Nanopore-Based, Multi-Parametric Characterization of Single, Unlabeled Proteins in Solution

Abstract

Most methods for characterizing proteins - including electron microscopy, NMR spectroscopy, and X-Ray crystallography - require either some sort of physical modification such as purifying, drying, freezing, or crystallizing proteins, or they require chemical modification such as fluorescently labeling or immobilizing proteins. Resistive pulse-based nanopore sensing of proteins is emerging as a powerful analysis technique; it has the potential to overcome these requirements while characterizing individual, unlabeled proteins in aqueous solution. We recently used lipid-coated synthetic nanopores to determine the volume, shape, charge, dipole moment, and rotational diffusion coefficient of single proteins that we tethered to the fluid lipid bilayer to slow their passage through the pore. Here, we compare two different non-adsorptive nanopore coatings combined with high bandwidth recordings to determine the shape and volume of freely translocating proteins in solution. The difficulty of this approach lies in the lateral diffusion of untethered proteins transiting a nanopore, which facilitates position-dependent, asymmetric disruptions in the electric field as proteins approach the nanopore walls. These ‘off-axis’ effects pose a challenge to multi-parametric protein characterization because they distort the observed resistive pulse magnitudes, leading to analysis artifacts. Using lipid-coated nanopores to minimize non-specific protein adhesion, we mitigated these off-axis effects by reducing the residence time of proteins at the pore wall. This approach made it possible to estimate shape and volume values for nine different native proteins free in solution. In contrast, pores coated with the detergent Tween-20 were not suitable for protein shape and volume characterization likely because of significant adhesion to the pore wall and concomitant off-axis effects. By examining unlabeled proteins in an aqueous sample on a single molecule level in lipid bilayer-coated nanopores, we enable new applications for nanopore-based protein sensing, especially those involving transient protein complexes or macromolecules.