• Ribonuclease A has eight cysteine residues, which form four disulfide bridges

• Mercaptoethanol provides a reducing environment that breaks these bridges

• Urea forms hydrogen bonding bridges that allow water to disrupt the hydrophobic interactions in the interior of the protein; it is a chaotropic agent

• The combination causes the ribonuclease to unfold, and completely lose its activity.

Subsequent experiments demonstrated that the denatured protein could spontaneously regenerate its native conformation:

• If the mercaptoethanol is removed and the urea retained, air oxidation regenerates disulfide bridges
  • the bridges reform randomly, producing a mixture of enzymes that still has no activity

• The incorrectly folded structure is placed in an enviroment with no urea and just a trace of mercaptoethanol
  • the enzyme rapidly returns to its original structure and activity

• The mercaptoethanol permits equilibria between the 105 possible disulfide bridges, and as these form, the lipophilic interactions reform as well

Native Bovine RNase A (1afu)

Structures like the inactive, disulfide-bridged form of ribonuclease, occasionally form when proteins fold inside a cell.

Anfinsen found an enzyme, called protein disulfide isomerase, that catalyzes reduction of inappropriate disulfide bridges by oxidizing its own two cysteines to a disulfide. This gives the misfolded protein a second chance to get it right.

Every living cell contains an enzyme with this activity; the one from E. coli is shown below.

• The cysteines of the reduced form and the cystines of the oxidized are picked out in gold, on the surface of the protein

• The conformation changes only slightly on oxidation, since the sulfurs in both oxidation states must be readily accessible to other proteins.

Reduced Form of DSBA	Oxidized Form of DBSA

Anfinsen made three suggestions:

• The protein itself contains all the information needed to get the fold right; the cellular enviroment is not necessary

• Since the refolding occurs rapidly, in a matter of seconds, the protein does not explore conformation space randomly

• Therefore, folding likely follows a specific pathway

Some problems with the simplistic interpretation of Anfinsen's experiments:

• The unfolded state is likely an ensemble of structures

• Folded proteins likewise may be an ensemble of closely related structures

• As demonstrated by ribonuclease, secondary minima may trap incorrectly folded structures

• Proteins may fold from structures that retain some secondary and even tertiary structure

• Significant evidence exists for some folding occurring while the protein is still on the ribosome [Cabrita et al., Curr. Opin. Struct. Biol., 2010, 20, 33]

• Thus, this picture might represent the folding of a small, single domain protein:
  

Non-functional, but conserved, residues are found in several protein families, all of which are hydrophobic, and form networks of contacts in the native structure:

• In cytochromes C: Gly-6, Phe-10, Leu-94, and Tyr-97

  Conserved Residues in Horse Cytochrome (1hrc)	Conserved Residues in Tuna Cytochrome (5cyt)

• In globins: Val-10, Trp-14, Ile-111, Leu-115

  Conserved Residues in Whale Myoglobin (1a6m)	Conserved Residues in Tuna Myoglobin (1myt)