Disulfide (biochemistry)
Disulfides commonly appear as post-translational modifications within proteins. The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived from two thiol groups. Disulfide bridges formed between thiol groups in two cysteine residues are an important component of the tertiary and quaternary structure of proteins. They are similar to organic disulfides in their structure.
Appearance in proteins
Disulfide bonds can be formed under oxidising conditions and 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 by the process of oxidative folding. 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. The structure of a disulfide bond can be described by its χss dihedral angle between the Cβ−Sγ−Sγ−Cβ atoms, which is usually close to ±90°.
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 links two segments of the protein chain, increases the effective local concentration of protein residues, and decreases 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 formation of 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 misfolded. As the number of cysteines increases, the number of nonnative species increases factorially.
Establishing disulfide position within protein chain
Multiple ways allow establishing how many and where do disulfide bridges appear within a given peptide or protein. Free cysteine residues can be selectively marked upon denaturation of the protein. Mass spectrometry can then be used to establish where these cysteines are. Mass spectrometry can also be used to determine the presence of disulfide bridges, because they are not fragmented under certain conditions. Proteins can thus be selectively cleaved in certain positions in order to find the positions of the disulfides.Occurrence
In bacteria and archaea
Disulfide bonds play an important protective role for bacteria as a reversible switch that turns a protein on or off when bacterial cells are exposed to oxidation reactions. Hydrogen peroxide in particular could severely damage DNA and kill the bacterium at low concentrations if not for the protective action of the SS-bond. Archaea typically have fewer disulfides than higher organisms.In eukaryotes
In eukaryotic cells, in general, stable disulfide bonds are formed in the lumen of the RER and the mitochondrial intermembrane space but not in the cytosol. This is due to the more oxidizing environment of the aforementioned compartments and more reducing environment of the cytosol. Thus disulfide bonds are mostly found in secretory proteins, lysosomal proteins, and the exoplasmic domains of membrane proteins.There are notable exceptions to this rule. For example, many nuclear and cytosolic proteins can become disulfide-crosslinked during necrotic cell death. Similarly, a number of cytosolic proteins which have cysteine residues in proximity to each other that function as oxidation sensors or redox catalysts; when the reductive potential of the cell fails, they oxidize and trigger cellular response mechanisms. The virus Vaccinia also produces cytosolic proteins and peptides that have many disulfide bonds; although the reason for this is unknown. Presumably they have protective effects against intracellular proteolytic machinery.
Disulfide bonds are also formed within and between protamines in the sperm chromatin of many mammalian species.
Disulfides in regulatory proteins
As disulfide bonds can be reversibly reduced and re-oxidized, the redox state of these bonds has evolved into a signaling element. In chloroplasts, for example, the enzymatic reduction of disulfide bonds has been linked to the control of numerous metabolic pathways as well as gene expression. The reductive signaling activity has been shown, thus far, to be carried by the ferredoxin-thioredoxin system, channeling electrons from the light reactions of photosystem I to catalytically reduce disulfides in regulated proteins in a light dependent manner. In this way chloroplasts adjust the activity of key processes such as the Calvin–Benson cycle, starch degradation, ATP production and gene expression according to light intensity. Additionally, It has been reported that disulfides plays a significant role on redox state regulation of Two-component systems, which could be found in certain bacteria including photogenic strain. A unique intramolecular cysteine disulfide bonds in the ATP-binding domain of SrrAB TCs found in Staphylococcus aureus is a good example of disulfides in regulatory proteins, which the redox state of SrrB molecule is controlled by cysteine disulfide bonds, leading to the modification of SrrA activity including gene regulation.In hair and feathers
Over 90% of the dry weight of hair comprises proteins called keratins, which have a high disulfide content, from the amino acid cysteine. The robustness conferred in part by disulfide linkages is illustrated by the recovery of virtually intact hair from ancient Egyptian tombs. Feathers have similar keratins and are extremely resistant to protein digestive enzymes. The stiffness of hair and feather is determined by the disulfide content. Manipulating disulfide bonds in hair is the basis for the permanent wave in hairstyling. Reagents that affect the making and breaking of S−S bonds are key, e.g., ammonium thioglycolate. The high disulfide content of feathers dictates the high sulfur content of bird eggs. The high sulfur content of hair and feathers contributes to the disagreeable odor that results when they are burned.Thiol–disulfide exchange
Thiol–disulfide exchange is the principal reaction by which disulfide bonds are formed and rearranged in a protein. The rearrangement of disulfide bonds within a protein generally occurs via intra-protein thiol–disulfide exchange reactions; a thiolate group of a cysteine residue attacks one of the protein's own disulfide bonds. This process of disulfide rearrangement does not change the number of disulfide bonds within a protein, merely their location. Disulfide reshuffling is generally much faster than oxidation/reduction reactions, which change the number of disulfide bonds within a protein. The oxidation and reduction of protein disulfide bonds in vitro also generally occurs via thiol–disulfide exchange reactions. Typically, the thiolate of a redox reagent such as glutathione, dithiothreitol attacks the disulfide bond on a protein forming a mixed disulfide bond between the protein and the reagent. This mixed disulfide bond when attacked by another thiolate from the reagent, leaves the cysteine oxidized. In effect, the disulfide bond is transferred from the protein to the reagent in two steps, both thiol–disulfide exchange reactions.The in vivo oxidation and reduction of protein disulfide bonds by thiol–disulfide exchange is facilitated by a protein called thioredoxin. This small protein, essential in all known organisms, contains two cysteine amino acid residues in a vicinal arrangement, which allows it to form an internal disulfide bond, or disulfide bonds with other proteins. As such, it can be used as a repository of reduced or oxidized disulfide bond moieties.
Commonly used reagents
In some cases, certain cysteine residues might undergo an unwanted oxidation to form disulfide bridges. This can be reversed by the use of thiols such as β-mercaptoethanol or dithiothreitol serve as reductants through thiol-disulfide exchange. These reagents are used in excess to drive the following equilibrium towards the products:tTris(2-carboxyethyl)phosphine can also be used. It is odorless, unlike β-ME and DTT, and it is selective, works at both alkaline and acidic conditions, it is more hydrophilic and more resistant to oxidation in air. Furthermore, it is often not needed to remove TCEP before modification of protein thiols.