Optimized green fluorescent protein fused to FoF1-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

TL;DR

This paper presents an advanced ABELtrap device with improved control and longer observation times for single-molecule fluorescence studies, enabling detailed real-time analysis of membrane protein conformational changes using optimized fluorescent fusion proteins.

Contribution

The authors develop a novel ABELtrap with electro-optical beam deflectors and FPGA control, achieving over 100 seconds of trapping and enhanced smFRET analysis of membrane proteins.

Findings

Trap duration exceeds 100 seconds for nanoparticles.

Enhanced signal-to-background ratio with mNeonGreen fusion.

Observation of multiple smFRET levels indicating conformational states.

Abstract

Observation times of freely diffusing single molecules in solution are limited by the photophysics of the attached fluorescence markers and by a small observation volume in the femtolitre range that is required for a sufficient signal-to-background ratio. To extend diffusion-limited observation times through a confocal detection volume, A. E. Cohen and W. E. Moerner have invented and built the ABELtrap - a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. 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 chip. This ABELtrap holds single fluorescent nanoparticles for more than 100 seconds, increasing the observation time of fluorescent nanoparticles compared to free diffusion by a factor of 10000. To monitor conformational changes of individual membrane proteins in real time, we record sequential distance changes between two specifically attached dyes using F\"orster resonance energy transfer (smFRET). Fusing the a-subunit of the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lower laser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different smFRET levels attributed to varying distances between the FRET donor (mNeonGreen) and acceptor (Alexa568) fluorophore attached at the a- and c-subunit of the FoF1-ATP synthase respectively.