• 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 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:

Chorionic Gonadotropin (1hrp)	The Disulfide Bonds

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

• All of the disulfide bonds (the orange residues) are within individual chains

• The chains themselves are held together by hydrogen bonds that are enabled by the unusual conformation caused by the disulfides
  • That is, the disulfide bonds enable a conformation with polar groups outside

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

The influenza virus haemaglutinin 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.

Influenza Haemaglutinin	The Disulfide Bonds

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.

A Single Chain of the Haemaglutinin Protein

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.

BPTI (1bpi)	The Disulfide Bonds

With six cysteines, BPTI in principle can form 2⁶ = 64 disulfide bonds. Only one combination produces an active protein: 30-51, 5-55, and 14-38.

• Creighton and Kim developed a method for trapping disulfide-bonded intermediates in protein folding

• They find at least five intermediate species when denatured BPTI folds

• All of these have the 30-51 disulfide, along with a variety of "incorrect" disulfides

• Evolution has taken care of this latter possibility by providing a protein disulfide isomerase enzyme

  The a Unit of Human Protein Disulfide Bond Isomerase (1mek)

• The intact protein consists of two a units and two b units, which have no cysteines and are therefore inactive

The best examples I know of are a family of knotted molecules, called knottins or cyclotides. Many of these are found in Australian spider venoms. They are categorized as either "bracelet" or Mobius structures.

Kalata B1 was the first cyclotide to be structurally characterized.

• Discovered in the 1970's in a native herbal medicine in the Congo

• Used to accelerate labor and childbirth; causes strong uterine contractions that often produce injury in mother or child

It is a 29-residue protein with a deceptively simple looking structure:

Cartoon of Kalata B1 (1nb1)	Backbone of Kalata B1

(NOTE: None of my molecule viewers shows the 1-29 connection when I use the cartoon display; you will see a gap.)

The molecule contains six conserved Cys residues which form three disulfides, making a topology called a cysteine knot:

The 1-15 Disulfide	The 5-17 Disulfide
The 10-22 Disulfide Makes the Knot	All Disulfides
The 10-22 SS Through 1-5, 15-17 Ring	Side View

Since the Cys fill the core of the protein, the hydrophobic residues make a surface patch.

• The hydrophobics are largely on one face (green)

• The face other is hydrophilic (white)

Hydrophobic Face of Kalata B1	Hydrophilic Face of Kalata B1

OK, what's the deal with the "bracelet" and "Mobius" categories.

• The bracelets have the normal peptide backbone - all trans peptide bonds

• The Mobius ones have one cis peptide bond, where residue 19 meets the highly conserved Pro20:

  The Cis Peptide Bond in Kalata B1

  (Remember, cis means the backbone leaves the peptide bond on the same side it came in.)

Here is the structure in a Jmol window so you can try to follow the chain yourself.

Mobius was a German topologist, who devised/discovered the Mobius strip, which has only one edge and one surface.