Engineering a Biological Nanopore for Monitoring Protein Dynamics and Conformational Changes at the Single-Molecule Level.

Biological nanopores can be used to monitor the conformational dynamics of enzymatic reactions in real time and at the single-molecule level. However, the molecular basis of this process and the requirements for efficiently engineering a nanopore for measuring conformational transitions in proteins remain poorly understood. Here, we combine all-atom molecular dynamics simulations and electrophysiology experiments to determine the molecular mechanism and improve the resolution for the recognition of glutamine by a substrate-binding domain (SBD) protein using ClyA nanopores through mutagenesis. By matching the simulated and experimental currents, we found that the SBD protein most likely resides very close to the constriction of the nanopore, where the open and closed conformations induce large current differences. To amplify the signal, we introduced bulky tryptophan residues near the likely location of the SBD protein. This effectively narrowed the nanopore and resulted in a 3-fold signal amplification. Molecular dynamics simulations suggest that prolonged residence of the SBD protein within ClyA is likely caused by an electro-osmotic flow that imparts a restoring force on the protein, counteracting its displacement from an equilibrium location. Our work shows how nanopore systems can be engineered in a rational way for the monitoring of biochemical reactions at the single-molecule level.