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. Author manuscript; available in PMC: 2017 Jul 24.
Published in final edited form as: Mol Cell. 2012 Jul 26;47(5):777–787. doi: 10.1016/j.molcel.2012.06.018

Figure 1. [H+]-Dependence of Substrate Affinity.

Figure 1

(A) Affinity of solubilized, purified MdfA for TPP as represented by dissociation constants. The line represents a nonlinear regression fit to an equation describing competitive binding between substrates and protons.

(B) Western blot showing the effect of 0.5 mM substrates on cysteine PEGylation of selected single-Cys mutants in membrane vesicles. Pyr = Pyronin. The PEGylated protein appears as a heavier adduct.

(C and D) Upper panel, substrate concentration dependence of the protection against A128C PEGylation in membrane vesicles. Lower panel: quantification of upper panel by densitometry. The line represents a nonlinear regression fit to a saturation binding function.

(E and F) [H+]-dependent affinity of single Cys MdfA(A128C) for TPP and pyronin, respectively, as represented by dissociation constants determined from the PEGylation of the protein in membrane vesicles. The lines represent a nonlinear regression fit to an equation describing competitive binding between substrates and protons. All the experiments were repeated at least three times and the error bars represent SD. See also Figure S1.