Disulfide

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Disulfide

In biochemistry, a disulfide (or disulphide in British English) refers to a functional group with the structure R−S−S−R′. 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 biology, disulfide bridges formed between thiol groups in two cysteine residues are an important component of the secondary and tertiary structure of proteins. Persulfide usually refers to R−S−S−H compounds.

In inorganic chemistry disulfide usually refers to the corresponding anion S²⁻
₂ (⁻S−S⁻).

Organic disulfides

A selection of disulfides:

 

Pyrite, FeS₂, "fool's gold"

 

Disulfur dichloride, S₂Cl₂, a common industrial chemical

 

cystine, crosslinker in many proteins

lipoic acid, a vitamin

Diphenyl disulfide, Ph₂S₂, a common organic disulfide

Symmetrical disulfides are compounds of the formula R₂S₂. Most disulfides encountered in organo sulfur chemistry are symmetrical disulfides. Unsymmetrical disulfides (also called heterodisulfides) are compounds of the formula RSSR'. They are less common in organic chemistry, but most disulfides in nature are unsymmetrical.

Properties

The disulfide bonds are strong, with a typical bond dissociation energy of 60 kcal/mol (251 kJ mol⁻¹). However, being about 40% weaker than C−C and C−H bonds, the disulfide bond is often the "weak link" in many molecules. Furthermore, reflecting the polarizability of divalent sulfur, the S−S bond is susceptible to scission by polar reagents, both electrophiles and especially nucleophiles (Nu):

${\displaystyle \text{RS}-\text{SR}+{\text{Nu}}^{-}⟶\text{RS}-\text{Nu}+{\text{RS}}^{-}}$

The disulfide bond is about 2.05 Å in length, about 0.5 Å longer than a C−C bond. Rotation about the S−S axis is subject to a low barrier. Disulfides show a distinct preference for dihedral angles approaching 90°. When the angle approaches 0° or 180°, then the disulfide is a significantly better oxidant.

Disulfides where the two R groups are the same are called symmetric, examples being diphenyl disulfide and dimethyl disulfide. When the two R groups are not identical, the compound is said to be an asymmetric or mixed disulfide.

Although the hydrogenation of disulfides is usually not practical, the equilibrium constant for the reaction provides a measure of the standard redox potential for disulfides:

${\displaystyle \text{RSSR}+{\text{H}}_2^{}⟶2\text{RSH}}$

This value is about −250 mV versus the standard hydrogen electrode (pH = 7). By comparison, the standard reduction potential for ferrodoxins is about −430 mV.

Synthesis

Disulfide bonds are usually formed from the oxidation of sulfhydryl (−SH) groups, especially in biological contexts. The transformation is depicted as follows:

${\displaystyle 2\text{RSH}\stackrel{-\rightharpoonup }{\leftharpoondown -}\text{RS}-\text{SR}+2{\text{H}}^{+}+2{\text{e}}^{-}}$

A variety of oxidants participate in this reaction including oxygen and hydrogen peroxide. Such reactions are thought to proceed via sulfenic acid intermediates. In the laboratory, iodine in the presence of base is commonly employed to oxidize thiols to disulfides. Several metals, such as copper(II) and iron(III) complexes affect this reaction. Alternatively, disulfide bonds in proteins often formed by thiol-disulfide exchange:

${\displaystyle \text{RS}-\text{SR}+{\text{R}}^{\prime }\text{SH}\stackrel{-\rightharpoonup }{\leftharpoondown -}{\text{R}}^{\prime }\text{S}-\text{SR}+\text{RSH}}$

Such reactions are mediated by enzymes in some cases and in other cases are under equilibrium control, especially in the presence of a catalytic amount of base.

The alkylation of alkali metal di- and polysulfides gives disulfides. "Thiokol" polymers arise when sodium polysulfide is treated with an alkyl dihalide. In the converse reaction, carbanionic reagents react with elemental sulfur to afford mixtures of the thioether, disulfide, and higher polysulfides. These reactions are often unselective but can be optimized for specific applications.

