Fig. 1.

Schematic representation of the kinesin constructs and the single-motor confocal fluorescence microscopy assay. (A) Two kinesin motor domains (PDB code 2kin) in a two motor domains MT bound state, too far apart for FRET, and a potential intermediate with motor domains close enough for FRET. The gray circles represent a single MT protofilament, plus-end pointing upwards. Green, orange, red, and black arrows represent excitation light, donor emission, acceptor emission, and FRET, respectively. (B) The four positions where cysteines were introduced for specific labeling (green spheres). (C) Experimental assay with fluorescently-labeled kinesin landing on a MT and walking through the confocal volume (orange), where it senses a Gaussian-shaped excitation intensity profile (green curve). Drawing not to scale; FWHM of the profile is ≈250 nm, corresponding to ≈30 kinesin steps.