Estimation of Shape, Volume, and Dipole Moment of Individual Proteins Freely Transiting a Synthetic Nanopore

Abstract

This paper demonstrates that high-bandwidth current recordings in combination with low-noise silicon nitride nanopores make it possible to determine the molecular volume, approximate shape, and dipole moment of single native proteins in solution without the need for labeling, tethering, or other chemical modifications of these proteins. The analysis is based on current modulations caused by the translation and rotation of single proteins through a uniform electric field inside of a nanopore. We applied this technique to nine proteins and show that the measured protein parameters agree well with reference values but only if the nanopore walls were coated with a nonstick fluid lipid bilayer. One potential challenge with this approach is that an untethered protein is able to diffuse laterally while transiting a nanopore, which generates increasingly asymmetric disruptions in the electric field as it approaches the nanopore walls. These "off-axis" effects add an additional noise-like element to the electrical recordings, which can be exacerbated by nonspecific interactions with pore walls that are not coated by a fluid lipid bilayer. We performed finite element simulations to quantify the influence of these effects on subsequent analyses. Examining the size, approximate shape, and dipole moment of unperturbed, native proteins in aqueous solution on a single-molecule level in real time while they translocate through a nanopore may enable applications such as identifying or characterizing proteins in a mixture, or monitoring the assembly or disassembly of transient protein complexes based on their shape, volume, or dipole moment.

Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation

“…(67,68) Although each monomer of wild-type streptavidin has a calculated dipole moment of 256 D according to the Weizmann server, (69,70) the individual moments of the monomers cancel each other out when aligned in the tetramer. (19,40,71) Such cancellation of the invidiual moments of the monomers does not occur for monovalent streptavidin. Slight variations in the structure of MSA compared to that of wild-type streptavidin introduce a dipole of around 80 D, (69,70) as seen in Figure 1a.…”

“…While the volume-based model in Equation 2 accurately describes streptavidin's translocation at low voltage, it does not address the effects of protein orientation (relative to the pore axis) or of conformational changes which become increasingly likely at higher voltages that favor protein shearing. (46,47) Recently, Yusko(19) and Houghtaling et al (40) developed a model linked to Maxwell's derivation:…”

Application of Solid-State Nanopore in Protein Detection

“…Although the first one is more accurate for shape estimation, the second one is the first strategy to estimate the shape and volume of a single protein molecule in situ in real time, and can also determine the rotational diffusion coefficient and dipole moment of a protein. In their later work, they successfully determined the ellipsoidal shape, volume, and dipole moment of single protein, which was not tethered to the lipid anchor [79]. This allowed free diffusion of the protein and increased the number of events available for analysis.…”

The applications of nanopores in studies of proteins

“…In 2017, Yusko et al [72] demonstrated the ability of bilayer-coated solid-state nanopores to characterize single proteins, providing estimations of parameters such as volume, shape, charge, dipole moment and rotational diffusion coefficient, for individual proteins based on current peaks. Furthermore, in 2019, the same group [73] utilized a solid-state nanopore without modification of the bilayer to characterize several parameters (including volume, shape and dipole moment) of various proteins.…”