Popular to Peculiar: 30+ Post-translational Modifications

We all know the two stages of information flow from nucleic acid to protein – transcription and translation. But it should be three stages, really where the last stage is post-translational modification (PTM) of proteins – covalent and usually enzymatic modification of proteins following protein biosynthesis. In fact, there is hardly any protein that doesn’t have a PTM, so though DNA encodes 20 primary amino acids, proteins contain more than 140 different residues. One of the most common modifications is glycosylation – the addition of sugar monomers and polysaccharide chains to proteins. The most dramatic is probably phosphorylation, attachment of the phosphate groups that makes kinases active, triggering regulatory cascades in the cells. [1]

The Importance of Post-translational Modification

For many proteins, especially eukaryotic, the absence of PTM makes them as useless as a Christmas tree without decorations. The absence of many types of PTMs characteristic to eukaryotic cells makes the production of functional eukaryotic proteins in E.coli difficult. For example, the first human hormone produced using recombinant technology, insulin, consists of two polypeptide chains connected by another common PTM, a disulfide bond that forms between two methionines. The chains are produced separately in E.coli, and after the purification, the disulfide bonds have to be chemically introduced [2]. PTMs occur on the amino acid side chains or at the protein’s C- or N- termini. Amino acids that receive post-translational modification usually have a functional group that can serve as a nucleophile in the reaction:

N- and C-termini that have free amine or carboxyl groups;
the amine forms of arginine, histidine, and lysine;
the hydroxyl groups of serine, threonine, and tyrosine;
the thiolate anion of cysteine or the carboxylates of aspartate and glutamate.

In addition, despite being a weak nucleophile, the amide of asparagine can act as an attachment point for glycans – an oligo or polysaccharide. Rarer PTMs take place at oxidized methionines and some methylenes in side chains. [3]

A-Z of Post-translational Modifications

Let’s walk through the alphabet of the post-translational modifications. You can award yourself a point for each one you know.

