In vitro motility-based tether-scanning of the kinesin motor domain

Kinesin-1 is a dimeric motor protein that moves toward the microtubule plus-end. However, its minimal motor domain-a single catalytic head-is sufficient to support directional motility <i>in vitro</i>, raising fundamental questions about how directionality and force generation are encoded within the motor domain. Here, we describe a method for tether-scanning the kinesin motor domain using an <i>in vitro</i> microtubule gliding assay. A cysteine-light kinesin-1 motor domain is covalently tethered to a glass surface through linkers differing in length and flexibility, such as PEG or DNA, attached at defined positions including the C-terminus or surface-exposed loops. Fluorescently-labelled microtubules glide over the kinesin-coated surface, allowing direct observation under fluorescence microscopy. By systematically altering tether geometry and mechanical properties, this method enables precise analysis of how spatial constraints affect motility parameters such as velocity and direction. The protocol has the potential to be adapted to other motor proteins, although such applications may require careful optimisation of labelling sites to preserve motor function. This approach provides a platform for studying the intrinsic motility of the motor domain. • <i>In vitro</i> method to study how tether geometry affects kinesin-1 motility. • Platform for analysing motor function; adaptable with careful optimisation.