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

in: SPIE Proceedings (2016)
Dienerowitz, Maria; Ilchenko, Mykhailo; Su, Bertram; Mayer, Günter; Deckers-Hebestreit, Gabriele; Henkel, Thomas; Heitkamp, Thomas ; Börsch, Michael
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 ABEL trap - a microfluidic device to actively counteract Brownian motion of single nanoparticles with an electrokinetic trap. Here we present a version of an ABEL trap with a laser focus pattern generated by electro-optical beam deflectors and controlled by a programmable FPGA chip. This ABEL trap 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 subunitof the FoF1-ATP synthase with mNeonGreen results in an improved signal-to-background ratio at lowerlaser excitation powers. This increases our measured trap duration of proteoliposomes beyond 2 s. Additionally, we observe different sm FRET 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.

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