Oxidative
protein folding
Protein folding is the physical process by which a protein chain is translated to its native three-dimensional structure, typically a "folded" conformation by which the protein becomes biologically functional. Via an expeditious and reproduc ...
is a process that is responsible for the formation of
disulfide bonds
In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure . The linkage is also called an SS-bond or sometimes a disulfide bridge and is usually derived by the coupling of two thiol groups. In ...
between
cysteine residues in
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s. The driving force behind this process is a
redox reaction
Redox (reduction–oxidation, , ) is a type of chemical reaction in which the oxidation states of substrate change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is the gain of electrons or a d ...
, in which
electrons
The electron ( or ) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family,
and are generally thought to be elementary particles because they have no ...
pass between several proteins and finally to a
terminal electron acceptor
An electron acceptor is a chemical entity that accepts electrons transferred to it from another compound. It is an oxidizing agent that, by virtue of its accepting electrons, is itself reduced in the process. Electron acceptors are sometimes mista ...
.
In prokaryotes
In
prokaryote
A prokaryote () is a single-celled organism that lacks a nucleus and other membrane-bound organelles. The word ''prokaryote'' comes from the Greek πρό (, 'before') and κάρυον (, 'nut' or 'kernel').Campbell, N. "Biology:Concepts & Conne ...
s, the mechanism of oxidative folding is best studied in
Gram-negative bacteria
Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the Gram staining method of bacterial differentiation. They are characterized by their cell envelopes, which are composed of a thin peptidoglycan cell wall ...
. This process is catalysed by protein machinery residing in the
periplasmic space
The periplasm is a concentrated gel-like matrix in the space between the inner cytoplasmic membrane and the bacterial outer membrane called the ''periplasmic space'' in gram-negative bacteria. Using cryo-electron microscopy it has been found tha ...
of bacteria. The formation of disulfide bonds in a protein is made possible by two related pathways: an oxidative pathway, which is responsible for the formation of the disulfides, and an isomerization pathway that shuffles incorrectly formed disulfides.
Oxidative pathway
The oxidative pathway relies, just like the isomerization pathway, on a protein relay. The first member of this protein relay is a small periplasmic protein (21 kDa) called
DsbA
DsbA is a bacterial thiol disulfide oxidoreductase (TDOR). DsbA is a key component of the Dsb (disulfide bond) family of enzymes. DsbA catalyzes intrachain disulfide bond formation as peptides emerge into the cell's periplasm.
Structurally, ...
, which has two cysteine residues that must be oxidized for it to be active. When in its oxidized state, the protein is able to form disulfide bonds between cysteine residues in newly synthesized, and yet unfolded proteins by the transfer of its own disulfide bond onto the folding protein. After the transfer of this disulfide bond, DsbA is in a reduced state. For it to act catalytically again, it must be reoxidized. This is made possible by a 21 kDa
inner membrane protein, called
DsbB
Disulfide bond formation protein B (DsbB) is a protein component of the pathway that leads to disulfide bond formation in periplasmic proteins of ''Escherichia coli'' () and other bacteria. In ''Bacillus subtilis'' it is known as ''BdbC'' ().
T ...
, which has two pairs of cysteine residues. A mixed disulfide is formed between a cysteine residue of DsbB and one of DsbA. Eventually, this cross-link between the two proteins is broken by a
nucleophilic attack
In chemistry, a nucleophile is a chemical species that forms bonds by donating an electron pair. All molecules and ions with a free pair of electrons or at least one pi bond can act as nucleophiles. Because nucleophiles donate electrons, they are ...
of the second cystein residue in the DsbA
active site. On his turn, DsbB is reoxidized by transferring
electron
The electron ( or ) is a subatomic particle with a negative one elementary electric charge. Electrons belong to the first generation of the lepton particle family,
and are generally thought to be elementary particles because they have no ...
s to oxidized
ubiquinone
Coenzyme Q, also known as ubiquinone and marketed as CoQ10, is a coenzyme family that is ubiquitous in animals and most bacteria (hence the name ubiquinone). In humans, the most common form is coenzyme Q10 or ubiquinone-10.
It is a 1,4-benzoq ...
, which passes them to
cytochrome
Cytochromes are redox-active proteins containing a heme, with a central Fe atom at its core, as a cofactor. They are involved in electron transport chain and redox catalysis. They are classified according to the type of heme and its mode of bi ...
oxidases, which finally reduce
oxygen
Oxygen is the chemical element with the symbol O and atomic number 8. It is a member of the chalcogen group in the periodic table, a highly reactive nonmetal, and an oxidizing agent that readily forms oxides with most elements as ...
; this is in
aerobic
Aerobic means "requiring air," in which "air" usually means oxygen.
