Factors governing protein stability and protein stability

Question 1

ΔG(U → F) = ?

Answer 1

Gf - Gu

Question 2

When is a protein stable and the margin?

Answer 2

ΔG < 0

ΔG = -5 to -15 kcal/mol

Question 3

ΔG = ?

Answer 3

ΔG = ΔH - TΔS

Question 4

Force working against protein folding

Answer 4

Forces counteracting decrease in entropy comes from the gain of non covalent interactions in the protein.

Question 5

Hydrophobic effect

Answer 5

Exposure of hydrophobic surface results in decrease entropy.

To minimize the exposed hydrophobic surface, the non polar molecules will aggregate.

Question 6

Cold denaturation

Answer 6

When low temperature more orded H2O lead to ΔS < 0

More effective vdW interactions between H2O and non-polar AA

Increase solubility of U → cold denaturation

LAGEST hydrophobic interactions and no hydration effect occur when ΔS = 0 → No entropic parameter that stabilizes the folded state

Hydrophobic interactions is driven by enthalpy and NOT entropy thats why cold denaturation occurs

Enthalpic is the driving force for protein folding.

Question 7

Find out ⟦N⟧ and ⟦U⟧ to calculate ΔG

Answer 7

1, Find biophysical parameter (y) of the protein that is different between N and U

2, Equilibrate the protein in various concentration of perturbant.

3, Measure the (y) at each concentration or temperature

4, Calculate the fraction of unfolded protein fu = (yf - yobs)/(yF - FU)

Question 8

Methods to measure biophysical parameter

Answer 8

Fluorescence: Trp fluorescence shift in emission spectrum when going from non-polar to polar environment.

Circular dichroism: Measure different in absorbance between right and left polarized light to probe changes in secondary and tertiary structures.

Question 9

Pertunbant

Answer 9

Denaturant (Urea or GuHcl)

Different PH

Temperature

Question 10

Unfolding curve

Answer 10

Make plot of fu versus perturbant

f = 0 for the folded protein
f = 1 for the unfolded protein

When the fraction of U = N = 0,5 then Keq = 1

If the curve only shows one transition → the protein unfolds in a cooperative process.

The reaction is described as a two state system, then the protein structure is formed / lost in one singel event

High slope of the transition = high cooperatively

Question 11

How to calculate Keq

Answer 11

N → U Keq= fu / ff = fu / (1-fu) = (yF - yobs) / (yobs - yu)

ΔG kan be measured at several points during the transition
ΔG = -RTln(Keq)
Gives different ΔG at end yobs

Question 12

Using denaturant to investigate protein stability

Answer 12

Make plot with ΔG versus ⟦Denaturant⟧ in the transition

Linear plot gives ΔG = ΔG(H2O) - m⟦Denaturant⟧

m = slope the line measure the cooperativity and is also related to how large surface that is exposed during unfolding of the protein.

ΔG = 0 → ⟦F⟧ = ⟦U⟧