Lecture 6 Flashcards
Actin monomers
Small globular proteins, bind ATP and hydrolyse it. Assemble into filaments.
Treadmilling
Preferentially assemble at + end (barbed end) and disassemble at - end (pointed end).
Monomer binding
There is no energetic difference between monomer binding at the barbed versus the
pointed end.
The reactants are the same and the products are the same, regardless of which end of
the filament the monomer binds to. The ∆G (change in free energy) is the same, as is
the Ccrit at both ends
When [ ] of actin monomer in solution is greater than the critical [ ]…
When the concentration of actin monomer in solution (C) is greater than Ccrit, you are
more likely to have collisions between monomers in solution and the filament → more likely to have binding occur
What controls actin filament assembly
monomer availability.
Kinetic differences in actin filaments
There exists structural asymmetry at the barbed + pointed ends, the actin filament is polar.
The kinetic differences arise because the monomer conformation is malleable and the probability of it being in the conformation that would allow for polymerization at the pointed end is lower than the probability of it being in the conformation that would allow for binding at the barbed ends.
In sum, this causes binding of monomers at the barbed end to occur more rapidly than at the pointed end.
Polymerisation motor
Does not require ATP hydrolysis to generate force.
Fmax = (kBT/d) ln(C/Ccrit)
For actin in a cell: ~5-10 pN
Why does actin hydrolyse ATP
Pays for actin tread milling; T form is added to barbed end fast, and T form polymerises very slowly to pointed end - so have growing at the barbed end and shrinking at the pointed end. Net staying the same length, moving in the direction of polymerisation.
Need ATP to maintain a dynamic system.
T vs D form actin polymer
Many actin-binding
proteins regulate
filament dynamics
and architecture
Including myosin.
Summary
*Actin polymerization generates force
*ATP hydrolysis pays for actin treadmilling
*Actin-binding proteins regulate filament dynamics and organization
*Biochemical reconstitution
*Correlation and causation experiments
evidence for actin-based motility in biology - correlation
Actin polymerization rates should correlate with cell motility rates.
evidence for actin-based motility in biology - necessity
Inhibition of actin polymerization should prevent cell movement.
evidence for actin-based motility in biology - sufficiency
Induction of actin polymerization, in the absence of any other factors, should drive cell movement.
Reconstitution of actin-based Listeria motility
in a “cell-free” system
Protein purification:
- Cell lysis
- Initial fractionation (centrifugation)
- Column-chromatography (charge, size, affinity)