Aerobic may also refer to
* Aerobic exercise, prolonged exercise of moderate intensity
* Aerobics, a form of aerobic exercise
* Aerobic respiration, the aerobic process of cel ...
conditions. As molecular oxygen serves as the terminal electron acceptor in aerobic conditions, oxidative folding is conveniently coupled to it through the
respiratory chain
An electron transport chain (ETC) is a series of protein complexes and other molecules that transfer electrons from electron donors to electron acceptors via redox reactions (both reduction and oxidation occurring simultaneously) and couples thi ...
. In anaerobic conditions however, DsbB passes its electrons to
menaquinone
Vitamin K2 or menaquinone (MK) () is one of three types of vitamin K, the other two being vitamin K1 (phylloquinone) and K3 ( menadione). K2 is both a tissue and bacterial product (derived from vitamin K1 in both cases) and is usually found in a ...
, followed by a transfer of electrons to
fumarate reductase Fumarate reductase is the enzyme that converts fumarate to succinate, and is important in microbial metabolism as a part of anaerobic respiration.
Succinate + acceptor fumarate + reduced acceptor
Fumarate reductases can be divided into two classe ...
or
nitrate reductase.
Isomerization pathway
Especially for proteins that contain more than one disulfide bond, it is important that incorrect disulfide bonds become rearranged. This is carried out in the isomerization pathway by the protein DsbC, that acts as a disulfide
isomerase
Isomerases are a general class of enzymes that convert a molecule from one isomer to another. Isomerases facilitate intramolecular rearrangements in which bonds are broken and formed. The general form of such a reaction is as follows:
A–B ...
. DsbC is a
dimer
Dimer may refer to:
* Dimer (chemistry), a chemical structure formed from two similar sub-units
** Protein dimer, a protein quaternary structure
** d-dimer
* Dimer model, an item in statistical mechanics, based on ''domino tiling''
* Julius Dimer ...
, consisting of two identical 23 kDa
subunits and has four cysteine residues in each subunit. One of these cysteines (Cys-98) attacks an incorrect disulfide in a misfolded protein and a mixed disulfide is formed between DsbC and this protein. Next, the attack of a second cysteine residue results in the forming of a more stable disulfide in the refolded protein. This may be a cysteine residue either from the earlier misfolded protein or one from DsbC. In the last case, DsbC becomes oxidized and must be reduced in order to play another catalytic role. There is also a second isomerase that can reorganize incorrect disulfide bonds. This protein is called DsbG and it is also a dimer that serves as a
chaperone. To fulfil their role as isomerases, DsbC and DsbG must be kept in a reduced state. This is carried out by DsbD, which must be reduced itself to be functional. Thioredoxin, which itself is reduced by
thioredoxin reductase
Thioredoxin reductases (TR, TrxR) () are enzymes that reduce thioredoxin (Trx). Two classes of thioredoxin reductase have been identified: one class in bacteria and some eukaryotes and one in animals. In bacteria TrxR also catalyzes the reduction ...
and
NADPH, ensures the reduction of the DsbD protein.
Because these two pathways coexist next to each other in the same periplasmic compartment, there must be a mechanism to prevent oxidation of DsbC by DsbB. This mechanism indeed exists as DsbB can distinguish between DsbA and DsbC because this latter has the ability to dimerize.
In eukaryotes
A very similar pathway is followed in
eukaryotes, in which the protein relay consists of proteins with very analogous properties as those of the protein relay in Gram-negative bacteria. However, a major difference between prokaryotes and eukaryotes is found in the fact that the process of oxidative protein folding occurs in the
endoplasmatic reticulum
The endoplasmic reticulum (ER) is, in essence, the transportation system of the eukaryotic cell, and has many other important functions such as protein folding. It is a type of organelle made up of two subunits – rough endoplasmic reticulum ...
(ER) in eukaryotes. A second difference is that in eukaryotes, the use of molecular oxygen as a terminal electron acceptor is not linked to the process of oxidative folding through the respiratory chain as is the case in bacteria. In fact, one of the proteins involved in the oxidative folding process uses a
flavin-dependent reaction to pass electrons directly to molecular oxygen.
A
homolog
In biology, homology is similarity due to shared ancestry between a pair of structures or genes in different taxa. A common example of homologous structures is the forelimbs of vertebrates, where the wings of bats and birds, the arms of prima ...
of DsbA, called
protein disulfide isomerase
Protein disulfide isomerase (), or PDI, is an enzyme in the endoplasmic reticulum (ER) in eukaryotes and the periplasm of bacteria that catalyzes the formation and breakage of disulfide bonds between cysteine residues within proteins as the ...