Synthesis of unsymmetrical disulfides (heterodisulfides)

Many specialized methods have been developed for forming unsymmetrical disulfides. Reagents that deliver the equivalent of "RS⁺" react with thiols to give asymmetrical disulfides:

${\displaystyle \text{RSH}+{\text{R}}^{\prime }{\text{SNR}}_2^{″}⟶\text{RS}-{\text{SR}}^{\prime }+{\text{HNR}}_2^{″}}$

where R″₂N is the phthalimido group. Bunte salts, derivatives of the type RSSO−3Na⁺are also used to generate unsymmetrical disulfides:

${\displaystyle \text{Na}[{\text{O}}_3^{}{\text{S}}_2^{}\text{R}]+{\text{NaSR}}^{\prime }⟶{\text{RSSR}}^{\prime }+{\text{Na}}_2^{}{\text{SO}}_3^{}}$

Reactions

The most important aspect of disulfide bonds is their cleavage, which occurs via reduction. A variety of reductants can be used. In biochemistry, thiols such as β-mercaptoethanol (β-ME) or dithiothreitol (DTT) serve as reductants; the thiol reagents are used in excess to drive the equilibrium to the right:

${\displaystyle \text{RS}-\text{SR}+2{\text{HOCH}}_2^{}{\text{CH}}_2^{}\text{SH}\stackrel{-\rightharpoonup }{\leftharpoondown -}{\text{HOCH}}_2^{}{\text{CH}}_2^{}\text{S}-{\text{SCH}}_2^{}{\text{CH}}_2^{}\text{OH}+2\text{RSH}}$

The reductant tris(2-carboxyethyl)phosphine (TCEP) is useful, beside being odorless compared to β-ME and DTT, because it is selective, working at both alkaline and acidic conditions (unlike DTT), is more hydrophilic and more resistant to oxidation in air. Furthermore, it is often not needed to remove TCEP before modification of protein thiols.

In organic synthesis, hydride agents are typically employed for scission of disulfides, such as sodium borohydride. More aggressive, alkali metals will effect this reaction:

${\displaystyle \text{RS}-\text{SR}+2\text{Na}⟶2\text{NaSR}}$

These reactions are often followed by protonation of the resulting metal thiolate:

${\displaystyle \text{NaSR}+\text{HCl}⟶\text{HSR}+\text{NaCl}}$

Thiol–disulfide exchange is a chemical reaction in which a thiolate group −S⁻ attacks a sulfur atom of a disulfide bond −S−S−. The original disulfide bond is broken, and its other sulfur atom is released as a new thiolate, carrying away the negative charge. Meanwhile, a new disulfide bond forms between the attacking thiolate and the original sulfur atom.

Thiol–disulfide exchange showing the linear intermediate in which the charge is shared among the three sulfur atoms. The thiolate group (shown in red) attacks a sulfur atom (shown in blue) of the disulfide bond, displacing the other sulfur atom (shown in green) and forming a new disulfide bond.

Thiolates, not thiols, attack disulfide bonds. Hence, thiol–disulfide exchange is inhibited at low pH (typically, below 8) where the protonated thiol form is favored relative to the deprotonated thiolate form. (The pK_a of a typical thiol group is roughly 8.3, but can vary due to its environment.)

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 (known as disulfide shuffling) does not change the number of disulfide bonds within a protein, merely their location (i.e., which cysteines are bonded). 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 or 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 (i.e., next to each other), 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.

Many specialized organic reactions have been developed for disulfides, again mainly associated with the scission of the S−S bond, which is usually the weakest bond in a molecule. In the Zincke disulfide cleavage reactions, disulfides are cleaved by halogens. This reaction gives a sulfenyl halide:

${\displaystyle \text{ArSSAr}+{\text{Cl}}_2^{}⟶2\text{ArSCl}}$

Occurrence in biology

Schematic of disulfide bonds crosslinking regions of a protein

Occurrence 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.

Cystine is composed of two cysteines linked by a disulfide bond (shown here in its neutral form).

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 χ_ss dihedral angle between the C^β−S^γ−S^γ−C^β atoms, which is usually close to ±90°.

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

1. 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.
2. 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.
3. Related to 1 and 2, the disulfide bond links two segments of the protein chain, 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 formation of a disulfide bond between them.

