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 α-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 toward nucleotides and enhances product binding. In doing so, we demonstrate the power and versatility of nanopore enzymology to scrutinize a critical post-translational modification.