Single-molecule enzymology with a ClyA nanopore

The goal of this thesis was to use nanopore measurements to study enzymes at the single-molecule level.
We started by developing a nanopore sensor based on a protein lodged inside the ClyA nanopore, so that the concentration of glucose and asparagine could be determined. The concentrations of these metabolites in pico-liters of untreated samples of blood, sweat and urine could be accurately determined simultaneously within seconds.

By making mutations to the glucose binding protein, we tried to understand the mechanism of binding of this protein. We propose that a hydrogen bond network of water molecules in the active site plays a key and dominant role in promoting the conformational change that induces the closing of the protein around glucose.
Next, the ligand-induced conformational changes of the enzyme dihydrofolate reductase (DHFR) was studied with the ClyA nanopore. The measurements revealed different DHFR blockades, all with distinctive affinities for its ligands. This showed that DHFR exists in at least four different ground-state conformers. Interconversions between the conformers were accelerated during the transition-state conformation of DHFR.

Additionally, the full reaction cycle of DHFR could be followed by sampling the exchange between five slightly different conformations. The single-molecule data revealed the precise hierarchy of binding and the affinity of the enzyme for the different reaction intermediates. The sampling of up to hundreds of consecutive reactions of a single enzyme revealed rare and transient intermediates along the reaction pathway as well as disorder in the enzyme.