A COMMONS seminar: Nicole Galenkamp

A COMMONS seminar by Postdoctoral researcher Nicole Galenkamp, University of Groningen, The Netherlands.

Title: Biological nanopores for biosensing and single-molecule enzymology

Abstract: Biological nanopores are a class of membrane proteins that form nanometer-size apertures on lipid membranes. Under an external applied potential, the ionic current passing through individual nanopores is emerging as a new powerful to identify molecules or to follow reactions at the single-molecule level. Nanopores current are advantageous because of the ability to sense single molecules in real-time without the need for labelling, immobilization or chemical modifications. Proteins can be trapped inside nanopores by exploiting electrophoretic forces. Using this approach, nanopores can be used as a sensor for quantification of metabolites.

Here we show that protein sensors in combination with a biological nanopore Cytolysin A (ClyA), acting as an electrical transducer, can accurately quantify metabolites from tiny amounts of bodily fluids with internalized substrate binding proteins (SBDs). The single-molecule nature of the sensor allows continuous sampling of multiple analytes simultaneously without the need of calibration for signal drift.

Nanopores can also be used as a nanoscale reactor for single-molecule enzymology studies. We will show the first single-molecule characterization of the full enzymatic reaction of dihydrofolate reductase (DHFR) with the ClyA nanopore. Sampling of hundreds of consecutive reactions from single enzymes revealed that DHFR populates frequent non-productive transition-state conformations and undergoes second-long catalytic pauses. We also found that the free-energy landscape of the enzyme is sculpted with multiple ground-state conformers with different affinity for substrate, cofactor and product that undergo hierarchical changes during the catalytic cycle.