Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Hendrik Sielaff; Jamieson A. L. Howard; Steven D. Quinn; Frank Dienerowitz; Mark C. Leake; Maria Dienerowitz

doi:10.1117/12.2657888

11 January 2023 Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap

Hendrik Sielaff, Jamieson A. L. Howard, Steven D. Quinn, Frank Dienerowitz, Mark C. Leake, Maria Dienerowitz

Author Affiliations +

Proceedings Volume 12333, Frontiers in Biophotonics and Imaging II; 1233306 (2023) https://doi.org/10.1117/12.2657888
Event: SPIE Photonex, 2022, Birmingham, United Kingdom

Abstract

Monitoring the dynamic behaviour of individual molecular motors in solution reveals insights into their catalytic activities. Single-molecule Förster resonance energy transfer (smFRET) allows us to study molecular motors in real time at high spatial and temporal resolution. Combining smFRET with molecular confinement enables us to increase the observation time up to several seconds without tethered surface attachment of the observed molecules. Here, we employed an Anti-Brownian ELectrokinetic trap (ABEL trap) that used variable homogeneous electric fields to actively confine single molecules to a femtolitre sized observation volume by acting on their surface charge. We present a non-invasive confinement method that allows trapping of molecular motors like the Rep helicase and the F_OF₁-ATP synthase. In Escherichia coli the ATP-dependent Rep DNA helicase facilitates the replisome progression and is necessary for the restart of the DNA replication machinery. We selectively trapped active Rep molecules based on their smFRET signal with sub-millisecond temporal resolution recording conformational switching events during observation times of up to several seconds. In addition, we observed the ATP-hydrolysis driven rotation of individual F_OF₁-ATP synthase molecules over numerous consecutive reaction cycles and extracted their kinetic rates.

Conference Presentation

Citation Download Citation

Hendrik Sielaff, Jamieson A. L. Howard, Steven D. Quinn, Frank Dienerowitz, Mark C. Leake, and Maria Dienerowitz "Single-molecule FRET dynamics of molecular motors in an anti-Brownian electrokinetic trap", Proc. SPIE 12333, Frontiers in Biophotonics and Imaging II, 1233306 (11 January 2023); https://doi.org/10.1117/12.2657888

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