Disulfide Bond - Occurrence in Proteins

Occurrence in Proteins

Disulfide bonds play an important role in the folding and stability of some proteins, usually proteins secreted to the extracellular medium. Since most cellular compartments are reducing environments, in general, disulfide bonds are unstable in the cytosol, with some exceptions as noted below, unless a sulfhydryl oxidase is present.

Disulfide bonds in proteins are formed between the thiol groups of cysteine residues. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. A disulfide bond is typically denoted by hyphenating the abbreviations for cysteine, e.g., when referring to Ribonuclease A the "Cys26-Cys84 disulfide bond", or the "26-84 disulfide bond", or most simply as "C26-C84" where the disulfide bond is understood and does not need to be mentioned. The prototype of a protein disulfide bond is the two-amino-acid peptide cystine, which is composed of two cysteine amino acids joined by a disulfide bond (shown in Figure 2 in its unionized form). The structure of a disulfide bond can be described by its dihedral angle between the atoms, which is usually close to ±90°.

The disulfide bond stabilizes the folded form of a protein in several ways:

1) It holds two portions of the protein together, biasing the protein towards the folded topology. That is, the disulfide bond destabilizes the unfolded form of the protein by lowering its entropy.

2) The disulfide bond may form the nucleus of a hydrophobic core of the folded protein, i.e., local hydrophobic residues may condense around the disulfide bond and onto each other through hydrophobic interactions.

3) Related to #1 and #2, the disulfide bond link two segments of the protein chain, the disulfide bond increases the effective local concentration of protein residues and lowers the effective local concentration of water molecules. Since water molecules attack amide-amide hydrogen bonds and break up secondary structure, a disulfide bond stabilizes secondary structure in its vicinity. For example, researchers have identified several pairs of peptides that are unstructured in isolation, but adopt stable secondary and tertiary structure upon forming a disulfide bond between them.

A disulfide species is a particular pairing of cysteines in a disulfide-bonded protein and is usually depicted by listing the disulfide bonds in parentheses, e.g., the "(26-84, 58-110) disulfide species". A disulfide ensemble is a grouping of all disulfide species with the same number of disulfide bonds, and is usually denoted as the 1S ensemble, the 2S ensemble, etc. for disulfide species having one, two, etc. disulfide bonds. Thus, the (26-84) disulfide species belongs to the 1S ensemble, whereas the (26-84, 58-110) species belongs to the 2S ensemble. The single species with no disulfide bonds is usually denoted as R for "fully reduced". Under typical conditions, disulfide reshuffling is much faster than the formation of new disulfide bonds or their reduction; hence, the disulfide species within an ensemble equilibrate more quickly than between ensembles.

The native form of a protein is usually a single disulfide species, although some proteins may cycle between a few disulfide states as part of their function, e.g., thioredoxin. In proteins with more than two cysteines, non-native disulfide species may be formed, which are almost always unfolded. As the number of cysteines increases, the number of nonnative species increases factorially. The number of ways of forming p disulfide bonds from n cysteine residues is given by the formula

For example, an eight-cysteine protein such as ribonuclease A has 105 different four-disulfide species, only one of which is the native disulfide species. Isomerases have been identified that catalyze the interconversion of disulfide species, accelerating the formation of the native disulfide species.

Disulfide species that have only native disulfide bonds (but not all of them) are denoted by des followed by the lacking native disulfide bond(s) in square brackets. For example, the des disulfide species has all the native disulfide bonds except that between cysteines 40 and 95. Disulfide species that lack one native disulfide bond are frequently folded, in particular, if the missing disulfide bond is exposed to solvent in the folded, native protein.

In order to analyze the structure of proteins, it is often necessary to break disulfide bonds. This reduction of disulfide bonds can be accomplished by treatment with 2-mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine.