Hetero-Oligomeric Protein Pores for Chemical and Biosensing

Abstract

Nanopores are used as single-molecule sensors to detect various complex biological and chemical molecules in a solution specifically for nucleic acid sequencing. In particular, pulling charged molecules through nanopores remains a very prominent research area. Here, we introduce a novel hetero-oligomeric porin protein, NfpAB, from the Gram-positive mycolata, Nocardia farcinica, which is composed of two subunits NfpA and NfpB. More specifically, we elucidate the transport of differently sized and charged cationic molecules such as polypeptides and cyclic oligosaccharides through this pore at a single-molecule level. Notably, we quantified the translocation kinetics as the applied potential acts as a driving force promoting the electrostatic interaction of charged molecules with the pore surface. Remarkably, we observed asymmetry in transport kinetics of molecules due to asymmetric geometry, shape and charge distribution of the pore and provide a quantitative description of relevant kinetic parameters required to control the transport of molecules through the pore. Furthermore, the controlled attachment of cyclodextrins to the pore wasapplied to modulate the molecular transport of differently charged peptidesto identify the discrete amino acid signals. The attached cyclodextrins aided in maintaining the peptides in the pore for a longer duration, thus increasing the resolution for sensing molecules effectively and accurately. We emphasize that theinbuilt cysteine residues in the pore lumen and strong charge distribution aids in the attachment of adapter molecules by chemical modification and electrostatic attraction. We suggest that hetero-oligomeric nanopores described in this work will be useful for sensing a wide variety of complex biopolymers.