Nature Journal Club

Clive R. Bagshaw

University of Leicester, UK

A biochemist is excited by a universal glue for molecular biology.

Investigating the dynamic properties of proteins at the level of a single molecule allows insight into properties that are masked in ensemble studies. I have often found that the hardest part of such studies is immobilizing the molecule on a silica surface in a ‘permanent’ way that retains the molecule’s function.

The proteins we investigate are usually prepared with a His-tag — comprising 6+ engineered histidine residues — that binds, via a chelated nickel ion, to nitriloacetic acid (NTA), aiding purification on an NTA affinity column. Immobilization through this tag would therefore be an attractive option. But alas, this is only partly successful using the standard NTA group because proteins have a significant probability of detaching from the silica support on the timescale of minutes.

But surely a chemist somewhere has improved on this technology? Thanks to Google, I found the work of Jacob Piehler who, in 2005, introduced tris-NTA, a cyclam ring with three groups attached to it. Tris-NTA shows a thousand-fold higher affinity for His-tags in the presence of nickel than NTA and a dissociation half-life of many hours.

Piehler and colleagues have gone on to exploit this technology as a general means of attaching fluorophores to His-tagged proteins and, most recently, as a convenient way of specifically conjugating proteins to streptavidin (A. Reichel et al. Anal. Chem. doi:10.1021/ac0714922; 2007).

The streptavidin-biotin complex is another widely-used ‘glue’ in biotechnology, but the use of an intermediate tris-NTA-biotin adaptor broadens its application to His-tagged proteins and renders the attachment reversible on addition of excess imidazole. I look forward to using this technology in our single-molecule studies, for which such a reversible glue has the same appeal as a Post-It note.


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