Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales 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 reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings contribute to on 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.

Conformational changes of proteins are essential to their functions. Yet it remains challenging to measure the amplitudes and timescales of protein motions. Here we show that the ClyA nanopore can be 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. The current measurements revealed three distinct ligand-bound states for MBP in the presence of reducing saccharides. Our biochemical and kinetic analysis reveal that these three states represented MBP bound to different isomers of reducing sugars. These findings shed light on 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.

Recasting temperature replica exchange (T-RE) as a special case of Gibbs sampling has led to a simple and efficient scheme for enhanced mixing (Chodera and Shirts, 2011). To critically examine if T-RE with independence sampling (T-REis) improves conformational sampling, we performed T-RE and T-REis simulations of ordered and disordered proteins using coarse-grained (CG) and atomistic models. The results demonstrate that T-REis effectively increase the replica mobility in temperatures space with minimal computational overhead, especially for folded proteins. However, enhanced mixing does not translate well into improved conformational sampling. The convergences of thermodynamic properties interested are similar, with slight improvements for T-REis of ordered systems. The study re-affirms the efficiency of T-RE does not appear to be limited by temperature diffusion, but by the inherent rates of spontaneous large-scale conformational re-arrangements. Due to its simplicity and efficacy of enhanced mixing, T-REis is expected to be more effective when incorporated with various Hamiltonian-RE protocols.