L11: Molecular motors I part 2 Flashcards
bead assay (this time myosin V) and without a bead method?
Bead Assay (Single Motor Protein Study):
Purpose: This assay focuses on watching the movement of a single motor protein interacting with a filament. It allows for precise tracking of the motor protein’s behavior.
Cargo Bead: A bead that has a cargo is attached to the motor protein, and the motor protein binds to the cargo domain. The cargo bead is then moved along the microtubule by the kinesin motor.
Motor Behavior: The position of the bead is tracked over time, which reveals how the motor protein moves along the microtubule.
Each step is typically 8 nm (the distance moved by kinesin with each ATP-driven step), and this can be visualized by plotting bead position (in nm) versus time (in seconds).
Processive Nature of Kinesin: A processive motor like conventional kinesin can move along a microtubule for many steps (e.g., many 8 nm steps) without detaching from the microtubule. This is important for efficient cargo transport.
Watching the Two Heads of a Motor Protein Move (Without the Bead):
Myosin V: In a similar experimental setup, Myosin V can be studied by labeling the two motor domains (heads) with different fluorophores (e.g., red and green).
Hand-Over-Hand Motion: When Myosin V moves along an actin filament:
The two motor domains alternate their motion. One head detaches from the actin filament and moves forward, while the other remains attached to the filament.
This results in hand-over-hand movement, where each motor domain takes turns stepping forward, allowing the motor to remain attached to actin with at least one head at all times.
Measuring Distance Traveled: By measuring the distance traveled by each motor domain over time, researchers can track how Myosin V moves in this processive manner, which allows the motor to travel long distances without detaching.
principles of motor protein mechanism?
Tracks are polar; polarity drives cargo movement in one direction.
Motors perform cyclical, asynchronous attachment and detachment cycles (one ATP used per cycle).
Processive motors: Stay attached for many steps (100s) without detaching (e.g., Myosin V).
Non-processive motors (e.g., muscle myosin): Detach after one step.
* Fuel: ATP + H20 ADP + Pi
Free energy from ATP hydrolysis (ΔG = 10^-19 J)
Work = force x step size
If efficiency is 50%: (efficiency of conversion of chemical energy into mechanical work)
0.5. 10-19 J = force x 10nm (order of magnitude displacement gen by motor in a single interaction?)→ force = ca 5pN (pN is 10-12 N)
Every motor uses 1 atp molecule in a single interaction
the atpase cycle of myosin?
- Myosin II is tightly bound to the actin filament in the absence of ATP
- The working stroke is linked to release of the hydrolysis products (ADP and Pi) from the
active site and drive filament sliding - Muscle myosin II is a ‘low duty ratio’ motor; for most of the ATPase cycle it is detached
from actin
Cyclical Interaction with Myosin Filament
In rigor condition (without ATP), myosin is strongly bound to actin but cannot generate force.
The next step: ATP binding triggers a loss of affinity between myosin and actin.
ATP hydrolysis occurs while the products (ADP + Pi) are still bound to the myosin motor domain, causing a conformational change and displacement of the myosin.
This displacement resets the motor, repriming it and getting it ready for the next force generation step.
The motor can then attach to actin, release Pi, and release ADP.
This release triggers the power stroke (or working stroke) — the step where the myosin motor generates force.
At the end of the power stroke, myosin returns to the rigor condition.
The different states (ADP + Pi and rigor) are elucidated by crystallizing myosin in the ADP + Pi state or rigor state.
atomic model for the working stroke in the myosin motor?
ATP hydrolysis is coupled with a conformational change in the converter domain, which causes a ~70° tilting of the lever-arm domain.
The lever arm tilts towards the top in the ADP + Pi state.
In the rigor state, there is no nucleotide bound to the myosin motor, and the motor has completed the working stroke.
The catalytic domain remains constant and does not change its conformation during these processes.
The lever arm and light chain are displaced by ~70°, with the end of the lever arm moving by about 11 nm. This is the crystallographic model of the working stroke.
