title: Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.
abstract: We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 μs, allows detection of interactions as brief as ∼100 μs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.
authors.affiliation: European Laboratory for Non-linear Spectroscopy, University of Florence, Sesto Fiorentino, Italy. capitan@lens.unifi.it
authors.forename: Marco
authors.initials: M
authors.lastname: Capitanio
authors.forename: Monica
authors.initials: M
authors.lastname: Canepari
authors.forename: Manuela
authors.initials: M
authors.lastname: Maffei
authors.forename: Diego
authors.initials: D
authors.lastname: Beneventi
authors.forename: Carina
authors.initials: C
authors.lastname: Monico
authors.forename: Francesco
authors.initials: F
authors.lastname: Vanzi
authors.forename: Roberto
authors.initials: R
authors.lastname: Bottinelli
authors.forename: Francesco Saverio
authors.initials: FS
authors.lastname: Pavone
mesh_terms: D000199:Actins / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004247:DNA / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D013057:Spectrum Analysis / Q000379:methods*
chemical_list: D000199:Actins; D000255:Adenosine Triphosphate; D004247:DNA; D009218:Myosins