Observing conformations of single FoF1-ATP synthases in a fast anti-Brownian electrokinetic trap

TL;DR

This paper presents an advanced ABELtrap device that significantly extends observation times of single membrane proteins, enabling real-time detection of conformational changes in FoF1-ATP synthase using FRET.

Contribution

We developed a laser focus pattern ABELtrap with FPGA control, vastly increasing observation times of single membrane proteins for conformational studies.

Findings

Extended observation of single proteins over 100 seconds

Detection of conformational changes in FoF1-ATP synthase

Observation time increased by over 1000 times

Abstract

To monitor conformational changes of individual membrane transporters in liposomes in real time, we attach two fluorophores to selected domains of a protein. Sequential distance changes between the dyes are recorded and analyzed by Forster resonance energy transfer (FRET). Using freely diffusing membrane proteins reconstituted in liposomes, observation times are limited by Brownian motion through the confocal detection volume. A. E. Cohen and W. E. Moerner have invented and built microfluidic devices to actively counteract Brownian motion of single nanoparticles in electrokinetic traps (ABELtrap). Here we present a version of an ABELtrap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA. This ABELtrap could hold single fluorescent nanobeads for more than 100 seconds, increasing the observation times of a single particle by more than a factor of 1000. Conformational changes of single FRET-labeled membrane enzymes FoF1-ATP synthase can be detected in the ABELtrap.