FoF1-ATP synthase is the essential membrane enzyme maintaining the cellular
level of adenosine triphosphate (ATP) and comprises two rotary motors. We
measure subunit rotation in FoF1-ATP synthase by intramolecular Foerster
resonance energy transfer (FRET) between two fluorophores at the rotor and at
the stator of the enzyme. Confocal FRET measurements of freely diffusing single
enzymes in lipid vesicles are limited to hundreds of milliseconds by the
transit times through the laser focus. We evaluate two different methods to
trap the enzyme inside the confocal volume in order to extend the observation
times. Monte Carlo simulations show that optical tweezers with low laser power
are not suitable for lipid vesicles with a diameter of 130 nm. A. E. Cohen
(Harvard) and W. E. Moerner (Stanford) have recently developed an Anti-Brownian
electrokinetic trap (ABELtrap) which is capable to apparently immobilize single
molecules, proteins, viruses or vesicles in solution. Trapping of fluorescent
particles is achieved by applying a real time, position-dependent feedback to
four electrodes in a microfluidic device. The standard deviation from a given
target position in the ABELtrap is smaller than 200 nm. We develop a
combination of the ABELtrap with confocal FRET measurements to monitor single
membrane enzyme dynamics by FRET for more than 10 seconds in solution.Comment: 12 pages, 10 figure