Probing protein structural dynamics at the single-molecule level by nanopore tweezers

Proteins are dynamic entities that sample multiple conformations over a range of time scales. Understanding how proteins work requires a full description of their conformational dynamics over time. Nuclear magnetic resonance (NMR) spectroscopy can measure protein dynamics at multiple time scales, however, application is laborious and limited to relatively small proteins that can be stably prepared at high concentrations. Conformational heterogeneity, such as multiple weakly populated conformational states, is challenging to resolve from ensemble methods such as NMR due to averaging effects. Integrative approaches and single-molecule techniques can be advantageous for revealing transient protein dynamics and functional heterogeneities. Nanopore tweezers have emerged as a powerful, single-molecule analytical tool to monitor the protein's conformational dynamics. Nanopore tweezers confine a single protein within the pore lumen and measure ionic current changes induced by the protein analyte's motion. Here we will present several studies of ClyA nanopore tweezers to showcase their utility in resolving ligand interaction modes, screening allosteric drugs and investigating intrinsically disordered protein interactions.