Creative Proteomics

Di-Sulfide Bond Localization Services

For many proteins and peptides, disulfide bridges are prerequisite for their proper biological function. Many commercialized proteins are cross-linked by disulfide bridges that increase their resistance to destructive effects of extreme environment used in industrial processes or protect protein-based therapeutics from rapid proteolytic degradation. Manufacturing of these products must take into account oxidative refolding—a formation of native disulfide bonds by specific pairs of cysteines located throughout a sequence of linear protein.

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.

Cystine is composed of two cysteines linked by a disulfide bond, disulfide bonds in proteins are formed between the thiol groups of cysteine residues by the process of oxidative folding. The other sulfur-containing amino acid, methionine, cannot form disulfide bonds. 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.

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

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.

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.

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.

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 mis-folded.

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