Fig 6. Functional features of BceAB after purification and reconstitution in liposomes.

(A) SDS-PAGE of the purified wild-type BceAB and catalytic mutants (K48A and E170Q). (B) ATPase activities of the transporters in LMNG detergent. In this experiment and in panels C-E, 5 mM of nucleotides and 300 μg/mL of bacitracin were used, where indicated. (C) ATPase activities of the transporters after reconstitution in liposomes. These activities were calculated according to the total amount of proteins added to the reconstitution mixture. (D) ATPase activities of the transporters after reconstitution in liposomes and further separation of the proteoliposomes by sucrose gradient. Where indicated, othovanadate (Vi) was used at 100 μM. (E) comparison of the ATPase and GTPase activities of the wild-type protein. (F) and (G), ATPase and GTPase activities of the wild-type transporter as a function of ATP and GTP concentrations, respectively. Data were fitted with positive cooperativity ((F), n_H = 2.4, K_M = 1 mM and (G), n_H = 1.9, K_M = 1.3 mM). Data shown are one representative experiment of at least two independent experiments and error bars indicate the standard deviation of triplicates. Statistical significance in panels C-E was calculated by Student´s t-test with Welch’s correction between the conditions indicated with brackets. Statistically significant differences are indicated with ** (p≤0.01) and *** (p≤0.001).