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Updated: Jun 24, 2026
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Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
Published on: October 17, 2014
A mobile kinesin-head intermediate during the ATP-waiting state
1Department of Physiology and Biophysics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Summary
Kinesin1 motor proteins coordinate their two heads for movement. This study reveals one head remains mobile in the ATP-waiting state, clarifying kinesin1
Area of Science:
- Molecular motor proteins
- Cellular transport mechanisms
- Biophysics of molecular motors
Background:
- Kinesin1 is a crucial motor protein for intracellular transport, utilizing ATP hydrolysis to move cargoes along microtubules.
- It possesses two identical motor domains (heads) that work in coordination for processive movement.
- The precise coordination mechanism and structural intermediates, especially in the ATP-binding state, are not fully understood.
Purpose of the Study:
- To investigate the coordination mechanism between Kinesin1 motor domains.
- To resolve the controversy regarding the number of bound heads in the ATP-waiting state.
- To elucidate the mobility and orientation of Kinesin1 heads during different ATP concentrations.
Main Methods:
- Utilized ensemble and single-molecule fluorescence polarization microscopy (FPM).
- Analyzed the mobility and orientation of Kinesin1 heads across varying ATP concentrations.
- Employed heterodimeric constructs with one impaired microtubule-binding head.
Main Results:
- Provided evidence for a mobile head in the Kinesin1 ATP-waiting state.
- Quantified head mobility and orientation dynamics under different biochemical conditions.
- Demonstrated functional insights using engineered heterodimeric kinesin constructs.
Conclusions:
- The study supports a model where one Kinesin1 head is mobile during the ATP-waiting state.
- The findings contribute to a refined understanding of Kinesin1 processive translocation.
- The developed model successfully integrates diverse experimental observations on kinesin motor function.
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