A disulfide species is a particular pairing of cysteines in a disulfide-bonded protein and is usually depicted by listing the disulfide bonds in parentheses, e.g., the "(26–84, 58–110) disulfide species". A disulfide ensemble is a grouping of all disulfide species with the same number of disulfide bonds, and is usually denoted as the 1S ensemble, the 2S ensemble, etc. for disulfide species having one, two, etc. disulfide bonds. Thus, the (26–84) disulfide species belongs to the 1S ensemble, whereas the (26–84, 58–110) species belongs to the 2S ensemble. The single species with no disulfide bonds is usually denoted as R for "fully reduced". Under typical conditions, disulfide reshuffling is much faster than the formation of new disulfide bonds or their reduction; hence, the disulfide species within an ensemble equilibrate more quickly than between ensembles.

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.

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 (H₂O₂) 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 (rough endoplasmic reticulum) 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 (see glutathione). 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 proteolysis 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 (TCSs), 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.

Inorganic disulfides

The disulfide anion is S²⁻
₂, or ⁻S−S⁻. In disulfide, sulfur exists in the reduced state with oxidation number −1. Its electron configuration then resembles that of a chlorine atom. It thus tends to form a covalent bond with another S⁻ center to form S²⁻
₂ group, similar to elemental chlorine existing as the diatomic Cl₂. Oxygen may also behave similarly, e.g. in peroxides such as H₂O₂. Examples:

• Hydrogen disulfide (S₂H₂), the simplest inorganic disulfide
• Disulfur dichloride (S₂Cl₂), a distillable liquid.
• Iron disulfide (FeS₂), or pyrite.

Related compounds

CS₂

 

MoS₂

Thiosulfoxides are orthogonally isomeric with disulfides, having the second sulfur branching from the first and not partaking in a continuous chain, i.e. >S=S rather than −S−S−.

Disulfide bonds are analogous but more common than related peroxide, thioselenide, and diselenide bonds. Intermediate compounds of these also exist, for example thioperoxides (also known as oxasulfides) such as hydrogen thioperoxide, have the formula R¹OSR² (equivalently R²SOR¹). These are isomeric to sulfoxides in a similar manner to the above; i.e. >S=O rather than −S−O−.

Thiuram disulfides, with the formula (R₂NCSS)₂, are disulfides but they behave distinctly because of the thiocarbonyl group.

Compounds with three sulfur atoms, such as CH₃S−S−SCH₃, are called trisulfides, or trisulfide bonds.

Misnomers

Disulfide is also used to refer to compounds that contain two sulfide (S²⁻) centers. The compound carbon disulfide, CS₂ is described with the structural formula i.e. S=C=S. This molecule is not a disulfide in the sense that it lacks a S-S bond. Similarly, molybdenum disulfide, MoS₂, is not a disulfide in the sense again that its sulfur atoms are not linked.

Applications

Rubber manufacturing

The vulcanization of rubber results in crosslinking groups which consist of disulfide (and polysulfide) bonds; in analogy to the role of disulfides in proteins, the S−S linkages in rubber strongly affect the stability and rheology of the material. Although the exact mechanism underlying the vulcanization process is not entirely understood (as multiple reaction pathways are present but the predominant one is unknown), it has been extensively shown that the extent to which the process is allowed to proceed determines the physical properties of the resulting rubber- namely, a greater degree of crosslinking corresponds to a stronger and more rigid material. The current conventional methods of rubber manufacturing are typically irreversible, as the unregulated reaction mechanisms can result in complex networks of sulfide linkages; as such, rubber is considered to be a thermoset material.

Covalent adaptable networks

Due to their relatively weak bond dissociation energy (in comparison to C−C bonds and the like), disulfides have been employed in covalent adaptable network (CAN) systems in order to allow for dynamic breakage and reformation of crosslinks. By incorporating disulfide functional groups as crosslinks between polymer chains, materials can be produced which are stable at room temperature while also allowing for reversible crosslink dissociation upon application of elevated temperature. The mechanism behind this reaction can be attributed to the cleavage of disulfide linkages (RS−SR) into thiyl radicals (2 RS•) which can subsequently reassociate into new bonds, resulting in reprocessability and self-healing characteristics for the bulk material. However, since the bond dissociation energy of the disulfide bond is still fairly high, it is typically necessary to augment the bond with adjacent chemistry that can stabilize the unpaired electron of the intermediate state. As such, studies usually employ aromatic disulfides or disulfidediamine (RNS−SNR) functional groups to encourage the dynamic dissociation of the S−S bond; these chemistries can result in the bond dissociation energy being reduced to half (or even less) of its prior magnitude.

