Purification and characterization of two monomeric kinesin constructs

Area of Science:

• Molecular and Cellular Biology
• Biochemistry
• Structural Biology

Background:

• Kinesins are motor proteins crucial for intracellular transport.
• Understanding the kinetics and structure of kinesin motor domains is key to elucidating their mechanism.
• Dimeric versus monomeric forms of kinesin may exhibit distinct functional properties.

Purpose of the Study:

• To kinetically and structurally compare two monomeric Drosophila kinesin constructs (K341, K366) with a dimeric construct (K401).
• To investigate the role of dimerization in kinesin's ATPase activity and microtubule interaction.
• To identify the rate-limiting step in microtubule-activated ATP hydrolysis.

Main Methods:

• Steady-state and pre-steady-state kinetic analyses of ATPase activity.
• Electron microscopy to study microtubule binding and structural periodicity.
• Rapid quench experiments to determine the rate-limiting step of ATP hydrolysis.

Main Results:

• Monomeric K341 and K366 showed significantly higher microtubule-activated ATPase rates (84 s-1 and 64 s-1, respectively) compared to dimeric K401 (20 s-1).
• All constructs bind microtubules with 8 nm axial periodicity; K366 induced microtubule aggregation.
• ADP release is rate-limiting in the absence of microtubules; ATP hydrolysis is rate-limiting in its presence for all constructs.

Conclusions:

• Kinesin motor domains K341 and K366 retain essential kinetic and structural properties for functional analysis.
• Monomeric kinesin constructs display enhanced microtubule-activated ATPase activity compared to the dimeric form.
• Dimerization may involve inhibitory interactions impacting ATP turnover coupling to force generation.