Abstract
Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and time scales of protein motions. Here we show that the cytolysin A (ClyA) nanopore was used as a molecular tweezer to trap a single maltose-binding protein (MBP) within its lumen, which allows conformation changes to be monitored as electrical current fluctuations in real time. In contrast to the current two state binding model, the current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our analysis reveals that these three states represented MBP bound to different isomers of reducing sugars. These findings contribute to the understanding of the mechanism of substrate recognition by MBP and illustrate that the nanopore tweezer is a powerful, label-free, single-molecule approach for studying protein conformational dynamics under functional conditions.