Protein Stability Flashcards
What is the difference between DG and DGo?
What is DG at equilibrium?
What does thermodynamics of protein folding show us?
What is RT a measure of?
What is protein stability a measure of?
What is the 2-state folding assumption?
Why is aggregation the most energetically favourable state?
DG = free energy & shows direction of a reaction
DGo = free energy of a system in standard conditions & related to equilibrium constant
DG = 0
If the process occurs - folding is spontaneous DGo>0 then K<1 so shifts to unfolded in an equilibrium
If DG (difference in free energy between U and F state) is lower than RT, then the folded structure is accessible via random fluctuations
Difference in free energy between folded & unfolded state
Proteins rapidly fluctuate between different unfolded conformations before reaching folded native state (to which undergoes different conformations too)
Exposure of hydrophobic residues in aqueous solution = high entropy state - so shifts equilibrium to left
What is cooperative 2-state unfolding/folding?
What are the conclusions of cooperative 2-state unfolding?
How does it shift the equilibrium?
What are the 4 assumptions of ligand binding?
Unfolding - segments of protein unfold & interact with other segments to destabilise & unfold them
Partially unfolded states are more stable than unfolded states
Prevents accumulation of unfolded states into aggregation
- total ligand concentration is free ligand + bound ligand
- we know [Lt] in in vitro experiments
- ligand only binds to folded state
- only 1 ligand binds to 1 protein
What is Tm? What are DGo and K at this?
What is Tmax/Tcold?
What is Ts?
What does thermal stability assume?
What does an increase in temperature do to protein stability?
What does the Gibbs-Helmholtz equation assume?
What is the correlation between protein size & difference in heat capacity & how curved the plot is?
What does a more curved plot mean?
When 50% [U] and 50% [F] such DGo = 0 and K = 1 (equilibrium)
Tm
Temp of maximum stability (folded state)
S entropy & H enthalpy are independent of temperature
Less stable
- Difference in heat capacity is independent of temperature
- If difference in heat capacity is 0, & H and S are independent of temperature, then DGo can be measured at any temperature (protein stability)
Larger protein (more AA) then larger DCPo & so more curved
Tm is smaller
Why do smaller proteins have a higher Tm?
How is a low entropy folded state compensated for?
How is thermal stability measured?
How does pressure/volume alter protein stability?
Smaller difference in specific heat capacity due to smaller change in entropy due to hydrophobic effect
Hydrophobic effect (entropy increase) and low enthalpy state
CD and fluorescence to measure shift & protein ligand binding shift
Higher pressure means denaturation occurs due to water entering interior of hydrophobic core
Low volume = high pressure
How do additives favour the unfolded state?
What do co-solutes do?
How do stabilisers favour the folded state?
Unfolded state has larger surface area - more interactions & lowers energy state & shifts equilibrium
Inhibit aggregation (due to lower surface area) so interacts favourably with unfolded protein to shift equilibrium from aggregation -> unfolded
free energy of unfolded & folded state increased but the unfolded state has a much higher energy state than the folded state - so folded state is more energetically favourable
How do additives favour the unfolded state?
What do co-solutes do?
How do stabilisers favour the folded state?
Unfolded state has larger surface area - more interactions & lowers energy state & shifts equilibrium
Inhibit aggregation (due to lower surface area) so interacts favourably with unfolded protein to shift equilibrium from aggregation -> unfolded
free energy of unfolded & folded state increased but the unfolded state has a much higher energy state than the folded state - so folded state is more energetically favourable
