Processive movement by a kinesin heterodimer with an inactivating mutation in one head

Area of Science:

• Molecular motor proteins
• Cellular transport mechanisms
• Biochemistry of kinesin-1

Background:

• Kinesin-1 is a motor enzyme crucial for intracellular transport.
• Processive movement of kinesin-1 relies on coordinated action of its two heads.
• ATP hydrolysis drives kinesin-1's step-wise motion along microtubules.

Purpose of the Study:

• Investigate the role of ATP hydrolysis in kinesin-1's processivity.
• Determine the effect of the R210K mutation on kinesin-1 function.
• Explore the mechanism of functional complementation in kinesin-1 heterodimers.

Main Methods:

• Expression and purification of wild-type and R210K mutant Drosophila kinesin heavy chain.
• Construction and analysis of R210K homodimers and R210K/wild-type heterodimers.
• Biochemical assays measuring microtubule-stimulated ATPase activity.
• Single-molecule motility assays to observe processive movement.

Main Results:

• The R210K mutation inactivates kinesin-1's ATPase activity and motility.
• R210K/wild-type heterodimers exhibit significant microtubule-stimulated ATPase activity (>50% of wild-type).
• Heterodimeric kinesin-1 displays high duty ratio processive movement, similar to wild-type.

Conclusions:

• Dimerization with a functional subunit can rescue the inactivating R210K mutation.
• Complementation suggests modulation of kinetic barriers in the mutant head by the functional head.
• Findings support a gating mechanism where one kinesin head influences the other's catalytic cycle.