Nanopore Passport Control for Substrate-Specific Translocation

Abstract

Membrane protein pores have demonstrated applications in nanobiotechnology and single-molecule chemistry for effective detection of biomolecules. Here, we define the molecular basis of carbohydrate polymers translocation through a substratespecific bacterial nanopore, CymA, which has a 15-residue N terminus segment inside the pore, restricting its diameter. Using single-channel recordings, we determined the kinetics of cationic cyclic oligosaccharide binding and elucidated the translocation mechanism across the pore in real-time. The cationic cyclic hexasaccharide binds to the densely packed negatively charged residues at the extracellular side of the pore with high affinity, facilitating its entry into the pore driven by the applied voltage. Further, the dissociation rate constant increased with increasing voltages, indicating unidirectional translocation toward the pore exit. Specifically, a larger cationic cyclic octasaccharide rapidly blocked the pore more effectively, resulting in the complete closure of the pore with increasing voltage, implying only strong binding. Further, we show that uncharged oligosaccharides exclusively bind to the extracellular side of the pore and the electroosmotic flow most likely drives their translocation. We propose that CymA favors selective translocation of cyclic hexasaccharide and linear maltooligosaccharides due to an asymmetrical charge pattern and the N terminus that regulates the substrate transport. We suggest that this substrate-specific nanopore with sophisticated geometry will be useful for complex biopolymer characterization.

The C2 entity of chitosugars is crucial in molecular selectivity of the Vibrio campbellii chitoporin

“…VhChiP/chitosugar interaction illustrates the evolution of molecular machinery with extraordinary functional performance and optimization of the cell membrane for bacterial survival. The study findings agree with recent conclusions on electrostatically driven pore permeation through porins in other microorganisms (10,(73)(74)(75). From the viewpoint of the nanotech industry they may be seen as guidance for construction of solid-state or genetically modified biological nanopores for technology and analytical chemistry applications.…”

“…The diameters of the dextran-filled J o u r n a l P r e -p r o o f liposomes varied from 100 to 1000 nm. The purified VhChiP (100 ng) was reconstituted into liposomes as described previously (10). The isotonic solute concentration was determined with different concentrations of raffinose solution (prepared in 20 mM HEPES buffer, pH 6.5 or 8.5) added into the proteoliposome suspensions.…”

“…[5][6][7][8]. Substrate-specificity was also verified for the bacterial nanopore CymA, from the outer membrane of Gram-negative bacteria Klebsiella oxytoca (9) and Vikraman et al described a chemical 'passport control' mechanism at the pore opening that regulated the passage of anionic and cationic linear and cyclic oligosaccharides through CymA, selectively (10). Another porin with high substrate specifity is the sucrose-permeable ScrY channel (11)(12)(13)(14).…”

Dynamic interaction of fluoroquinolones with magnesium ions monitored using bacterial outer membrane nanopores

“…Here we introduce outer membrane nanopores, which are pore forming proteins in the OM and especially general diffusion channels also called "porins", e.g., OmpF and OmpC from E. coli, facilitate the diffusion of hydrophilic nutrients and are slightly cation selective as found in both in situ as well as in silico characterization. 6,20,21 These porins are known to have a signicant role in the inux of several classes of antibiotics, as shown by recent studies based on viability assays, 22 electrophysiological characterization [23][24][25] and in silico studies. 26,27 Therefore, down-regulating the expression of these porins is oen a prime mechanism chosen by bacteria to achieve resistance to many drugs.…”

Dynamic Interaction of Norfloxacin to Magnesium Monitored by Bacterial Outer Membrane Nanopores

“…3 Compared to Gram-positive bacteria lacking the OM, this additional layer acts as a significant barrier for hydrophilic solutes. Pore forming proteins in the OM and especially the general diffusion channels also called "porins", e.g., OmpF and OmpC from E. coli, facilitate the diffusion of hydrophilic nutrients and are slightly cation selective from both in situ and in silico characterization [4][5][6] .These porins are known to have a significant role in the influx of several classes of antibiotics, as shown by recent studies based on viability assays 7 , electrophysiological characterizations [8][9][10] and in silico studies 11,12 . Therefore, down-regulating the expression of these porins is often a prime mechanism chosen by bacteria to achieve resistance to many drugs 4,13 .…”