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

Maria Dienerowitz; Mykhailo Ilchenko; Bertram Su; Gabriele Deckers-Hebestreit; Günter Mayer; Thomas Henkel; Thomas Heitkamp; Michael Börsch

doi:10.1117/12.2209592

1 March 2016 Optimized green fluorescent protein fused to F_oF₁-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap

Maria Dienerowitz, Mykhailo Ilchenko, Bertram Su, Gabriele Deckers-Hebestreit, Günter Mayer, Thomas Henkel, Thomas Heitkamp, Michael Börsch

Author Affiliations +

Proceedings Volume 9714, Single Molecule Spectroscopy and Superresolution Imaging IX; 971402 (2016) https://doi.org/10.1117/12.2209592
Event: SPIE BiOS, 2016, San Francisco, California, United 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 Förster resonance energy transfer (smFRET). Fusing the a-subunit of the F_oF₁-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_oF₁-ATP synthase respectively.

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Maria Dienerowitz, Mykhailo Ilchenko, Bertram Su, Gabriele Deckers-Hebestreit, Günter Mayer, Thomas Henkel, Thomas Heitkamp, and Michael Börsch "Optimized green fluorescent protein fused to F_oF₁-ATP synthase for single-molecule FRET using a fast anti-Brownian electrokinetic trap", Proc. SPIE 9714, Single Molecule Spectroscopy and Superresolution Imaging IX, 971402 (1 March 2016); https://doi.org/10.1117/12.2209592

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