In chemistry, a disulfide bridge , disulfide bond or disulfide bond denotes a covalent bond between two sulfur atoms whose only free valence is saturated with an organyl radical . In biochemistry , the disulfide bond is the covalent bond (an atomic bond ) between the sulfur atoms of two cysteine molecules that appear in the amino acid side chain of a protein .
Two cysteine residues in proteins linked by means of a disulfide bond are also known as cystine bridges.
Disulfide bridges form and stabilize the three-dimensional protein structure (tertiary structure) through the formation of loops within the amino acid chains or link several amino acid chains to form a functional protein . As covalent bonds, disulphide bridges have a significantly more fixing effect than, for example, the hydrogen bonds which also occur in the molecule and which are among the secondary valence bonds .
Disulfide bond formation can be a separate step in the folding of a protein without delaying subsequent folding steps .
Disulfide bonds are typical for secretory proteins because they cannot form in the cytosol.
The proteohormone insulin consists of two different amino acid chains, which are called A and B chains, the A chain being connected intrachenarically by one and interchenarly with the B chain by two disulfide bonds.
G-protein-coupled receptors are membrane-bound proteins that are stabilized by a disulfide bridge between the third transmembrane helix and the second extracellular loop. Mutation studies show that the receptor is no longer expressed when one of the cysteines involved is mutated to serine .
The functional groups involved in the formation of a disulfide bond are called thiol groups (mercapto groups). In simplified terms, the formation of such a SS bond can be understood as oxidation (release of hydrogen or electrons):
Oxidation: R-SH + HS-R '→ RSSR' + 2 H + + 2e -
Reduction: 2 Fe 3+ + 2 e - → 2 Fe 2+
In biochemistry, R and R 'denote the cysteines on the peptide / protein. The two excess hydrogen atoms are bound by a hydrogen acceptor (the notation [H] makes it clear that they are not released as hydrogen gas). They can ultimately be transferred to oxygen , for example .
- 4 [H] + O 2 → 2 H 2 O
Disulfide bridges are inserted into the proteins during translation , if parts of them are already in the endoplasmic reticulum (ER) (in the eukaryotic) , or afterwards, if they are completely in the ER or another membrane-encased cell organelle , what then represents a post-translational modification . In the case of prokaryotes, this applies analogously to translation into the periplasm.
The formation of disulfide bonds is not a spontaneous process; it is a redox reaction that requires an appropriate reaction partner for electron transfer. The formation is enzyme-catalyzed. If a protein also has more than two cysteines, it is possible that linking the “wrong” cysteines will result in disulfide bridges that do not correspond to the native state of the protein. The wrong disulfide bridges must be re-tied ( reshuffling ).
Protein disulfide isomerases
Eukaryotes have protein disulfide isomerases (PDI) in the endoplasmic reticulum (ER ). The progressive folding slowly brings cysteines that belong together into spatial proximity, which makes correct connections increasingly likely.
The prokaryotic counterpart to the protein disulfide isomerases is the periplasmic and inner-membrane-permanent Dsb system (dsb from disulfide-bond), which controls disulfide formation and isomerization.
GSH / GSSG system
Glutathione (GSH) is an isopeptide that is present in the cytoplasm of both prokaryotes and eukaryotes and participates in the formation of disulfide bridges. It reacts in a disulfide exchange reaction:
R and R 'are again the cysteines in the protein backbone, GSSG is the GSH dimer with a disulfide bridge (expressed by the sulfur atoms “SS” written next to each other).
- R-SH + GSSG → RSSG + GSH
The left of the two products is called the mixed disulfide. It will continue to be implemented:
- RSSG + HS-R '→ RSSR' + GSH
In the cytosol it is (enzymatically) kept in a reduced form (GSH). One speaks of “reducing conditions”.
These conditions can be illustrated by the relative concentration ratios of GSH and the corresponding disulfide-bridged dimer GSSG:
The conditions in the ER correspond to the extracellular milieu in the presence of oxygen (the lumen of the ER is topologically equivalent to the outer space).
GSH also plays a role in oxidative stress .
Importance for recombinant protein expression
In eukaryotes, disulfide bridges are formed in the endoplasmic reticulum. However, expression systems are often prokaryotes that do not have an ER. If the protein is translated into the cytosol, no disulfide bridges can form (see GSH / GSSG system).
Formation of inclusion bodies
Without disulfide bridges, the folding of the protein is disturbed. In addition to proteolytic degradation, excessive production (overexpression) of the protein, which is desired for reasons of yield, can lead to the formation of inclusion bodies ( protein aggregation to form the so-called inclusion body ). The interior of the inclusion body is protected from reduction and therefore forms arbitrary disulfide bridges to other proteins. Both misfolding and the formation of inclusion bodies make further work steps necessary for the purification of the protein and in some cases only provide a limited amount of functional protein.
Resolubilization with reducing agents
DTT (dithiothreitol) , mercaptoethanol , tris (2-carboxyethyl) phosphine and DTE (dithioerythritol) are reducing agents for disulfide bonds. They are used in biochemistry to break up the arbitrarily formed disulfide bonds in inclusion bodies.
This is done by reducing them, as shown in the picture. The individual proteins are thereby separated from one another and dissolve again. One speaks of the resolubilization (English soluble = soluble) of the proteins.
Reoxidation with glutathione
The proteins from this process contain only reduced disulfide bonds. In order to get functional protein, the protein must be correctly folded. To do this, the disulfide bonds must be re-established and this in a controlled manner so that only the “desired” cysteine pairs bond with one another.
To achieve this, the proteins are treated with glutathione (GSH). The disulfide bonds are reoxidized to their native state (= "back-oxidation" to the bond state of this protein provided by nature). During this process, various conditions must be adapted and adhered to in order to avoid renewed protein aggregation (see formation of inclusion bodies). To do this, the protein and GSH concentrations, pH value of the solution, temperature and reaction times are varied and optimized.
It is also possible to add folding additives such as arginine, which also support the correct formation of disulfide bonds. With the so-called pulse renaturation , not all of the protein is added to the renaturation solution at the beginning of the renaturation . Instead, after adding small portions, there is a short wait to give the proteins already in the solution time to fold. Folded proteins no longer aggregate, which reduces the risk that unfolded proteins will collide and form inclusion bodies with one another.
Expression in the periplasm
In the periplasm of prokaryotes, unlike in the cytosol, oxidizing conditions prevail. A GSG / GSSG system does not exist here, for the outer membrane is proteins that are smaller than 500 Da, permeable (GSH has a molar mass of only 307.3 g / mol). Oxygen can also diffuse through the outer membrane. The recombinant expression of proteins with disulfide bonds is therefore also being researched with the periplasm as a target.
- Hans-Dieter Jakubke, Hans Jeschkeit: amino acids, peptides, proteins , Verlag Chemie, Weinheim, p 101, 1982, ISBN 3-527-25892-2 .
- Creighton, TE. Protein folding coupled to disulphide bond formation. Biol Chem . 1997 Aug, 378 (8): 731-744.
- W. Thieman, M. Palladino: Biotechnology. Pearson Studium, Germany, 2007, ISBN 978-3-8273-7236-9