Subunit rotation in a single F o F 1 -ATP synthase in a living bacterium monitored by FRET

Abstract

F o F 1 -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 ΔpH plus an electric potential ΔΨ across the lipid membrane. Single-molecule in vitro experiments have confirmed that proton-driven subunit rotation within F o F 1 -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 ΔpH ~ 4 and an unknown ΔΨ. In contrast, in living bacteria the maximum ΔpH across the plasma membrane is likely 0.75, and ΔΨ 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 F o F 1 -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 F o F 1 -ATP synthases in vitro and in vivo, which was enabled by a new labeling approach for single-molecule FRET measurements.

Twisting and subunit rotation in single FOF1-ATP synthase

“…Finally, single-molecule rotation measurements of F O F 1 -ATP synthases need to be established in living cells [108]. The present in vitro rotation experiments for ATP synthesis required artificially high DpH .…”

Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

“…Enzymatic dye labeling schemes have been used for single-molecule microscopy in eukaryotes [ 70 ], but applications of such schemes to proteins inside live bacteria are limited to date because it is hard to get dye into these cells [ 98 , 99 ]. Recent examples include single extracellular membrane proteins in the Bacteroides thetaiotaomicron gut symbiont using both enzymatic and antibody dye labeling [ 97 ].…”

Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

“…Cells were transferred from a glycerol stock, stored at -80°C, to an agar plate with appropriate antibiotics (ampicillin), and grown for 1 day at 37°C. To reduce autofluorescence a small amount of cells were taken from the plate and grown for 9 hours at 30°C in 3 ml M9 minimal medium [20] in 15 ml tubes with gentle shaking for sufficient oxygen access. The M9 medium contained ampicillin.…”