title: Observation of transient disorder during myosin subfragment-1 binding to actin by stopped-flow fluorescence and millisecond time resolution electron cryomicroscopy: evidence that the start of the crossbridge power stroke in muscle has variable geometry.
abstract: The mechanism of binding of myosin subfragment-1 (S1) to actin in the absence of nucleotides was studied by a combination of stopped-flow fluorescence and ms time resolution electron microscopy. The fluorescence data were obtained by using pyrene-labeled actin and exhibit a lag phase. This demonstrates the presence of a transient intermediate after the collision complex and before the formation of the stable "rigor" complex. The transient intermediate predominates 2-15 ms after mixing, whereas the rigor complex predominates at time >50 ms. Electron microscopy of acto-S1 frozen 10 ms after mixing revealed disordered binding. Acto-S1 frozen at 50 ms or longer showed the "arrowhead" appearance characteristic of rigor. The most likely explanation of the disorder of the transient intermediate is that the binding is through one or more flexible loops on the surfaces of the proteins. The transition from disordered to ordered binding is likely to be part of the force-generating step in muscle.
authors.affiliation: School of Biomedical Sciences, Leeds University, Leeds LS2 9JT, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Walker
authors.forename: X Z
authors.initials: XZ
authors.lastname: Zhang
authors.forename: W
authors.initials: W
authors.lastname: Jiang
authors.forename: J
authors.initials: J
authors.lastname: Trinick
authors.forename: H D
authors.initials: HD
authors.lastname: White
mesh_terms: D000199:Actins / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D009132:Muscles / Q000502:physiology*; D015879:Myosin Subfragments / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D011721:Pyrenes / Q000378:metabolism
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D011721:Pyrenes; C030984:pyrene