Single-molecule FRET dynamics of molecular motors in an ABEL Trap

TL;DR

This paper demonstrates how the ABEL trap can be combined with smFRET to observe molecular motors like Rep over extended periods, revealing their conformational dynamics without surface tethering.

Contribution

It introduces a method for integrating the ABEL trap with smFRET to enable long-duration, high-resolution observation of molecular motor dynamics in vitro.

Findings

Extended observation of Rep molecules up to 6 seconds

Captured multiple conformational switching events

Provided a detailed guide for ABEL trap implementation

Abstract

Single-molecule F\"orster resonance energy transfer (smFRET) of molecular motors provides transformative insights into their dynamics and conformational changes both at high temporal and spatial resolution simultaneously. However, a key challenge of such FRET investigations is to observe a molecule in action for long enough without restricting its natural function. The Anti-Brownian ELectrokinetic Trap (ABEL trap) sets out to combine smFRET with molecular confinement to enable observation times of up to several seconds while removing any requirement of tethered surface attachment of the molecule in question. In addition, the ABEL trap's inherent ability to selectively capture FRET active molecules accelerates the data acquisition process. Here we exemplify the capabilities of the ABEL trap in performing extended timescale smFRET measurements on the molecular motor Rep, which is crucial for removing protein blocks ahead of the advancing DNA replication machinery and for restarting stalled DNA replication. We are able to monitor single Rep molecules up to 6 s with 1 ms time resolution capturing multiple conformational switching events during the observation time. Here we provide a step-by-step guide for the rational design, construction and implementation of the ABEL trap for smFRET detection of Rep in vitro. We include details of how to model the electric potential at the trap site and use Hidden Markov analysis of the smFRET trajectories.