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Updated: Feb 20, 2026
07:25
Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins
Published on: October 17, 2014
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ATPγS substantially defeats the biasing mechanism for kinesin steps.
Vishakha Karnawat1, Algirdas Toleikis1, Nicholas J Carter1
1Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK.
Nature Communications
|February 18, 2026
Summary
Kinesin-1 motors use ATP to transport cargo along microtubules. A specific ATP analogue, ATPγS, at high concentrations disrupts this motor
Area of Science:
- Molecular motor function
- Cellular transport mechanisms
Background:
- Kinesin-1 motors are ATP-powered dimers that move along microtubules.
- They exhibit load-dependent directional bias during processive stepping.
Purpose of the Study:
- To investigate the role of ATP hydrolysis and nucleotide binding in kinesin-1 stepping bias.
- To elucidate the mechanism underlying load-dependent directional bias in kinesin-1 motors.
Main Methods:
- Single-molecule optical trapping assays.
- Utilized ATPγS, a slowly hydrolyzed ATP analogue, at varying concentrations (1 mM and 1 µM).
Main Results:
- 1 mM ATPγS significantly impaired the kinesin-1 biasing mechanism, while 1 µM ATPγS supported it.
- Identified a novel 'Await-Isomerisation' (AI) state induced by nucleotide binding, which is overpopulated by ATPγS and causes slow backsteps.
- Proposed a model where load-dependent neck-linker docking potentiates exit from the AI state, enabling hydrolysis and forward stepping.
Conclusions:
- ATPγS overpopulates the AI state, highlighting its critical role in the kinesin-1 biasing mechanism.
- The biasing mechanism optimizes forward stepping under load by coupling steered diffusion, neck-linker docking, and nucleotide hydrolysis.
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