title: Myosin monomer density and exchange in synthetic thick filaments investigated using fluorescence microscopy with single molecule sensitivity.
abstract: The number of myosin molecules in synthetic thick filaments, prepared by dialysis at 0.12 M NaCl and pH 7.0, was estimated to be between 400 and 800 molecules per micrometre under conditions appropriate for in vitro motility assays. This estimate was based on a number count of Cy3-labelled myosin molecules incorporated into filaments at a nominal ratio of 1:1000. At this dilution, single fluorescent spots were resolved corresponding to individual labelled myosins. The spots usually bleached with a one- or two-step profile but, in around 30% of the cases, fluctuations were observed indicating that additional photophysical or photochemical events had occurred. Myosin molecules were shown to exchange between filaments in suspension on a time-scale of several hours at 4 degrees C, but the reaction was only 75% complete after 48 h, suggesting a non-exchangeable core. However, myosin exchange does not appear to be the predominant source of the fluctuations in fluorescence intensity in the single molecule assays.
authors.affiliation: Department of Biochemistry, University of Leicester, UK. crb5@le.ac.uk
authors.forename: P B
authors.initials: PB
authors.lastname: Conibear
authors.forename: C R
authors.initials: CR
authors.lastname: Bagshaw
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal / Q000737:chemistry; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D011817:Rabbits
chemical_list: C112962:2'(3')-O-(N-(2-((Cy3)amido)ethyl)carbamoyl)adenosine triphosphate; D002232:Carbocyanines; D005456:Fluorescent Dyes; C085321:cyanine dye 5; D000255:Adenosine Triphosphate; D009218:Myosins