1 research outputs found
Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET
FoF1-ATP synthase is the ubiquitous membrane-bound enzyme in mitochondria,
chloroplasts and bacteria which provides the 'chemical energy currency'
adenosine triphosphate (ATP) for cellular processes. In Escherichia coli ATP
synthesis is driven by a proton motive force (PMF) comprising a proton
concentration difference {\Delta}pH plus an electric potential {\Delta}{\Psi}
across the lipid membrane. Single-molecule in vitro experiments have confirmed
that proton-driven subunit rotation within FoF1-ATP synthase is associated with
ATP synthesis. Based on intramolecular distance measurements by single-molecule
fluorescence resonance energy transfer (FRET) the kinetics of subunit rotation
and the step sizes of the different rotor parts have been unraveled. However,
these experiments were accomplished in the presence of a PMF consisting of a
maximum {\Delta}pH ~ 4 and an unknown {\Delta}{\Psi}. In contrast, in living
bacteria the maximum {\Delta}pH across the plasma membrane is likely 0.75, and
{\Delta}{\Psi} has been measured between -80 and -140 mV. Thus the problem of
in vivo catalytic turnover rates, or the in vivo rotational speed in single
FoF1-ATP synthases, respectively, has to be solved. In addition, the absolute
number of functional enzymes in a single bacterium required to maintain the
high ATP levels has to be determined. We report our progress of measuring
subunit rotation in single FoF1-ATP synthases in vitro and in vivo, which was
enabled by a new labeling approach for single-molecule FRET measurements.Comment: 9 pages, 6 figure