Model for Kinesin Motion Flashcards
What is the general structure of kinesin?
- 2 x 120kDa heavy chains
- 2 x 70kDa light chains
- Coiled coil domain
Is kinesin a dimer?
Yes
How many regions on kinesin can ATP bind?
2
Where does ATPase action occur in kinesin?
N-terminal globular heads
Where does cargo bind on kinesin?
On the C-terminal globular cargo binding and selection domain
How was a movement assay done for kinesin?
- Isolated purified motor proteins stick on glass via tails with heads sticking upwards
- microtubule attaches to motorhead and is moved towards minus end
How was observation of kinesin ‘walking’ first done?
- Microtubule attached to glass cover strip and latex particle moved along
- Dilute motor protein enough so each particle only has 1 motor attached
What are the problems associated with the direct observation of kinesin walking?
- Latex particle subject to Brownian motion so never know exactly where particle is on microtubule
- Difficult to see individual steps
How can the problems with observing kinesin be solved?
Using an optical trap
How do optical traps work?
- Moves at the same rate as the motor
- Focussed beam of laser light traps particle (stops movement due to Brownian motion0
What profile does kinesin produce with an optical trap?
- Staircase profile characteristic of a processive motor
- 8nm steps between stable states
How does FIONA work?
- Label one motor head with flurophore
- microtubule on cover slip and fluro labelled kinesins added
- Collect enough images of flurophores that the centre of the spot can be found accurately
Why was FIONA used in analysis of kinesin?
Can differetiate between 8nm and 16nm steps
What model does kinesin use to move along microtubules?
Hand over Hand
When does the power stroke occur in kinesin?
When ATP is bound
Is kinesin a high duty bound or low duty bound motor?
High duty bound
What happens in kinesin when ATP binds?
Neck linker docks to core motor and now faces forwards
What happens after the power stroke in kinesin?
ATP hydrolysis
What happens when ADP is bound in kinesin?
- Head releases tubulin, neck linker unlocks
- Motor is weakly bound to track
What happens when ADP is released in kinesin?
- Binds new tubulin site
- Neck linker facing backwards
When happens in kinesin when the neck-linker docking is completed by the leading head?
The partner head is thrown forward by 16nm
What does the processive hand-over-hand stepping of kinesin 1 require?
The ATPase cycles of each head are co-ordinated
How are the ATPase cycles of the heads in kinesin co-ordinated?
- Core ATPases do not contact each other directly
- Long range allostery
- Mechanical tension transferred between 2 heads via neck linkers
