Kinesin's step dissected with single-motor FRET

Area of Science:

• Cellular Biology
• Biophysics
• Molecular Motors

Background:

• Kinesin-1 is a vital motor protein responsible for intracellular transport along microtubules.
• It moves in a processive, hand-over-hand manner using its two motor domains.
• The precise motion of individual motor domains during stepping remains poorly understood.

Purpose of the Study:

• To investigate the movement dynamics of a single Kinesin-1 motor domain during processive stepping.
• To resolve the relative distance and orientation between Kinesin-1 motor domains on a submillisecond timescale.

Main Methods:

• Utilized Förster resonance energy transfer (FRET) between fluorescent labels on both motor domains of single kinesin molecules.
• Monitored FRET efficiency to infer changes in motor domain distance and orientation.
• Analyzed data on a submillisecond timescale to capture rapid conformational changes.

Main Results:

• Observed distinct transitions between high and low FRET values, indicative of conformational changes.
• These FRET changes were dependent on the specific labeling positions on the kinesin constructs.
• Demonstrated that individual kinesin motor domains reside in a stable intermediate position for approximately 3 milliseconds during each step.

Conclusions:

• The study provides novel insights into the stepping mechanism of Kinesin-1 at the single-molecule level.
• Reveals a previously uncharacterized dwell time in an intermediate conformation for individual motor domains.
• This finding refines our understanding of how Kinesin-1 achieves processive movement.