In practical terms, disulfide-containing CANs can be used to impart recyclability to polymeric materials while still exhibiting physical properties similar to that of thermosets. Typically, recyclability is restricted to thermoplastic materials, as said materials consist of polymer chains which are not bonded to each other at the molecular level; as a result, they can be melted down and reformed (as the addition of thermal energy allows the chains to untangle, move past each other, and adopt new configurations), but this comes at the expense of their physical robustness. Meanwhile, conventional thermosets contain permanent crosslinks which bolster their strength, toughness, creep resistance, and the like (as the bonding between chains provides resistance to deformation at the macroscopic level), but due to the permanence of said crosslinks, these materials cannot be reprocessed akin to thermoplastics. However, due to the dynamic nature of the crosslinks in disulfide CANs, they can be designed to exhibit the best attributes of both of the aforementioned material types. Studies have shown that disulfide CANs can be reprocessed multiple times with negligible degradation in performance while also exhibiting creep resistance, glass transition, and dynamic modulus values comparable to those observed in similar conventional thermoset systems.

Translation (biology)

From Wikipedia, the free encyclopedia
https://en.wikipedia.org/wiki/Translation_(biology)

Overview of the translation of eukaryotic messenger RNA

 

Diagram showing the translation of mRNA and the synthesis of proteins by a ribosome

 

Initiation and elongation stages of translation as seen through zooming in on the nitrogenous bases in RNA, the ribosome, the tRNA, and amino acids, with short explanations.

 

The three phases of translation initiation polymerase bind to the DNA strand and move along until the small ribosomal subunit binds to the DNA. Elongation is initiated when the large subunit attaches and termination ends the process of elongation.

In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.

In translation, messenger RNA (mRNA) is decoded in a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later folds into an active protein and performs its functions in the cell. The ribosome facilitates decoding by inducing the binding of complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome.

Translation proceeds in three phases:

1. Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached at the start codon.
2. Elongation: The last tRNA validated by the small ribosomal subunit (accommodation) transfers the amino acid. It carries to the large ribosomal subunit which binds it to the one of the preceding admitted tRNA (transpeptidation). The ribosome then moves to the next mRNA codon to continue the process (translocation), creating an amino acid chain.
3. Termination: When a stop codon is reached, the ribosome releases the polypeptide. The ribosomal complex remains intact and moves on to the next mRNA to be translated.

In prokaryotes (bacteria and archaea), translation occurs in the cytosol, where the large and small subunits of the ribosome bind to the mRNA. In eukaryotes, translation occurs in the cytoplasm or across the membrane of the endoplasmic reticulum in a process called co-translational translocation. In co-translational translocation, the entire ribosome/mRNA complex binds to the outer membrane of the rough endoplasmic reticulum (ER), and the new protein is synthesized and released into the ER; the newly created polypeptide can be stored inside the ER for future vesicle transport and secretion outside the cell, or immediately secreted.

Many types of transcribed RNA, such as transfer RNA, ribosomal RNA, and small nuclear RNA, do not undergo a translation into proteins.

Several antibiotics act by inhibiting translation. These include anisomycin, cycloheximide, chloramphenicol, tetracycline, streptomycin, erythromycin, and puromycin. Prokaryotic ribosomes have a different structure from that of eukaryotic ribosomes, and thus antibiotics can specifically target bacterial infections without any harm to a eukaryotic host's cells.

Basic mechanisms

Further information: Bacterial translation, Archaeal translation, and Eukaryotic translation

 

A ribosome translating a protein that is secreted into the endoplasmic reticulum. tRNAs are colored dark blue.

