Single molecule studies of protein folding hold keys to unveiling protein folding pathways and elusive intermediate folding states—attractive pharmaceutical targets. Although conventional single-molecule approaches can detect folding intermediates, they presently lack throughput and require elaborate labeling. Here, we theoretically show that measurements of ionic current through a nanopore containing a protein can report on the protein’s folding state. Our all-atom molecular dynamics simulations show that the unfolding of a protein lowers the nanopore ionic current, an effect that originates from the reduction of ion mobility in proximity to a protein. Using a theoretical model, we show that the average change in ionic current produced by a folding-unfolding transition is detectable despite the orientational and conformational heterogeneity of the folded and unfolded states. By analyzing millisecond-long all-atom MD simulations of multiple protein transitions, we show that a nanopore ionic current recording can detect folding-unfolding transitions in real time and report on the structure of folding intermediates.

Nanopore sensing has emerged as a versatile approach to detection and identification of biomolecules. Presently, researchers rely on experience and intuition for choosing or modifying the nanopores to detect a target analyte. The field would greatly bene t from a computational method that could relate the atomic-scale geometry of the nanopores and analytes to the blockade nanopore currents they produce. Existing computational methods are either computationally too expensive to be used routinely in experimental laboratories or not sensitive enough to account for the atomic structure of the pore and the analytes. Here, we demonstrate a robust and inexpensive computational approach-the steric exclusion model (SEM) of nanopore conductance-that is orders of magnitude more efficient than all-atom MD and yet is sensitive enough to account for the atomic structure of the nanopore and the analyte. The method combines the computational efficiency of a finite element solver with the atomic precision of a nanopore conductance map to yield unprecedented speed and accuracy of ionic current prediction. We validate our SEM approach through comparison with the current blockades computed using the all-atom molecular dynamics method for a range of proteins confined to a solid-state nanopore, biological channels embedded in a lipid bilayer membranes and blockade currents produced by DNA homopolymers in MspA. We illustrate potential applications of SEM by computing blockade currents produced by nucleosome proteins in a solid-state nanopore, individual amino acids in MspA and by testing the effect of point mutations on amino acid distinguishability. We expect our SEM approach to become an integral part of future development of the nanopore sensing field. * These authors contributed equally to the manuscript Supplementary Information Available The following files are available free of charge: SI Wilson2019.pdf. Simulated dependence of KCl, K + and Cl − conductivity from the center of carbon, hydrogen, nitrogen, oxygen atoms; comparison of the nanopore currents computed using the resistor and SEM models; comparison of SEM and MD blockade currents in MspA done using nominal conductivity of KCl and for 3'-trans systems; supplementary methods detailing MD simulations of biological nanopores; and a table summarizing conditions for the biological nanopores simulations.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semi-quantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

Ion transport through nanopores is a process of fundamental significance in nature and in engineering practice. Over the past decade, it has been found that the ion conductivity in nanopores could be drastically enhanced and different mechanisms have been proposed to explain this observation. To date, most reported studies have been carried out with relatively dilute electrolytes while ion transport in nanopores under high electrolyte concentrations (>1 M) has been rarely explored. Through systematic experimental and atomistic simulation studies with NaCl solutions, here we show that at high electrolyte concentrations, ion mobility in small nanopores could be significantly reduced from the corresponding bulk value. Subsequent molecular dynamics studies indicate that in addition to the low mobility of surface-bound ions in the Stern layer, enhanced pairing and collisions between partially dehydrated ions of opposite charges also make important contributions to the reduced ion mobility. Furthermore, we show that the extent of mobility reduction depends on the association constant between cations and anions in different electrolytes with a more drastic reduction for a larger association constant.

Optical nanopore methods are widely believed to be practical approaches to address the flicker noise and the throughput limitations of nanopore detection. However, most optical nanopore methods in the literature are based on fluorescent labeling, which perplexes the detection operation and limits their applications. In this paper, we demonstrate a brand new optical nanopore technique named “scattering nanopore method”. This method achieves single nanopore readouts without the introduction of dyes by detecting scattering light through a single subwavelength aperture on Au film. The single particle induced optical fluctuations could be detected simultaneously with the nanopore ionic current readouts. The method we demonstrated here opens a new direction for nanopore sensing of single particles, even single molecules in the near future.