Optimized green fluorescent protein fused to F_oF₁-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

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 Förster resonance energy transfer (smFRET). Fusing the asubunit of the F o F 1 -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 F o F 1 -ATP synthase respectively.

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

“…By applying electric fields, the fluorescent molecule is pushed back to the center of the laser focus pattern. In preliminary experiments, FRET-labeled EcF O F 1 could be held and analyzed for up to 4 s in the absence of ATP until photobleaching of the dyes [156,157,158,159]. The limiting 1 µm thickness of the microfluidics and the uniform illumination by a fast moving laser focus yields constant fluorescence intensity from a single trapped molecule.…”

Observing monomer: dimer transitions of neurotensin receptors 1 in single SMALPs by homoFRET and in an ABELtrap

“…For the SMALP measurements with NTSR1-mRuby3 in our ABELtrap [21][22][23][24] , the 491 nm laser (Calypso; Cobolt, Hübner Photonics, Kassel, Germany) was replaced by a new combined 4-line laser operated at 561 nm (Skyra, Cobolt; a temporary loan from Cobolt, Solna, Sweden, and initiated by von Gegerfelt Photonics, Bensheim, Germany). The schematic of the ABELtrap setup is shown in Figure 1 below.…”

Confining Brownian motion of single nanoparticles in an ABELtrap

“…Yet, the drawbacks of these initial measurements are short observation times and large intensity fluctuations inherent to single molecules freely diffusing through a confocal detection volume. Our first experiments using an ABELtrap to hold single enzymes in place during the smFRET measurements extended the observation time to hundreds of milliseconds and up to seconds [38][39][40][41][42][43] . We expect a significant increase in precision of distance measurements within the enzyme in the experiments to follow.…”