 

Tertiary structure of tRNA. CCA tail in yellow, Acceptor stem in purple, Variable loop in orange, D arm in red, Anticodon arm in blue with Anticodon in black, T arm in green.

The basic process of protein production is the addition of one amino acid at a time to the end of a protein. This operation is performed by a ribosome. A ribosome is made up of two subunits, a small subunit, and a large subunit. These subunits come together before the translation of mRNA into a protein to provide a location for translation to be carried out and a polypeptide to be produced. The choice of amino acid type to add is determined by an mRNA molecule. Each amino acid added is matched to a three-nucleotide subsequence of the mRNA. For each such triplet possible, the corresponding amino acid is accepted. The successive amino acids added to the chain are matched to successive nucleotide triplets in the mRNA. In this way, the sequence of nucleotides in the template mRNA chain determines the sequence of amino acids in the generated amino acid chain. The addition of an amino acid occurs at the C-terminus of the peptide; thus, translation is said to be amine-to-carboxyl directed.

The mRNA carries genetic information encoded as a ribonucleotide sequence from the chromosomes to the ribosomes. The ribonucleotides are "read" by translational machinery in a sequence of nucleotide triplets called codons. Each of those triplets codes for a specific amino acid.

The ribosome molecules translate this code to a specific sequence of amino acids. The ribosome is a multisubunit structure containing rRNA and proteins. It is the "factory" where amino acids are assembled into proteins. tRNAs are small noncoding RNA chains (74–93 nucleotides) that transport amino acids to the ribosome. The repertoire of tRNA genes varies widely between species, with some Bacteria having between 20 and 30 genes while complex eukaryotes could have thousands. tRNAs have a site for amino acid attachment, and a site called an anticodon. The anticodon is an RNA triplet complementary to the mRNA triplet that codes for their cargo amino acid.

Aminoacyl tRNA synthetases (enzymes) catalyze the bonding between specific tRNAs and the amino acids that their anticodon sequences call for. The product of this reaction is an aminoacyl-tRNA. In bacteria, this aminoacyl-tRNA is carried to the ribosome by EF-Tu, where mRNA codons are matched through complementary base pairing to specific tRNA anticodons. Aminoacyl-tRNA synthetases that mispair tRNAs with the wrong amino acids can produce mischarged aminoacyl-tRNAs, which can result in inappropriate amino acids at the respective position in the protein. This "mistranslation" of the genetic code naturally occurs at low levels in most organisms, but certain cellular environments cause an increase in permissive mRNA decoding, sometimes to the benefit of the cell.

The ribosome has two binding sites for tRNA. They are the aminoacyl site (abbreviated A), and the peptidyl site/ exit site (abbreviated P/E). Concerning the mRNA, the three sites are oriented 5’ to 3’ E-P-A, because ribosomes move toward the 3' end of mRNA. The A-site binds the incoming tRNA with the complementary codon on the mRNA. The P/E-site holds the tRNA with the growing polypeptide chain. When an aminoacyl-tRNA initially binds to its corresponding codon on the mRNA, it is in the A site. Then, a peptide bond forms between the amino acid of the tRNA in the A site and the amino acid of the charged tRNA in the P/E site. The growing polypeptide chain is transferred to the tRNA in the A site. Translocation occurs, moving the tRNA to the P/E site, now without an amino acid; the tRNA that was in the A site, now charged with the polypeptide chain, is moved to the P/E site and the tRNA leaves, and nothinger aminoacyl-tRNA enters the A site to repeat the process.

After the new amino acid is added to the chain, and after the tRNA is released out of the ribosome and into the cytosol, the energy provided by the hydrolysis of a GTP bound to the translocase EF-G (in bacteria) and a/eEF-2 (in eukaryotes and archaea) moves the ribosome down one codon towards the 3' end. The energy required for translation of proteins is significant. For a protein containing n amino acids, the number of high-energy phosphate bonds required to translate it is 4n-1. The rate of translation varies; it is significantly higher in prokaryotic cells (up to 17–21 amino acid residues per second) than in eukaryotic cells (up to 6–9 amino acid residues per second).

