The Major Factors Affecting Protein Stability

1. hydrophobic interactions
2. hydrogen bonds
3. conformational entropy

The literature is in general agreement that the two types of interaction that are most prevalent in proteins are (i) hydrophobic interactions and (ii) hydrogen bonds. The reaction of these bonds upon going from the unfolded to the folded state is summarized in the cartoon below.

Diagram showing the burial of hydrophobic moities and formation of intramolecular H-bonds upon protein folding. Note the release of water molecules upon folding. (Adapted from Pace et al., 1996.)

Although both these interactions have small free energies per residue, they are important because there are so many of them. The same is true for those interactions which stabilize the unfolded state. The most important of which is conformational entropy. Thus, the overall free energy of a folded protein is given by the small difference between two large numbers. This is a major reason for the difficulty of quantitative computational calculation of protein stability. In a recent analysis of the factors contributing to the stability of RNase T1, the stabilizing and destabilizing interactions were estimated at 271 and 286 kcal/mol, respectively (Pace et al., 1996). Hydrogen bonding and hydrophobic interactions were estimated to contribute 260 kcal/mol to the stabilizing interactions, while the bulk of the destabilizing factors were attributed to loss of conformational entropy on folding, and unfavourable burial of peptide and polar groups. See table.

Destabilising	Free Energy (kcal/mol)
Conformational Entropy	-177
Peptide Groups Buried	-81
Polar Groups Buried	-28
Total Destabilising	-286
Stabilising
Histidine Ionisation	+4
Disulphide Bonds	+7
Hydrophobic Groups Buried	+94
Hydrogen Bonding	+166
Total Stabilising	+271
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G (estimate)	-15
G (measured)	+9

Table taken from Pace et al., 1996. Note that the while the difference between the experimental and estimated values is small, the estimated value would yield an unfolded protein.

The Unfolded State The Hydrophobic EffectBeginning