Protein stability

Define protein stability

It is the net thermodynamic balance that determine whether a protein remains in its functional folded 3D structure (native state) or unfolds (denatured state)

• Denoted by ΔG, which represents the balance between enthalpy (ΔH) and entropy (ΔS)

• Negative ΔG → Native state favoured

• Positive ΔG → Unfolded state favoured

What factors affect protein stability?

• Hydrophobic effect: nonpolar side chains bury themselves inside the protein core to avoid water

• Temperature:

  • Excess heat breaks weak non-covalent bonds by providing kinetic energy, shifting the equilibrium toward unfolding

  • Extreme cold can trigger cold denaturation when water solvation of hydrophobic residues becomes energetically favorable

• pH: alters the ionisation states of amino acid side chains; extreme pH levels disrupt crucial ionic bonds and electrostatic interactions

Methods to study protein stability

• Circular Dichroism (CD) Spectroscopy

• Tryptophan/Intrinsic Fluorescence Spectroscopy

• Differential Scanning Calorimetry (DSC)

What drives protein unfolding?

THE HYDROPHOBIC EFFECT, WHICH IS DRIVEN BY THE ENTHALPY (ΔG), THIS IS WHY WE HAVE COLD DENATURATION

• Hydrophobic effect: nonpolar side chains bury themselves inside the protein core to avoid water

• If entropy (ΔS) drove folding, higher temperature would mean more stable protein, but the opposite is true because heat denatures proteins

• At maximum stability, ΔS = 0, which rules out entropy as the driver

• At low temps, water forms more ordered structures, making it enthalpically favourable to surround non-polar residues → protein unfolds → this is cold denaturation

Steps for calculating protein stability (ΔG) and K_eq

1. Find biophysical parameter (y) that differs between the states (folded vs unfolded).

   • This can be tryptophan fluorescence for example

2. Protein will be put in different concentrations of perturbant (denaturant), like Urea or GluHCl, or pH/temp

3. Parameter y is measured at each pertubant concentration or temperature 

4. Fraction of unfolded protein can be calculated for each y-value, using the equation

5. Plot y versus perturbant

6. From plot, unfolded and folded states can be noted at the plateaus

Steps for calculating Protein stability parameters (ΔG_H2O, m-value, C_m)

Accuracy evaluation of protein stability parameters.

• C_M: Highly accurate; directly read from the center of the experimental transition zone

• m-value: Moderately accurate; relies heavily on having clean, well-defined pre- and post-transition baselines to calculate the slope

• ΔG_H2O: Least accurate; it is an extrapolated value calculated far outside the experimental transition window back to 0 M denaturant, any minor error in the m-value slope becomes amplified

What does the steepness of the graph represent?

Steepness of slope in graphs indicates cooperativity.

• The steeper the slope (the higher the m-value), the higher the cooperativity