Myosin undergoes a conformational change in these two states, resulting in the displacement of the lever arm by ~70°.
The key structural change is in the position of the converter domain and the light chain domain (long alpha helix), while the catalytic domain does not change its conformation.
atomic vs lever arm model vs progressive mechanism for working stroke in the myosin motor?
Atomic Model for the Working Stroke in the Myosin Motor
Thick filament (left) and actin filament (right).
Myosin binds to actin with one motor domain and adopts a conformation where the lever arm is “up.”
ATP is split, and Pi is released first.
Then, ADP is released in a sequential manner.
After this, the final conformation of the lever arm occurs, with the light chain domain tilting by ~70°.
This tilting causes the actin filament to slide towards the M-line (center of the sarcomere).
Lever Arm Model
The motor domain binds to actin and remains strongly bound without moving, while the light chain domain is the part that moves.
The lever arm tilts by ~70°, resulting in a displacement of about 5 to 10 nm (depending on the length of the lever arm).
A longer lever arm leads to a larger displacement.
Myosin 5 has a longer light chain than Myosin 2 (5-10 nm swing of lever arm), which results in a longer lever arm and greater displacement. (30-40 nm wing of lever arm for myosin V). So myosin V takes longer steps on actin filament than myosin 2 although tilting is the same. The amplitude of the working stroke depends on the length of the lever arm.
Progressive Mechanism: Asynchronous
Communication between the two motor domains enables the motor to move processively along the actin filament.
Two heads:
Leading head (front)
Lagging head (back)
One head is in the ADP state, while the other is in the rigor state.
ATP binding causes the lagging head to detach from actin.
The working stroke propels the lagging head forward.
The lagging head hydrolyzes ATP, transitioning to the ADP-Pi state, which causes it to strongly bind to actin and generate the next working stroke.
The newly bound head becomes the lagging head, and the cycle continues as it releases Pi and ADP.
The hand-over-hand mechanism relies on the coordination of the ATPase cycles between the leading and lagging heads.
kinesin-1 atpase cycle and processive movement on the microtubule?
The Kinesin-1 ATPase Cycle and Processive Movement on the Microtubule
Kinesin-1 motor domain is tightly bound to tubulin in the presence of ATP.
Exchange of ADP for ATP in the leading head throws the lagging head forward.
Coupling of the activity of the two motor domains.
One head is always bound to the microtubule – high processivity.
Kinesin also has two domains that can split ATP and generate motion. It has a leading and lagging head. Unlike myosin, ATP binding increases the affinity of kinesin for the microtubule. The opposite happens in myosin, where ATP decreases the affinity for the actin filament.
ATP binding: The leading head with ADP binds ATP, causing it to throw the lagging head forward.
ATP hydrolysis: The lagging head, now forward, hydrolyzes ATP to ADP and Pi.
Pi release: Pi is released from the lagging head.
ADP release: ADP is released from the leading head.
ATP exchange: The leading head exchanges ADP for ATP, and the cycle repeats.
This results in asynchronous, hand-over-hand motion along the microtubule.
*Asynchronous Motion: The two motor domains (the leading and lagging heads) act asynchronously. This is a key difference from myosin, where the affinity for the filament is decreased in the presence of ATP. For kinesin, ATP binding increases the affinity of the motor for the microtubule.
Processivity: Since one head is always attached to the microtubule at any given time, kinesin-1 moves processively along the microtubule, meaning it can take multiple steps without falling off. The hand-over-hand motion of the two motor domains ensures that kinesin can move over long distances without detaching.
adp-vi conditions and pi
ADP-Vi and Pi Conditions
cytoplasmic dynein: rotation of the microtubule-binding stalk
ADP-Vi condition: In this state, the motor is “frozen” or blocked in a specific conformation, which prevents the motor from proceeding further in its cycle.
Pi condition: When Pi is released, it triggers a conformational change that leads to the motor’s movement, particularly in the power stroke.
