The final part of my kinase detection ‘trilogy’ is now out in ACS Nano. This is work I developed during my PhD in the lab of Hagan Bayley at the University of Oxford.

In the first two publications of this series, we described how we used a novel engineering strategy to generate nanopore sensors for protein kinases. These publications covered the fundamental characterisation of kinase–substrate interactions for the Pim family of protein kinases, such as unusually fast, electrostatically enhanced association.

The second publication described how we could observe synergistic binding between nucleotides and peptide substrates, and how we then were able to apply this to develop a nanopore-based assay for protein kinase inhibitors.

The final part, now out, fills in the final missing piece of the jigsaw, which is the characterisation of the enzymatic activity of kinases. We were able to observe the phosphorylation of a single substrate molecule. This could also be reversed by use of a protein phosphatase.

In the course of investigating these phenomena we also uncovered several mechanistic insights regarding the effect of divalent metal ions on product and substrate binding.

This draws a close to my work on the detection of protein kinases with nanopores. It should be apparent that there is still a lot to do to develop ‘nanopore enzymology’. As we discuss in the conclusion of our paper, one of the most promising areas for further investigation is the detection of conformational dynamics at the single molecule level.

There are now protein pores in use that are able to capture proteins within their pore lumens, instead of being sensed at a distance. The hope is that such systems will allow a much higher scrutiny of protein dynamics during catalysis, for example. Early efforts have already been made and I hope others will be able to realise this in the coming years.