Protein Folding and Thermodynamics
Folding occurs in an aqueous environment surrounded by water and other polar molecules like Na+ and Cl–. Interactions between protein and these aqueous elements occur transiently because they’re tumbling in solution.
When unfolded or denatured, the protein will have many folding options, leading to a high conformational entropy S. Once folded into its native state, it’ll have much lower entropy. This is a major force against folding: the loss in entropy.
Yet, this is where these aqueous polar elements become critical. As they tumble, they form transient hydrogen bonds, which have high entropy; whereas, the interactions of water with the hydrophobic molecules of an unfolded molecule lowers the entropy.
Hydrophobic Effect: The folded protein will bury its hydrophobic core to prevent lingering interactions with surrounding water. This raises the overall S of the surrounding aqueous environment. So in fact, folding raises the entropy because of these hydrophobic interactions. Go figure!
The Entropy of Folding: Reduction in S from fold: +220 kJ/mol. Hydrophobic effect: -140 kJ/mol. Other non-covalent interactions: -150 kJ/m. Net S: -70 kJ/m < 0
Note: some of the exterior R chains are also hydrophobic, but it’s because they have a particular purpose (interacting with substrates, membrane proteins, etc.)
The Thermodynamic Hypothesis: the most stable conformation thermodynamically is the native fold.
Proposed and later experimentally confirmed by Christian Anfisen using RNaseA, a nuclease with 124 amino acids including four disulfide bonds (of cysteine). He added highly polar Urea, which at high concentrations alters the hydrogen bonding patterns of water by lowering the hydrophobic effects that drives folding. Without this, the protein denatures. He also broke the disulfide bonds with reducing agent called β-mercaptoethanol. Upon removing the Urea (by adding dialyze), the protein refolded!
Anfisen also performed an important control experiment in which he re-oxidized the protein while still in Urea, which prevented the disulfide bonds from reforming correctly (why?). When he added the dialyze, the protein did not fold (well, only 1% of it did). He repeated this experiment, but this time by adding more of the reducing agent to the 99% mess. 10 hours later it folded correctly. This is because the β-mercaptoethanol re-broke the randomly formed disulfide bonds, allowing it to refold naturally.
Conclusion: the sequence => spontaneous folding=> native fold. In theory, the folding of the protein should be predicted by the primary sequence alone, but this has yet to happen (though certain protein models are getting close).