Subunit rotation in a single FoF1-ATP synthase in a living bacterium monitored by FRET

摘要

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 ATPsynthesis is driven by a proton motive force (PMF) comprising a protonconcentration difference {\Delta}pH plus an electric potential {\Delta}{\Psi}across the lipid membrane. Single-molecule in vitro experiments have confirmedthat proton-driven subunit rotation within FoF1-ATP synthase is associated withATP synthesis. Based on intramolecular distance measurements by single-moleculefluorescence resonance energy transfer (FRET) the kinetics of subunit rotationand the step sizes of the different rotor parts have been unraveled. However,these experiments were accomplished in the presence of a PMF consisting of amaximum {\Delta}pH ~ 4 and an unknown {\Delta}{\Psi}. In contrast, in livingbacteria 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 ofin vivo catalytic turnover rates, or the in vivo rotational speed in singleFoF1-ATP synthases, respectively, has to be solved. In addition, the absolutenumber of functional enzymes in a single bacterium required to maintain thehigh ATP levels has to be determined. We report our progress of measuringsubunit rotation in single FoF1-ATP synthases in vitro and in vivo, which wasenabled by a new labeling approach for single-molecule FRET measurements.