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 FOF1-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 FOF1-ATP synthase molecules over numerous consecutive reaction cycles and extracted their kinetic rates.
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