Journal
BIOPHYSICAL JOURNAL
Volume 97, Issue 1, Pages 173-182Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2009.02.073
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Funding
- Research Council for Earth and Life Sciences
- Netherlands Foundation for Fundamental Research on Matter
- Deutsche Forschungsgemeinschaft
- Elite Network of Bavaria
- Protein Dynamics in Health and Disease
- Laserlab Europe
- European Commission
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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 Forster 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 similar to 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.
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