Protein motors are remarkable biological machines that convert chemical energy into mechanical work, driving a wide range of essential biological processes. Despite extensive study, the fundamental physical principles underlying their motion are not fully understood. Constructing and characterising artificial protein motors would help us to advance toward this goal.
We describe a single-molecule fluorescence microscopy assay designed to support development and functional characterisation of artificial walking proteins. The assay is designed to provide quantitative information on binding affinities between motor constructs and their tracks under varying conditions, and, in a gliding-motility regime, to enable direct visualisation of track movement driven by the motors. We employ fluorescently labelled actin filaments with actin-binding constructs and fluorescently labelled DNA nanotubes with DNA-binding domains. Key steps in assay development include surface passivation and protein immobilisation, a microfluidic platform allowing rapid exchange of chemical environments, and high-resolution fluorescence imaging for precise tracking of filament motion. Together, these tools aim to establish a general experimental framework for studying synthetic protein motors at the single-molecule level.