Apo State (No Nucleotide Bound)
Rigor state: This is a condition where no nucleotide (ATP, ADP, or Pi) is bound to the motor, causing it to be in a strongly bound state to actin or microtubules, often preventing movement.
Merged State (Regions Overlapped)
In this state, certain regions of the motor protein are merged or overlapped. However, due to different conformations, not all regions can overlap, leading to specific shifts and tilting that are part of the stalk region.
The tilting in one domain induces a shift in the position of the stalk region, contributing to the motor’s movement.
Tail Domain and Stalk
Ring/AAA domains: These are found in the motor head and are involved in the ATPase cycle, coordinating with the movement of the stalk.
Stalk region: This part of the motor protein attaches to the microtubule and rotates, driven by the motor protein’s ATP hydrolysis.
Rotation in the ring induces tilting in the stalk, which shifts the microtubule by approximately 8nm. This movement is then amplified by the lever arm, generating a linear displacement of about 8nm per cycle.
need to know similarities and differences in mechanisms and their adaptation for cellular role!!
the 3 models (clearer)
- Atomic Model:
The atomic model focuses on the molecular details of the interaction between myosin and actin at a very fine scale.
Key Concept: It’s about the conformational changes in the myosin head that occur when it binds to actin.
How it works:
When myosin binds to actin, the myosin head undergoes a conformational change, which tilts the lever arm (the part of myosin that sticks out and is responsible for generating movement).
This tilt is about 70 degrees and it’s a critical part of the power stroke, the event where myosin pulls actin to cause contraction.
Key Point: The lever arm acts like a mechanical lever that amplifies the motion of the myosin head.
The longer the lever arm, the greater the displacement of actin, allowing for more movement with less energy input. The tilt and displacement help drive the sliding of actin filaments, which is key in muscle contraction.
2. Lever Arm Model:
The lever arm model focuses on how the lever arm of myosin (the extended region of the myosin head) amplifies small movements to produce larger-scale displacement of actin.
Key Concept: It explains the mechanics behind myosin’s ability to generate movement at the actin filament.
How it works:
When ATP is bound to the myosin head, it causes a conformational change, causing myosin to detach from actin. When ATP is hydrolyzed, myosin reattaches and the lever arm tilts.
The tilt (about 70 degrees) results in a large displacement of actin in a short period of time, much like a long lever arm amplifies the movement of a short handle.
The length of the lever arm is important because a longer lever arm generates a greater displacement, meaning myosin can move actin more efficiently.
3. Processive Model:
The processive model focuses on how myosin can take multiple steps along the actin filament without detaching from it, which is important for efficient movement.
Key Concept: Processivity refers to myosin’s ability to continue moving along actin in a stepwise manner, without detaching after each step.
How it works:
When ATP binds to the myosin head, it induces a conformational change that causes myosin to release actin. The hydrolysis of ATP leads to the reattachment of myosin to actin, but now in a new position, and the process repeats.
This stepwise process is repeated multiple times as myosin moves along the actin filament.
Myosin heads work in a coordinated manner, where one head detaches and moves ahead while the other remains attached to the actin, which allows for continuous movement along the filament without losing grip.
This processivity is essential for processes like muscle contraction, where myosin needs to walk along actin to generate force over an extended distance.
Putting It Together:
Atomic Model: Describes the fine molecular mechanics of myosin’s interaction with actin and the tilt of the lever arm that causes movement.
Lever Arm Model: Focuses on the mechanical advantage provided by the long lever arm of myosin, which amplifies the movement of the actin filament.
Processive Model: Focuses on how myosin can continuously move along the actin filament without detaching, allowing for multiple steps to be taken in one cycle.
So in summary:
The atomic model explains the molecular changes during myosin-actin binding.
The lever arm model explains how these changes translate into displacement via a long lever arm.
The processive model explains how myosin can continue stepping along actin without detaching, which is important for processes that require sustained movement.
