Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization

High-Voltage Biomolecular Sensing Using a Bacteriophage Portal Protein Covalently Immobilized Within a Solid-State Nanopore

“…Our observed behavior in the low-voltage regime is also consistent and a former study on membrane-embedded portal with a similar portal mutant which showed that β-cyclodextrin transport does not occur in the crown-to-clip direction for the voltage range studied (<140 mV). 37 In contrast to the low voltage behavior, at voltages higher than 220 mV the scatter plots reveal a new population of events with deeper blockades and dwell times that decrease with increasing voltage. To study the dwell time change as a function of voltage, we fitted exponential functions to the dwell time distribution shown in Figure S4.…”

“…G20c portal protein from the thermostable bacteriophage G20c oligomerizes to form a dodecameric assembly with an internal channel containing 1.8 nm constriction, as shown in Figure 1a . In common with the wild-type protein, its CD/N mutant, where four aspartic acid residues (D) were substituted by asparagine (N) to alter the internal surface charge for enhanced sensing of negatively charged molecules, 36 in addition to substitution of an externally facing leucine residue to cysteine for chemical labeling, 37 also forms 12-mers ( Figure S1 ). In contrast to the most successfully implemented proteins as nanopore sensors that assemble to form channels upon insertion into the lipid bilayer or synthetic polymer membranes, the hydrophilic G20c portal protein is water-soluble and fully assembles in an aqueous solution.…”

“…As we have shown in our previous hybrid system, 36 successful portal protein insertion and corking into a SiN x nanopore with desired orientation required maintaining a voltage bias across the membrane to electrokinetically drive the protein to the SiN x nanopore whose geometry is commensurate with the protein’s structure and size ( Figure 1d ). The charge bipolarity of the portal protein’s external surface 37 prompts its orientation in the direction of the electric field, but the overall net force on the portal is such that reversing the bias results in ejection of the portal from the pore, limiting the applicability of our hybrid system at various applied voltages. In our current study, we have implemented the exact mechanism for the formation of the hybrid nanopore system along with chemically fixing the protein to the SiN x nanopore to inhibit its uncorking in reverse voltage and attain permanent immobilization of the portal protein within the support membrane.…”

“…In contrast to the most successfully implemented proteins as nanopore sensors that assemble to form channels upon insertion into the lipid bilayer or synthetic polymer membranes, the hydrophilic G20c portal protein is water-soluble and fully assembles in an aqueous solution. So far, our efforts have resulted in two distinct methods to utilize this protein as a nanopore sensor: 1. insertion into the lipid bilayer through a hydrophobic conjugate 37 and 2: corking into solid-state nanopore to form a lipid-free hybrid nanopore. 36 However, none of these methods have been efficient enough to deliver high-yield, full-range sensing capability in various experimental conditions, particularly failing at a high applied voltage.…”

“…Site-specific, reproducible, and uniformly oriented protein immobilization within a synthetic membrane in a manner that preserves the protein conformation and functionality requires an understanding of the membrane's surface chemistry, protein's properties, and the nature of their interaction to eliminate non-specific binding. We recently hacked the G20c portal protein into insertion into a lipid bilayer environment (unnatural for the portal protein) 37 and later demonstrated voltage-induced "corking" of the portal into a solid-state nanopore. 36 This protein has shown high stability and tunability.…”