Fig 2

Effect of porphyrins on reconstituted ABCB10.

(A) Size exclusion chromatography elution profiles of empty nanodiscs (dashed line) and nanodiscs containing either ABCB10 (dark gray) or MsbA (light gray). The shoulder around 11 ml corresponds to nanodiscs that contain ABC transporter. The inset shows a Coomassie-stained 10% polyacrylamide gel of the nanodisc preparations, with the low molecular band corresponding to MSP1D1 (MSP arrowhead) and the larger molecular weight band to the respective ABC transporter (additional information can be found in S2 Fig). (B) ATPase activity of ABCB10 in the absence (apo) or presence of 5 μM porphyrin. The increase induced by ZnMP was significant (p = 0.05; n = 7). (C) Activation of ABCB10 by increasing concentrations of ZnMP, and fitting to a Michaelis-Menten equation (black line). Inset shows the normalized ATPase activity of MsbA in the absence or presence of 5 μM ZnMP. Data represented as the mean and standard deviation of at least three independent experiments. (D) Enzyme-coupled assay of ABCB10 in presence of ZnCl₂. The activity of ABCB10 (dark gray curve) was increased by 5 μM ZnMP (red line, with steeper decay), but ZnCl₂ (20 μM, light gray line) did not induce activation of ABCB10. Inset shows the calculated ATPase rates relative to the apo protein. Data from three independent experiments, with a significant difference between the three conditions (p = 0.05).