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Probing the kinesin reaction cycle with a 2D optical force clamp
Steven M Block1, Charles L Asbury, Joshua W Shaevitz
1Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA. sblock@stanford.edu
Summary
Kinesin motor movement is highly sensitive to backward forces, stalling under loads that only weakly affect forward or sideways motion. This directional bias indicates the motor
Area of Science:
- Molecular motor function
- Biophysics
- Cellular mechanics
Background:
- Kinesin motors are crucial for intracellular transport, utilizing ATP hydrolysis to generate force and movement along microtubules.
- Understanding the mechanochemical cycle of kinesin is essential for deciphering cellular transport mechanisms.
Purpose of the Study:
- To investigate the directional dependence of forces on kinesin motor velocity and mechanochemical cycle.
- To elucidate the role of load direction in kinesin stepping and ATP hydrolysis.
Main Methods:
- Utilizing specialized apparatus to apply controlled forces in forward, backward, and sideways directions to single kinesin molecules.
- Employing fluctuation analysis to determine the number of transitions within the kinesin mechanochemical cycle.
Main Results:
- Kinesin motors stall under backward loads up to 8 pN, while forward and sideways loads have a weaker effect.
- Asymmetric slowing of kinesin by sideways loads at high ATP levels suggests load-dependent transitions.
- Fluctuation analysis reveals at least four transitions in the cycle, with hydrolysis tightly coupled to stepping.
Conclusions:
- The strong directional bias in kinesin's response to load suggests its working stroke is aligned with the microtubule axis.
- Additional load-dependent transitions occur late in the kinesin cycle.
- Current models of kinesin motion require revision to account for these findings.