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Getting a permanent grip on proteins

Manipulation of proteins is at the foundation of biochemical research; short protein ('peptide') tags give us the grip we need to study proteins in our scientific playgrounds.

However, the small size and flexibility of peptides makes them ‘slippery’ as molecular tools. For many applications that require long-term monitoring of proteins, such as single molecule imaging or linking proteins together, we often need to find better tools.

Spontaneous isopeptide bond formation between a peptide and its binding partner, for use in irreversible monitoring of proteins

Spontaneous isopeptide bond formation between a peptide and its binding partner, for use in irreversible monitoring of proteins

To address these issues, Bijan Zakeri in Dr Mark Howarth’s group has been trying to develop peptide tags that can bind their targets through an irreversible covalent bond. The pair took inspiration from an unlikely source – the flesh eating-bacterium Streptococcus pyogenes.

S. pyogenes belongs to a group of bacteria that cause many human diseases. Their potency partly stems from their ability to bind to host tissues through pili - long, thin cell surface attachments. Pili also help the bacteria to colonise the host and to form a biofilm (produced by bacterial aggregates), and are therefore considered as a major virulence factor.

Bijan Zakeri and Mark Howarth exploited a recently discovered feature of amino acid chemistry found in the pili of S. pyogenes that has been used by these bacteria to cope inside a hostile host. They have published their work in the Journal of the American Chemical Society (1).

Pili are constructed through covalent polymerization of a single major pilin subunit. The pilin used in this study has been shown to be the most mechanically stable protein ever characterised. The protein gets its incredible strength through strategically placed peptide bonds that occur outside of the main chain of the protein, giving an extra dimension to the linking of protein domains. These so-called isopeptide bonds occur spontaneously when the side chains of a lysine and an asparagine residue are placed in close proximity with a nearby glutamate residue to induce the reaction.

The researchers used this isopeptide bond forming scaffold to develop a new peptide tag which they have named isopeptag. They split the pilin protein into a 16 amino acid peptide and a binding partner. This peptide-protein pair was able to reconstitute spontaneously through a covalent bind. Surprisingly, the reaction proceeds equally well from 4°C to 37°C and can take place at a range of pHs and in various buffers. Bijan went on to demonstrate that when the peptide and protein are synthesised in E. coli, the pair will covalently reconstitute within the cell. He also demonstrated the potential of this technology for use in fluorescent microscopy, showing that he could use the isopeptag to specifically label mammalian cells.

This new technology will give biochemists an additional tool to manipulate proteins with an iron grip, enabling them to study interactions which were not previously tractable.

  1. Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting. Zakeri B. and Howarth, M. J Am Chem Soc 2010 Apr 7;132(13):4526-7



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