Even though the ribosomes are usually considered accurate and processive machines, the translation process is subject to errors that can lead either to the synthesis of erroneous proteins or to the premature abandonment of translation, either because a tRNA couples to a wrong codon or because a tRNA is coupled to the wrong amino acid. The rate of error in synthesizing proteins has been estimated to be between 1 in 10⁵ and 1 in 10³ misincorporated amino acids, depending on the experimental conditions. The rate of premature translation abandonment, instead, has been estimated to be of the order of magnitude of 10⁻⁴ events per translated codon. The correct amino acid is covalently bonded to the correct transfer RNA (tRNA) by amino acyl transferases. The amino acid is joined by its carboxyl group to the 3' OH of the tRNA by an ester bond. When the tRNA has an amino acid linked to it, the tRNA is termed "charged". Initiation involves the small subunit of the ribosome binding to the 5' end of mRNA with the help of initiation factors (IF). In bacteria and a minority of archaea, initiation of protein synthesis involves the recognition of a purine-rich initiation sequence on the mRNA called the Shine-Dalgarno sequence. The Shine-Dalgarno sequence binds to a complementary pyrimidine-rich sequence on the 3' end of the 16S rRNA part of the 30S ribosomal subunit. The binding of these complementary sequences ensures that the 30S ribosomal subunit is bound to the mRNA and is aligned such that the initiation codon is placed in the 30S portion of the P-site. Once the mRNA and 30S subunit are properly bound, an initiation factor brings the initiator tRNA-amino acid complex, f-Met-tRNA, to the 30S P site. The initiation phase is completed once a 50S subunit joins the 30 subunit, forming an active 70S ribosome. Termination of the polypeptide occurs when the A site of the ribosome is occupied by a stop codon (UAA, UAG, or UGA) on the mRNA, creating the primary structure of a protein. tRNA usually cannot recognize or bind to stop codons. Instead, the stop codon induces the binding of a release factor protein (RF1 & RF2) that prompts the disassembly of the entire ribosome/mRNA complex by the hydrolysis of the polypeptide chain from the peptidyl transferase center of the ribosome. Drugs or special sequence motifs on the mRNA can change the ribosomal structure so that near-cognate tRNAs are bound to the stop codon instead of the release factors. In such cases of 'translational readthrough', translation continues until the ribosome encounters the next stop codon.

The process of translation is highly regulated in both eukaryotic and prokaryotic organisms. Regulation of translation can impact the global rate of protein synthesis which is closely coupled to the metabolic and proliferative state of a cell. In addition, recent work has revealed that genetic differences and their subsequent expression as mRNAs can also impact translation rate in an RNA-specific manner.

Clinical significance

Translational control is critical for the development and survival of cancer. Cancer cells must frequently regulate the translation phase of gene expression, though it is not fully understood why translation is targeted over steps like transcription. While cancer cells often have genetically altered translation factors, it is much more common for cancer cells to modify the levels of existing translation factors. Several major oncogenic signaling pathways, including the RAS–MAPK, PI3K/AKT/mTOR, MYC, and WNT–β-catenin pathways, ultimately reprogram the genome via translation. Cancer cells also control translation to adapt to cellular stress. During stress, the cell translates mRNAs that can mitigate the stress and promote survival. An example of this is the expression of AMPK in various cancers; its activation triggers a cascade that can ultimately allow the cancer to escape apoptosis (programmed cell death) triggered by nutrition deprivation. Future cancer therapies may involve disrupting the translation machinery of the cell to counter the downstream effects of cancer.

Mathematical modeling of translation

Figure M0. Basic and the simplest model M0 of protein synthesis. Here, *M – amount of mRNA with translation initiation site not occupied by assembling ribosome, *F – amount of mRNA with translation initiation site occupied by assembling ribosome, *R – amount of ribosomes sitting on mRNA synthesizing proteins, *P – amount of synthesized proteins.

 

Figure M1'. The extended model of protein synthesis M1 with explicit presentation of 40S, 60S and initiation factors (IF) binding.

The transcription-translation process description, mentioning only the most basic ”elementary” processes, consists of:

1. production of mRNA molecules (including splicing),
2. initiation of these molecules with help of initiation factors (e.g., the initiation can include the circularization step though it is not universally required),
3. initiation of translation, recruiting the small ribosomal subunit,
4. assembly of full ribosomes,
5. elongation, (i.e. movement of ribosomes along mRNA with production of protein),
6. termination of translation,
7. degradation of mRNA molecules,
8. degradation of proteins.

