title: Orientation and rotational motions of single molecules by polarized total internal reflection fluorescence microscopy (polTIRFM).
abstract: In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.
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: D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D009038:Motion; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012235:Rhodamines / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D000199:Actins; D002147:Calmodulin; D005456:Fluorescent Dyes; D012235:Rhodamines; D009218:Myosins