title: Fluorescent labeling of myosin V for polarized total internal reflection fluorescence microscopy (polTIRFM) motility assays.
abstract: 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 how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (∼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.
authors.forename: John F
authors.initials: JF
authors.lastname: Beausang
authors.forename: Yujie
authors.initials: Y
authors.lastname: Sun
authors.forename: Margot E
authors.initials: ME
authors.lastname: Quinlan
authors.forename: Joseph N
authors.initials: JN
authors.lastname: Forkey
authors.forename: Yale E
authors.initials: YE
authors.lastname: Goldman
mesh_terms: D000818:Animals; D002645:Chickens; D005456:Fluorescent Dyes / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D009038:Motion; D024701:Myosin Type V / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes; D012235:Rhodamines; D024701:Myosin Type V