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

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

ACS nano (2019-04-18)
Jared Houghtaling, Cuifeng Ying, Olivia M Eggenberger, Aziz Fennouri, Santoshi Nandivada, Mitu Acharjee, Jiali Li, Adam R Hall, Michael Mayer
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
Alcohol Dehydrogenase from Saccharomyces cerevisiae, ≥300 units/mg protein, lyophilized powder (contains buffer salts), Mw 141-151 kDa
Sigma-Aldrich
Ferritin from human liver, Type IV, 10 μg/mL
Sigma-Aldrich
Glucose-6-phosphate Dehydrogenase from Leuconostoc mesenteroides, lyophilized powder, >= 550 units/mg protein (biuret)
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α-Amylase from Bacillus licheniformis, lyophilized powder, 500-1,500 units/mg protein, 93-100% (SDS-PAGE)
Sigma-Aldrich
Streptavidin from Streptomyces avidinii, affinity purified, lyophilized from 10 mM potassium phosphate, ≥13 U/mg protein