(PDI), is responsible for the formation of the disulfide bonds in unfolded eukaryotic proteins. This protein has two thioredoxine-like active sites, which both contain two cysteine residues. By transferring the disulfide bond between these two cysteine residues onto the folding protein it is responsible for the latter's oxidation. In contrast to bacteria, where the oxidative and isomerization pathways are carried out by different proteins, PDI is also responsible for the reduction and isomerization of the disulfide bonds. For PDI to catalyse the formation of disulfide bonds in unfolded proteins, it must be reoxidized. This is carried out by an ER membrane-associated protein, Ero1p, which is no homolog of DsbB. This Ero1p protein forms a mixed disulfide with PDI, which is resolved by a nucleophilic attack of the second cystein residue in one of the active sites of PDI. As result, oxidized PDI is obtained. Ero1p itself is oxidized by transferring electrons to molecular oxygen. As it is an
FAD
A fad or trend is any form of collective behavior that develops within a culture, a generation or social group in which a group of people enthusiastically follow an impulse for a short period.
Fads are objects or behaviors that achieve short- ...
-binding protein, this transfer of electrons is strongly favoured when Ero1p is bound to FAD. Also a transport system that imports FAD into the ER lumen has been described in eukaryotes. Furthermore, it has been shown that the ability to reduce or rearrange incorrect disulfide bonds in missfolded proteins is provided by the oxidation of reduced
glutathione
Glutathione (GSH, ) is an antioxidant in plants, animals, fungi, and some bacteria and archaea. Glutathione is capable of preventing damage to important cellular components caused by sources such as reactive oxygen species, free radicals, pe ...
(GSH) to oxidized glutathione (GSSG).
ROS and diseases
Because of the property of Ero1p to transfer electrons directly to molecular oxygen via a flavin-dependent reaction, its activity may produce
reactive oxygen species
In chemistry, reactive oxygen species (ROS) are highly reactive chemicals formed from diatomic oxygen (). Examples of ROS include peroxides, superoxide, hydroxyl radical, singlet oxygen, and alpha-oxygen.
The reduction of molecular oxygen () p ...
(ROS). In bacteria, this problem is solved by coupling oxidative folding to the respiratory chain. There, the reduction of molecular oxygen to
water
Water (chemical formula ) is an Inorganic compound, inorganic, transparent, tasteless, odorless, and Color of water, nearly colorless chemical substance, which is the main constituent of Earth's hydrosphere and the fluids of all known living ...
is carried out by a complex series of proteins, which catalyse this reaction very efficiently. In eukaryotes, the respiratory chain is separated from oxidative folding since
cellular respiration
Cellular respiration is the process by which biological fuels are oxidised in the presence of an inorganic electron acceptor such as oxygen to produce large amounts of energy, to drive the bulk production of ATP. Cellular respiration may be des ...
takes place in the
mitochondria and the formation of disulfide bonds occurs in the ER. Because of this, there is much more risk that ROS are produced in eukaryotic cells during oxidative folding. As is known these ROS may cause many diseases such as
atherosclerosis
Atherosclerosis is a pattern of the disease arteriosclerosis in which the wall of the artery develops abnormalities, called lesions. These lesions may lead to narrowing due to the buildup of atheromatous plaque. At onset there are usually no s ...
and some
neurodegenerative diseases.
Examples
Classical examples of proteins in which the process of oxidative folding is well studied are bovine pancreatic trypsin inhibitor (
BPTI) and
ribonuclease A
Pancreatic ribonuclease family (, ''RNase'', ''RNase I'', ''RNase A'', ''pancreatic RNase'', ''ribonuclease I'', ''endoribonuclease I'', ''ribonucleic phosphatase'', ''alkaline ribonuclease'', ''ribonuclease'', ''gene S glycoproteins'', ''Ceratit ...
(RNaseA). These two proteins have multiple disulfide bonds and so they are very useful to follow and understand the process of oxidative folding. Another example is
alkaline phosphatase, which contains two essential disulfides. It was used as an indicator protein to screen the effect of
mutations in DsbA.
References
* Jean-François Collet and James C. A. Bardwell.(2002). Oxidative protein folding in bacteria. Molecular Microbiology 44, 1-8
* Benjamin P. Tu and Jonathan S. Weissman.(2004). Oxidative protein folding in eukaryotes: mechanisms and consequences. The journal of Cell Biology 164, 341-346
* Benjamin P. Tu, Siew C. Ho-Schleyer, Kevin J. Travers, Jonathan S. Weissman.(2000). Biochemical basis of Oxidative Folding in the Endoplasmatic Reticulum. Science 290, 1571-1574
* Martin Bader, Wilson Muse, David P. Ballou, Christian Gassner and James C. A. Bardwell.(1999). Oxidative Protein Folding Is Driven by the Electron Transport System. Cell 98, 217-227
* Lawrence K. Low, Hang-Cheol Shin and Harold A. Sheraga.(2002). Oxidative Folding of Bovine Pancreatic Ribonuclease A: Insight into the Overall Catalysis of the Refolding Pathway by Phosphate. Journal of Protein Chemistry 21, 19-27
Protein structure