PTM	Details	Frequency of modification (from experimental data)[4]
A
Acetylation	The addition of an acetyl group. This can occur at lysine residues or at the protein’s N-terminus. The reverse is called deacetylation. Lysine acetylation of histones neutralizes lysine electrical charge, loosening the chromatin	6,775
Acylation	O-acylation (esters), N-acylation (amides), S-acylation (thioesters).	NR
Alkylation	The addition of an alkyl group such as methyl, ethyl	NR
Amino acid addition (not mediated by the ribosome)
•Arginylation	A tRNA-mediation addition	NR
•Polyglutamylation	The addition of multiple glutamic acid residues to certain proteins (e.g. tubulin) via the N-terminus	NR
•Polyglycylation	The addition of one or more glycine residues to the C-terminal tail of alpha or beta tubulin	NR
Amide bond formation	Also known as amidation. at protein C-terminus. Formed by oxidative dissociation of a C-terminal glycine residue	2,852
AMPylation	Also, known as Adenylylation. The covalent attachment of adenosine monophosphate (AMP) molecule to tyrosine, threonine, or serine residues. Common in GTPases	NR
B
Butyrylation	Adding butyric acid residue or one of its derivatives	NR
C
Carbonylation	Modification of histidine, cysteine, and lysine in proteins to aldehydes and ketones. While most of the rest of PTMs are “by design” to enable the protein function, carbonylation is a result of oxidative stress. It damages the proteins	NR
D
Disulfide bond formation	Formation of the covalent bond between two cysteines. Usually stabilizes the 3D structure of the protein. Unlike most of the covalent bonds, it is reversible by oxidoreductases	NR
F
Flavin moiety (FMN or FAD) attachment	(FMN or FAD) attachment that is the catalytic center of the oxidoreductases, components of the succinate dehydrogenase complex, correspondingly.	NR
G
Gamma-carboxylation	Of glutamic acid residues. Found in clotting factors and other proteins of the coagulation cascade and other proteins	450
Glycosylation	The addition of a glucose derivative, glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan resulting in a glycoprotein. One of the most abundant post-translational modifications	5,593 -N-linked glycosylation 1,189 - O-linked glycosylation 148 - C-linked glycosylation
Glycation	Non-enzymatic attachment of sugars	NR
H
Heme attachment	The significance of heme as a protein cofactor goes far beyond the most famous heme-containing protein, hemoglobin. Hemes are found in a number of other important hemoproteins such as myoglobin, cytochromes, catalases, heme peroxidase	NR
I
(Iso)prenylation	The addition of an isoprenoid group farnesol or geranylgeraniol to C-terminal cysteine(s) of the target protein.	NR
Isopeptide bond formation	This amide bond involves at least one amino acid side chain and can result in stably linked protein dimers/mulitmers. Common in surface proteins of Gram-positive bacteria	NR
L
Lipidation	Adding lipids to the proteins, for example, lipid-anchored proteins - prenylated protein (see above), fatty acylated proteins and glycosylphosphatidylinositol-linked proteins	NR
Lipoylation	Attachment of a lipoate (C8)	NR
M
Malonylation	Adding malonyl group to the amino acid lysine, changing its charge from +1 to −1	NR
Methylation	Involved the addition of a methyl group, at various residues (commonly lysine or arginine). The reverse reaction is called demethylation. This is another important modification of chromatin remodeling	1,545
Myristoylation	Attachment of myristate, a C14 saturated acid	179
N
Nucleotide addition	Such as ADP-ribosylation	NR
O
O-linked β-N-acetylglucosamination	The addition of N-acetylglucosamine to serine or threonine residues of nucleocytoplasmic proteins	NR
Palmitoylation	Attachment of palmitate, a C16 saturated acid	314
Phosphorylation	Addition of a phosphate group to a residue. The main residues for phosphorylation are serine, threonine, and tyrosine (O-linked), or histidine (N-linked). Possibly the most well known PTM. It sometimes seems that half of the cell proteins phosphorylate and dephosphorylate each other	58,525
Phosphate ester	(O-linked) or phosphoramidate (N-linked) formation	NR
Propionylation	Adding the propyl group, for example, lysine in histones	NR
Pyroglutamate formation	Formation of pyroglutamate (a cyclic amino acid) from either glutamic acid or glutamine – can occur spontaneously or catalyzed by glutaminyl cyclase	NR
Polysialylation	The addition of polysialic acid, PSA	NR
S
Signal sequence processing	Signal sequences are N-terminal extensions of newly synthesized secretory and membrane proteins from 16 up to 50 amino acid residues and have C-terminal region with the cleavage site for signal peptidase. In eukaryotes, signal sequences direct the insertion of proteins into the membrane of the endoplasmic reticulum and are usually cleaved off by signal peptidase	NR
S-modification of cysteine residue
•S-glutathionylation	Addition of glutathione, a tripeptide with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. Glutathione is a major antioxidant	NR
•S-nitrosylation	Formation of S-nitrosothiol (SNO). Occurs when a nitric oxide group is attached to a cysteine thiol. S-nitrosylation has diverse regulatory roles in bacteria, yeast, and plants and in all mammalian cells	63
•S-sulfenylation	Reversible covalent addition of an oxygen atom to the thiol group of a cysteine	NR
•S-sulfinylation	Covalent attachment of two oxygen atoms to the thiol group of a cysteine	NR
•S-sulfonylation	Covalent attachment of three oxygen atoms to the thiol group of a cysteine, leading to the formation of a cysteic acid	NR
Succinylation	Attachment of a succinyl group to lysin	NR
Sulfation	The addition of a sulfate group to a tyrosine	523
Sumoylation	Attachment of SUMO (small ubiquitin-like protein modifiers). Sumoylation can affect a protein’s structure and subcellular localization	422
U
Ubiquitylation/ ubiquitination	Attachment of a 8.6K protein, ubiquitin (from Latin ubique - “everywhere”). The ubiquitination can either consist of a single ubiquitin protein or a chain of ubiquitin (polyubiquitylation). The first ubiquitin molecule is covalently bound through its C-terminal carboxylate group to a particular lysine, cysteine, serine, threonine or N-terminus of the target protein. Ubiquitination can direct proteins for degradation via the proteasome, alter their cellular localization, affect their activity, and promote or prevent protein-protein interactions	905
Uridylylation	The addition of a uridylyl-group (i.e. uridine monophosphate, UMP), usually to tyrosine	NR

Summary of Post-translational Modifications

I bet that’s more post-translational modifications than you knew about. Being aware of all these “decorations” will help you in appreciating any unexpected traits of your proteins. For example, if your protein on western blot shows as multiple bands and you are sure it’s not degradation products or non-specific binding in your western blots, this might be an indication of post-translational modifications that change the molecular mass or charge of your protein, for example, glycosylation. These modifications are usually biologically relevant and point out research avenues to explore. Do let us know if we missed some important PTMs or need to update the article, by leaving a comment below.