The process of amino acid building to create protein in translation is a subject of various physic models for a long time starting from the first detailed kinetic models such as or others taking into account stochastic aspects of translation and using computer simulations. Many chemical kinetics-based models of protein synthesis have been developed and analyzed in the last four decades. Beyond chemical kinetics, various modeling formalisms such as Totally Asymmetric Simple Exclusion Process (TASEP),^[23]Probabilistic Boolean Networks (PBN), Petri Nets and max-plus algebra have been applied to model the detailed kinetics of protein synthesis or some of its stages. A basic model of protein synthesis that takes into account all eight 'elementary' processes has been developed, following the paradigm that "useful models are simple and extendable". The simplest model M0 is represented by the reaction kinetic mechanism (Figure M0). It was generalised to include 40S, 60S and initiation factors (IF) binding (Figure M1'). It was extended further to include effect of microRNA on protein synthesis. Most of models in this hierarchy can be solved analytically. These solutions were used to extract 'kinetic signatures' of different specific mechanisms of synthesis regulation.

Genetic code

Main article: Genetic code

It is also possible to translate either by hand (for short sequences) or by computer (after first programming one appropriately, see section below); this allows biologists and chemists to draw out the chemical structure of the encoded protein on paper.

First, convert each template DNA base to its RNA complement (note that the complement of A is now U), as shown below. Note that the template strand of the DNA is the one the RNA is polymerized against; the other DNA strand would be the same as the RNA, but with thymine instead of uracil.

DNA -> RNA
 A  ->  U
 T  ->  A
 C  ->  G
 G  ->  C
 A=T-> A=U

Then split the RNA into triplets (groups of three bases). Note that there are 3 translation "windows", or reading frames, depending on where you start reading the code. Finally, use the table at Genetic code to translate the above into a structural formula as used in chemistry.

This will give you the primary structure of the protein. However, proteins tend to fold, depending in part on hydrophilic and hydrophobic segments along the chain. Secondary structure can often still be guessed at, but the proper tertiary structure is often very hard to determine.

Whereas other aspects such as the 3D structure, called tertiary structure, of protein can only be predicted using sophisticated algorithms, the amino acid sequence, called primary structure, can be determined solely from the nucleic acid sequence with the aid of a translation table.

This approach may not give the correct amino acid composition of the protein, in particular if unconventional amino acids such as selenocysteine are incorporated into the protein, which is coded for by a conventional stop codon in combination with a downstream hairpin (SElenoCysteine Insertion Sequence, or SECIS).

There are many computer programs capable of translating a DNA/RNA sequence into a protein sequence. Normally this is performed using the Standard Genetic Code, however, few programs can handle all the "special" cases, such as the use of the alternative initiation codons which are biologically significant. For instance, the rare alternative start codon CTG codes for Methionine when used as a start codon, and for Leucine in all other positions.

Example: Condensed translation table for the Standard Genetic Code (from the NCBI Taxonomy webpage).

 AAs    = FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG
 Starts = ---M---------------M---------------M----------------------------
 Base1  = TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG
 Base2  = TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG
 Base3  = TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG

The "Starts" row indicate three start codons, UUG, CUG, and the very common AUG. It also indicates the first amino acid residue when interpreted as a start: in this case it is all methionine.

Translation tables

Main articles: List of genetic codes and Genetic code § List of alternative codons

Even when working with ordinary eukaryotic sequences such as the Yeast genome, it is often desired to be able to use alternative translation tables—namely for translation of the mitochondrial genes. Currently the following translation tables are defined by the NCBI Taxonomy Group for the translation of the sequences in GenBank:

