), yielding the indicated Kd. (c) Inhibition of [3H]-TPP binding to MdfA by increasing Dq concentrations. The results were analysed by non-linear regression fitting (line), yielding the indicated KI. Error bars indicate s.d. of triplicate measurements. (d) Dq-mediated photoinduced cross-linking of MdfA. Membranes expressing functional split-MdfA (C-terminally tagged N8-His6 and C4-His6, left lanes) or wild-type MdfA-His6 (right lanes) were incubated in the absence or presence of 100 μM Dq and irradiated with ultraviolet. Proteins were separated by SDS–PAGE and western blotting against the His tag. Cross-linked product of N8 and C4 is indicated by a star.
