The polarized total internal reflection fluorescence microscopy (polTIRFM) processive motility assay for myosin V.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes the processive motility assay for investigating the motility of myosin V in vitro. Biotin-Alexa actin filaments are fixed to a slide by biotin/streptavidin linkages and aligned with the microscope x-axis by fluid flow. The orientation of a rhodamine-calmodulin (CaM) probe bound to a single myosin V molecule is determined as it moves along an actin filament. Excess wild-type calmodulin (WT-CaM) is present in the buffer solution to replenish lost CaM from the myosin lever arm. The techniques for myosin V should be generally applicable to other single-molecule experiments where angular changes have an important mechanistic role in their biological function.