Monitoring single membrane protein dynamics in a liposome manipulated in solution by the ABELtrap

TL;DR

This paper demonstrates a method combining the ABELtrap with confocal FRET to observe single membrane protein dynamics in solution for over 10 seconds, overcoming previous temporal limitations.

Contribution

The study introduces a novel approach integrating the ABELtrap with confocal FRET to extend observation times of single membrane proteins in solution.

Findings

Extended observation of single enzyme dynamics beyond hundreds of milliseconds.

Successful immobilization of lipid vesicles using the ABELtrap for FRET measurements.

Potential for detailed real-time analysis of membrane protein function.

Abstract

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.