Disulfide bond

A disulfide bond, disulfide bond or disulfide bond in chemistry is a covalent bond between two sulfur atoms of which each single free valence is saturated with a organyl. In biochemistry, the disulfide bond is covalent bond ( an atomic bonding) between the sulfur atoms of two cysteine ​​molecules are present in the amino acid side chain of a protein.

Two linked by disulfide cysteine ​​residues in proteins is referred to here as a cystine bridge.

• 3.1 reaction
• 3.2 time

• 4.1 protein disulfide
• 4.2 thio -disulfide oxidoreductases

• 6.1 the formation of inclusion bodies
• 6.2 resolubilization with dithiothreitol and dithioerythritol
• 6.3 reoxidation with glutathione
• 6.4 expression into the periplasm

Function

Form disulfide bonds and stabilize the protein three-dimensional structure ( tertiary structure ) by the formation of loops within the amino acid chain or a plurality of amino acid chains linked to a functional protein. The covalent bond acts clearly fixative, as for example, a dimer formation due to non-covalent interactions, such as van der Waals forces.

The formation of disulfide bridges may represent a separate step in the folding of a protein, without which subsequent folding steps delayed.

Examples

Disulfide bonds are typical of secretory proteins since they can not form in the cytosol.

Insulin

Thus, the proteohormone insulin from two different chains of amino acids, referred to as A- and B - chain, the A chain is linked by a intrachenar interchenar and with the B-chain by two disulfide bonds.

Lipase

A lipase, a lipolytic enzyme from pancreas (pancreas) of the pig has seven disulfide bonds.

GPCRs

G- protein-coupled receptors are membrane-bound proteins that are stabilized by a disulfide bond between the third transmembrane helix and the second extracellular loop. Mutational studies demonstrate that the receptor is not expressed when one of the cysteines involved is mutated to serine.

Bond formation

Reaction

The involved in the formation of a disulfide bond functional groups is called thiol ( mercapto ). In simple terms, the formation of such a SS bond as oxidation (release of hydrogen or electrons) understand:

Oxidation: R -SH HS -R '→ RSSR ' 2H 2e -

Reduction: 2Fe3 2e - → 2Fe2

R and R ' denote the cysteines in biochemistry at the peptide / protein. The two excess hydrogen atoms are bound by a hydrogen acceptor ( The notation [H ] shows that they are not released as hydrogen gas). You can ultimately be transferred, for example, oxygen.

Time

Disulfide bridges are still inserted during translation into proteins when parts are by them in respect of their synthesis already in ( the eukaryotes ) endoplasmic reticulum ( ER), or afterwards, when they completely reside in the ER or other membrane-enclosed organelle, which then represents a post-translational modification. In prokaryotes, this similarly applies for translation into the periplasm.

Enzymes

The formation of disulfide bonds is not a spontaneous process. It is a redox reaction, which requires a corresponding reaction partner to the electron transfer. The training is enzyme-catalyzed. Has a protein also has more than two cysteines, then there is the possibility that the "wrong" by linking the cysteine ​​disulfide bridges found that do not correspond to the native state of the protein. There must be a Umknüpfung incorrect disulfide bonds (English reshuffling ) take place.

Protein disulfide

Eukaryotes have in the endoplasmic reticulum protein disulfide (PDI). The progressive folding leads belong together slowly cysteines in close proximity, making correct connections increasingly likely.

Thio -disulfide oxidoreductases

The prokaryotic counterpart of the protein disulfide is the periplasmic and inner membrane constant Dsb system ( dsb of di - sulfide -bond ), the disulfide formation and isomerization controlled.

GSH / GSSG system

Glutathione (GSH) is an isopeptide that is present in the cytoplasm of both prokaryotic and eukaryotic cells and participates in the formation of disulfide bridges. It reacts in a disulfide exchange reaction:

R and R ' are, in turn, the cysteines in the protein backbone is the GSSG GSH dimer with disulfide bond (as indicated by the adjacent written sulfur atoms "SS" ).

The left of the two products is called a mixed disulfide. It is further reacted:

In the cytosol it is ( enzymatic) kept in the 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 ratios in the ER correspond to the extracellular milieu in the presence of oxygen ( the lumen of the ER is topologically equivalent to the exterior ).

GSH also plays a role in oxidative stress.

Importance for recombinant protein expression

Disulfide bridges in proteins limit their ability recombinant expression, ie their biotechnological production.

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum. However, expression systems are commonly prokaryotes, which do not have the ER. Is the translated protein into the cytosol, no disulfide bonds may occur (see the GSH / GSSG system).

Formation of inclusion bodies

Without the disulfide bonds of the protein folding is disturbed. In addition to the proteolytic degradation, it can be especially desired when, for reasons of efficiency, excessive production ( over-expression ) of the protein to form inclusion bodies come ( so-called inclusion body of protein aggregation ). Here, the inclusion body is protected from reduction in the interior thereof and therefore forms disulfide bonds with other proteins from random. Both misfolding and the formation of inclusion bodies make further steps in the purification of the protein and provide necessary part only limited functional protein.

Resolubilization with dithiothreitol and dithioerythritol

DTT ( dithiothreitol ) and DTE ( dithioerythritol ) are reducing agent for disulfide bonds. They are used in molecular biology in order to dissolve the arbitrarily formed disulfide bonds again in the inclusion bodies.

This is the same as shown in the picture by reduction. The individual proteins are thus separated from each other and dissolve again. One speaks of the resolubilization ( engl. = soluble soluble) of the proteins.

Reoxidation with glutathione

The proteins from this process contain only reduced disulfide bonds. In order to obtain a functional protein, the protein must be correctly folded. These disulfide bonds must be formed again and this in a controlled manner, so that only the " desired " cysteine ​​pairs binding enter another.

To achieve this, the proteins are added to glutathione ( GSH). There is a reoxidation of the disulfide bonds in the native state ( = " Back - oxidation " to the intended nature of the bonding state of this protein ). In this process, several conditions have to be adapted and adhered to (see formation of inclusion bodies ) to avoid re- aggregation of the protein. To protein and GSH concentrations, pH value of the solution, temperature, and reaction times can be varied and optimized.

In addition, it is possible to add additives such as convolutional arginine, supporting the correct Disulfidbindungsausbildung also. In the so-called pulse renaturation the entire protein is added to the renaturation solution is not right at the beginning of the restoration. Instead, wait a little after the addition of small portions to give the already in the solution time for folding proteins. Folded proteins do not aggregate more, reducing the risk that unfolded proteins meet and together form inclusion bodies is reduced.

Expression into the periplasm

In the periplasm of prokaryotes reign, unlike in the cytosol, oxidizing conditions. A GSG / GSSG system there is not, as the outer membrane proteins that are smaller than 500 Da, permeable ( GSH has a molar mass of only 307.3 g / mol). Through the outer membrane and oxygen may diffuse. Recombinant expression of proteins with disulfide bonds is therefore researched with the periplasm as a destination.