1. The standard code
2. The vertebrate mitochondrial code
3. The yeast mitochondrial code
4. The mold, protozoan, and coelenterate mitochondrial code and the mycoplasma/spiroplasma code
5. The invertebrate mitochondrial code
6. The ciliate, dasycladacean and hexamita nuclear code
7. The kinetoplast code
9. The echinoderm and flatworm mitochondrial code
10. The euplotid nuclear code
11. The bacterial, archaeal and plant plastid code
12. The alternative yeast nuclear code
13. The ascidian mitochondrial code
14. The alternative flatworm mitochondrial code
15. The Blepharisma nuclear code
16. The chlorophycean mitochondrial code
21. The trematode mitochondrial code
22. The Scenedesmus obliquus mitochondrial code
23. The Thraustochytrium mitochondrial code
24. The Pterobranchia mitochondrial code
25. The candidate division SR1 and gracilibacteria code
26. The Pachysolen tannophilus nuclear code
27. The karyorelict nuclear code
28. The Condylostoma nuclear code
29. The Mesodinium nuclear code
30. The peritrich nuclear code
31. The Blastocrithidia nuclear code
33. The Cephalodiscidae mitochondrial code

Opioid peptide

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Opioid_peptide 

 

Vertebrate endogenous opioids neuropeptide
Identifiers
Symbol	Opiods_neuropep
Pfam	PF01160
InterPro	IPR006024
PROSITE	PDOC00964

Available protein structures:

Structural correlation between met-enkephalin, an opioid peptide, (left) and morphine, an opiate drug, (right)

Opioid peptides or opiate peptides are peptides that bind to opioid receptors in the brain; opiates and opioids mimic the effect of these peptides. Such peptides may be produced by the body itself, for example endorphins. The effects of these peptides vary, but they all resemble those of opiates. Brain opioid peptide systems are known to play an important role in motivation, emotion, attachment behaviour, the response to stress and pain, control of food intake, and the rewarding effects of alcohol and nicotine.

Opioid-like peptides may also be absorbed from partially digested food (casomorphins, exorphins, and rubiscolins). Opioid peptides from food typically have lengths between 4–8 amino acids. Endogenous opioids are generally much longer.

Opioid peptides are released by post-translational proteolytic cleavage of precursor proteins. The precursors consist of the following components: a signal sequence that precedes a conserved region of about 50 residues; a variable-length region; and the sequence of the neuropeptides themselves. Sequence analysis reveals that the conserved N-terminal region of the precursors contains 6 cysteines, which are probably involved in disulfide bond formation. It is speculated that this region might be important for neuropeptide processing.

Endogenous

The human genome contains several homologous genes that are known to code for endogenous opioid peptides.

• The nucleotide sequence of the human gene for proopiomelanocortin (POMC) was characterized in 1980. The POMC gene codes for endogenous opioids such as β-endorphin and γ-endorphin.
• The human gene for the enkephalins was isolated and its sequence described in 1982.
• The human gene for dynorphins (originally called the "Enkephalin B" gene because of sequence similarity to the enkephalin gene) was isolated and its sequence described in 1983.
• The PNOC gene encoding prepronociceptin, which is cleaved into nociceptin and potentially two additional neuropeptides.
• Adrenorphin, amidorphin, and leumorphin were discovered in the 1980s.
• The endomorphins were discovered in the 1990s.
• Opiorphin and spinorphin, enkephalinase inhibitors (i.e., prevent the metabolism of enkephalins).
• Hemorphins, hemoglobin-derived opioid peptides, including hemorphin-4, valorphin, and spinorphin, among others.

While not peptides, codeine and morphine are also produced in the human body.

Exogenous

Exogenous opioid substances are called exorphins, as opposed to endorphins. Exorphins include opioid food peptides like Gluten exorphin and opioid food peptides and are mostly contained in cereals and animal milk. Exorphins mimic the actions of endorphins by binding to an activating opioid receptors in the brain.

Common exorphins include:

• Casomorphin (from casein found in milk of mammals, including cows)
• Gluten exorphin (from gluten found in cereals wheat, rye, barley)
• Gliadorphin/gluteomorphin (from gluten found in cereals wheat, rye, barley)
• Soymorphin-5 (from soybean)
• Rubiscolin (from spinach)

Amphibian

• Deltorphin
• Deltorphin I
• Deltorphin II
• Dermorphin

Synthetic

• Zyklophin – semisynthetic KOR antagonist derived from dynorphin A