• Since the environment inside a cell is rarely oxidizing, disulfide bonds are unusual in intracellular proteins

• The oxidizing extracellular environment leads to formation of disulfides whenever the free cysteine side chains can assume the necessary configuration

• The extra bonding, about 4 kcal/mol per disulfide bridge, stabilizes conformations that otherwise would not exist

One example is human chorionic gonadotropin (1hrp; the hormone detected by the most common tests for pregnancy), which is a heterodimer with a very extensive set of disulfide bonds:

The hormone consists of two chains, colored green and blue in the figure, which have very similar folds, giving the structure almost two-fold symmetry.

• All of the disulfide bonds (the yellow rods) are within individual chains

• The chains themselves are held together by hydrogen bonds that are enabled by the unusual conformation caused by the disulfides

• The configuration of the individual chains sometimes is referred to as a "cystine knot"

The influenza virus haemagglutinin protein is another structure in which disulfide bonds stabilize a conformation wildly different than expected.

• The molecule is a trimer of identical polypeptide chains, each of which has a globular "head" containing the host-receptor binding site and the antigenic determinants

• The head lies at the top of a long coiled-coil structure built from two helices from each of the three subunits.

The picture below represents a segment of the coiled-coil region that has been extracted; it makes the disulfide bonds more visible. Again, these seem chiefly to stabilize the individual units, with hydrogen bonds holding together the trimer.

The looped-out sections of chain contain the antigenic regions; amino acid mutations in these regions alter the antigenic character of the molecule, resulting in the recurring influenza epidemics with which we all are familiar.

A final example is bovine pancreatic trypsin inhibitor, which has three disulfide links in just 58 residues.

This is one of the most conformationally stable proteins known; it does not denature thermally even at 100^o, unless the pH is lowered to about 2. A reducing environment, of course, cleaves the disulfide bonds and destroys the native conformation.

Wrong disulfide bonds occasionally form when proteins fold inside a cell.

• An enzyme, called protein disulfide isomerase, 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.

Reduced Form of DSBA	Oxidized Form of DBSA

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

The conformation changes only slightly on oxidation; this is reasonable since the sulfurs in both oxidation states must be readily accessible to other proteins.