Abstract:
The rotation of the central subunits of ATP synthase relative to the surrounding parts has been observed by several techniques, however, most previous studies were carried out using the purified protein immobilized on glass slides or incorporated into liposomes. In this project, we labeled the enzyme using site-specific fluorescent tags, which would allow the observation of the enzyme's rotation in vivo. This system might also reveal if ATP synthase displays a favored resting position. To carry out these experiments, the a-subunit of the enzyme was fused to cyan fluorescent protein (CFP) while the γ-subunit was labeled with fluorescein arsenical hairpin binder (FlAsH), a small fluorescent probe. Fluorescence resonance energy transfer (FRET) experiments were carried out using inverted membrane vesicles and whole cells of Escherichia coli. Labeling with the site-specific fluorophores was successful and the labeled enzyme was functional. Moreover, FRET was observed between the two fluorescent labels in inverted membrane vesicles. The FRET efficiency was dependent on the nucleotides present, which may be due to a rotation-dependent FRET efficiency, but may also be explained by direct interaction between ATP and one of the used fluorophores. ATP synthase also represents a promising drug target for the diarylquinolines (DARQs) which can efficiently kill drug-sensitive and drug-resistant Mycobacterium tuberculosis. However, it is not known how DARQs interfere with the catalytic cycle of ATP synthase, a question which may be addressed using single-molecule detection techniques. E. coli, the most used bacterial model system, is resistant to DARQs at whole cell level. However, it is not known if this resistance stems from the lack of binding of DARQs to ATP synthase or from the possibility that ATP synthesis may not be essential for E. coli compared to M. tuberculosis. We tested if ATP synthesis in E. coli is susceptible to two different DARQs. Neither of the two DARQs showed high affinity for E. coli ATP synthase. However, one of them inhibited ATP synthesis at elevated concentrations (50% inhibitory concentration [IC50] ~ 25 μM). These results will allow the use of single-molecule FRET experiments in E. coli to further investigate the mechanism of DARQ action.
