title: Alternating-site mechanism of kinesin-1 characterized by single-molecule FRET using fluorescent ATP analogues.
abstract: Kinesin-1 motor proteins move along microtubules in repetitive steps of 8 nm at the expense of ATP. To determine nucleotide dwell times during these processive runs, we used a Förster resonance energy transfer method at the single-molecule level that detects nucleotide binding to kinesin motor heads. We show that the fluorescent ATP analog used produces processive motility with kinetic parameters altered <2.5-fold compared with normal ATP. Using our confocal fluorescence kinesin motility assay, we obtained fluorescence intensity time traces that we then analyzed using autocorrelation techniques, yielding a time resolution of approximately 1 ms for the intensity fluctuations due to fluorescent nucleotide binding and release. To compare these experimental autocorrelation curves with kinetic models, we used Monte-Carlo simulations. We find that the experimental data can only be described satisfactorily on the basis of models assuming an alternating-site mechanism, thus supporting the view that kinesin's two motor domains hydrolyze ATP and step in a sequential way.
authors: Department of Physics and Astronomy, VU University, Amsterdam, The Netherlands.
authors: Sander
authors: S
authors: Verbrugge
authors: Bettina
authors: B
authors: Lechner
authors: Günther
authors: G
authors: Woehlke
authors: Erwin J G
authors: EJ
authors: Peterman
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D003198:Computer Simulation; D000242:Cyclic AMP / Q000031:analogs & derivatives; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D016547:Kinesins / Q000737:chemistry*; D007700:Kinetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D009010:Monte Carlo Method; D009038:Motion; D009711:Nucleotides / Q000737:chemistry*; D013997:Time Factors
chemical_list: D009711:Nucleotides; C067297:8-aminohexylamino cAMP; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D016547:Kinesins