Isopeptag is a 16-amino acid peptide tag (TDKDMTITFTNKKDAE) that can be genetically linked to

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s without interfering with protein folding. What makes the isopeptag different from other peptide tags is that it can bind its binding protein through a permanent and irreversible

covalent bond A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atom ...

. Other peptide tags generally bind their targets through weak non-covalent interactions, thus limiting their use in applications where molecules experience extreme forces. The isopeptag's covalent binding to its target overcomes these barriers and allows target proteins to be studied in harsher molecular environments.

Development

The isopeptag was developed by dissecting the

pilin Pilin refers to a class of fibrous proteins that are found in pilus structures in bacteria. These structures can be used for the exchange of genetic material, or as a cell adhesion mechanism. Although not all bacteria have pili or fimbriae, bact ...

protein (Spy0128) from ''Streptococcus pyogenes''. Spy0128 contains two intramolecular

isopeptide bond An isopeptide bond is a type of amide bond formed between a carboxyl group of one amino acid and an amino group of another. An isopeptide bond is the linkage between the side chain amino or carboxyl group of one amino acid to the α-carboxyl, α- ...

s,Kang,H.J., Coulibaly,F., Clow,F., Proft,T., and Baker,E.N. (2007). Stabilizing isopeptide bonds revealed in gram-positive bacterial pilus structure. Science 318, 1625-1628. and to generate the isopeptag one of these bonds was split by removing the last β-strand in the protein.

Mode of action

When the isopeptag is bound to a target protein, it spontaneously binds its binding partner through an

, an amide bond formed autocatalytically. The reaction is robust and occurs at various temperatures from 4-37 °C, a pH range of 5–8, and in the presence of commonly used detergents. Also, the reaction is independent of the

redox Redox ( , , reduction–oxidation or oxidation–reduction) is a type of chemical reaction in which the oxidation states of the reactants change. Oxidation is the loss of electrons or an increase in the oxidation state, while reduction is t ...

state of the environment and can occur equally well in both reducing and oxidizing conditions.

Applications

The covalent binding of the isopeptag to its binding partner can be used to permanently link proteins together in the complex environment of a bacterial cell, to target proteins of interest for cellular imaging, and to develop new protein structures.

References

{{Protein tag Protein methods Molecular biology techniques