Motor Proteins Flashcards
Types of Motion Generated by Motor Proteins
- linear motion
- -myosis
- -kinesins
- -dyneins
- rotational motion
- -bacterial flagella
- motor for ATP synthesis
Motility Assays
Fluorescent Labelling
- fluorescently label the protein
- watch how it moves on an immobilised surface by looking at the motion of the fluorescent label
- e.g. label dyneins and watch them walk on immobilised microtubules
- e.g. label actin fibres and watch them gliding on a carpet of immobilised myosins
Mobility Assays
Optical Tweezer
- motility assays with optical tweezers
- quantification of nanoscale motor movements: step length and duration, processivity at varied force
Kinesin 1
- processive motor, takes ~100 steps before detaching
- step size: 8nm
- maximum speed: 800nm/s (10ms/step)
Reynold’s Number
inertial forces / viscous forces
Can kinesin 1 walk against mechanical load?
- load slows the motion
- the motor stops at 7pn load
- larger load causes motion in the opposite direction
How does kinesin generate force and translate the 8nm steps?
- proteins operate at low Reynold’s numbers:
- -viscous drag dominates over inertia
- -mechanical equilibrium at every instance
- thermal noise is large:
- nanoscale motions are best described as random walks
- random walks are modulated by barriers in the energy landscape
- chemical energy input biases the random walk and drives directed motion
Nanoscale Motions and Biased Random Walks
- nanoscale motions are based on biased random walks
- directed motion is generated only if motion is NOT invariant under time reversal
- without energy input this does not happen
Models of Directed Motion
Rectified-Diffusion Model
-the motor diffuses along the microtubule surface
-ATP binding and hydrolysis rectify diffusion
-maximal force the model predicts the protein could work against:
Fmax = 2kbT/d ~ 1pN
-this model was tested with a upside-down motility assay, moors attached to a surface, microtubules in solution
-viscosity modulate drag force on microtubules
-experiments demonstrate that kinesin can work against forces larger than 1pN
Models of Directed Motion
Flashing-Ratchet Model
- potential felt by the motor alternates between an asymmetric sawtooth profile and a flat profile
- ATP hydrolysis controls switching between profiles
- the model predicts that 2 ATP are required per motor movement step with 1 ATP hydrolysed per switch of potential
- tested by comparing motor velocity and ATP hydrolysis rate compared under identical conditions
- experiments demonstrate only 1 ATP is consumed per 8nm of motion
Models of Directed Motion
Thermal-Ratchet Model
- the motor domains contains an elastic element that fluctuates thermally
- the motor domain can only bind when the spring is strained
- binding and unbinding are coupled to ATP hydrolysis
Model of Directed Motion
Powerstroke Motion
- similar to the thermal-ratchet model but strain is developed by a conformational change of the motor domain
- the conformational change is coupled to ATP hydrolysis
Thermal-Ratchet and Powerstroke Models in Relation to the Transition State
- both models can be understood in terms of a transition-state model, the difference is the location of the transition state
- for the thermal ratchet model, the transition state is close to the final state
- for the powerstroke model, the transition state is close to the initial state
Models of Directed Motion
Testing the Force Dependence With a Motility Assay
- kinesis motion and force build-up were measured in an optical trap
- force was also externally varied
- this provides statistics of the dwell time as a function of force
Models of Directed Motion
Testing the Force Dependence With a Motility Assay Under Force
- a short distance to the transition state, 1nm compared with the full step size, 8nm
- this matches better with the powerstroke model than the thermal-ratchet model
