The Anfinsen Experiment in Protein Folding

Disulfide bridges can be disrupted by treating a protein with 2-mercaptoethanol (HS-CH₂-CHOH). The bond between the two sulfurs in the protein is broken and a new bond is created between two sulfurs at the end of two molecules of 2-mercaptoethanol. (2-mercaptoethanol used to be called β-mercaptoethanol or βME.) Treatment with 2-mercaptoethanol is now standard procedure for denaturing proteins. For example, 2ME is always included when proteins are prepared for SDS polyacrylamide gel electrophoresis.

Anfinsen wanted to show that the information for protein folding resided entirely within the amino acid sequence of the protein. He choose ribonuclease A as his model for folding but he couldn't completely denature the protein unless he treated it with the denaturant urea plus 2ME to break the disulfide bridges.

Under those conditions, the protein unfolded. It would refold spontaneously once he removed urea and 2ME from the folding solution. Ribonuclease A regained biological activity under those conditions. This demonstrated that refolding could take place in vitro.

Anfinsen discovered that removing 2ME but not urea led to recovery of 1% of the activity. This is attributed to the formation of random disulfide bridges between the 8 cysteines present in the protein. There are 105 different possibilities (7x5x3x1) so the 1% recovery makes sense. It also shows that the correct three-dimensional conformation must be achieved fairly rapidly when urea is removed since most of the protein under those conditions becomes active.

However, recovery is not 100%. Mistakes are made in vitro and presumably in vivo as well. This led to the discovery of an enzyme called protein disulfide isomerase (PDI)—an enzyme that catalyzes reduction of incorrect disulfide bonds and allows a protein trapped in an incorrect conformation to unfold and try again.

PDI is a ubiquitous enzyme as expected from its important role in proper folding. The active site of the enzyme contains a disulfide (shown as two yellow sulfur atoms in the figure). Thus, the enzyme acts very much like 2-mercaptoethanol, catalyzing a disulfide exchange reaction where the incorrect disulfide bridge in the misfolded protein is reduced and PDI is oxidized. (The correct name of the protein is "thiol-disulfide oxidoreductase. Oxidoreductases form a large class of very important enzymes.)

The enzyme preferentially recognizes incorrect disulfide bridges since these tend to be exposed on the surface of the misfolded protein, whereas correct disulfide bridges are usually buried in the hydrophobic interior of the correctly folded protein.