Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 106, Issue 14, Pages 5657-5662Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0808355106
Keywords
cytoskeleton; fluorescence; microtubule; single-molecule; polarization
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Funding
- National Institutes of Health [RO1-AR48620]
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Kinesin1 is a motor protein that uses the energy from ATP hydrolysis to move intracellular cargoes along microtubules. It contains 2 identical motor domains, or heads, that coordinate their mechano-chemical cycles to move processively along microtubules. The molecular mechanism of coordination between head domains remains unclear, partly because of the lack of structural information on critical intermediates of the kinesin1 mechano-chemical cycle. A point of controversy has been whether before ATP binding, in the so called ATP-waiting state, 1 or 2 motor domains are bound to the microtubule. To address this issue, here we use ensemble and single molecule fluorescence polarization microscopy (FPM) to determine the mobility and orientation of the kinesin1 heads at different ATP concentrations and in heterodimeric constructs with microtubule binding impaired in 1 head. We found evidence for a mobile head during the ATP-waiting state. We incorporate our results into a model for kinesin translocation that accounts well for many reported experimental results.
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