Single-molecule protein phosphorylation and dephosphorylation by nanopore enzymology

Reversible protein phosphorylation plays a crucial and ubiquitous role in the control of almost all cellular processes. The interplay of protein kinases and phosphatases acting in opposition ensures tight dynamic control of protein phosphorylation states within the cell. Previously, engineered alpha-hemolysin pores bearing kinase substrate peptides have been developed as single-molecule stochastic sensors for protein kinases. Here, we have used these pores to observe, label-free, the phosphorylation and dephosphorylation of a single substrate molecule. Further, we investigated the effect of Mg2+ and Mn2+ upon substrate and product binding and found that Mn2+ relaxes active site specificity towards nucleotides and enhances product binding. In doing so, we demonstrate the power and versatility of nanopore enzymology to scrutinise a critical post-translational modification.