Electrokinetic detection of single-molecule phosphorylation

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

Abstract

We developed a method to sense phosphorylation by measuring charge modulation on single molecules. Kinase catalyzed phosphoryl transfer from ATP to target substrate adds two negative charges, while phosphatase catalyzed dephosphorylation process removes two negative charges (i.e. effectively adds two positive charges) on the substrate. This modulation of single-molecule charge affects the electrokinetic response under a driving electric field and can be inferred by measuring electrokinetic mobility. In fact, ensemble assays based on this simple principle has been developed in capillary electrophoresis. Here, we use the anti-Brownian ELecktrokinetic trap (ABEL) platform to measure single-molecule electrokinetic mobility, thus resolving the phosphorylation state of individual biomolecules. The ABEL trap uses high-speed single-molecule tracking and feedback electrokinetic motion to restrict the diffusion radius of individual molecules, allowing them to be observed in solution for extended periods of time. We demonstrated a few ground-breaking measurements on single-molecule phosphorylation and its dynamics. First, we resolve the phosphorylation state of single peptide molecules. Second, we determine the degree of phosphorylation on an intrinsically disordered FUS peptide. Individual molecules were observed to be either singly, doubly or on rare occasions, triply phosphorylated. Further, we visualize the phosphorylation and dephosphorylation cycles, driven by kinases and phosphatases in solution. We resolve individual steps of kinase binding, turn over, product release, as well as phosphatase catalyzed dephosphorylation steps.