Sensing PEGylated Peptide Conformations Using a Protein Nanopore

Abstract

Membrane pores are exploited for the stochastic sensing of various analytes, and here, we use electrical recordings to explore the interaction of PEGylated peptides of different sizes with a protein pore, CymA. This wide-diameter natural pore comprises densely filled charged residues, facilitating electrophoretic binding of polyethylene glycol (PEG) tagged with a nonaarginine peptide. The small PEG 200 peptide conjugates produced monodisperse blockages and exhibited voltage-dependent translocation across the pores. Notably, the larger PEG 1000 and 2000 peptide conjugates yielded heterogeneous blockages, indicating a multitude of PEG conformations hindering their translocation through the pore. Furthermore, a much larger PEG 5000 peptide occludes the pore entrance, resulting in complete closure. The competitive binding of different PEGylated peptides with the same pore produced specific blockage signals reflecting their identity, size, and conformation. Our proposed model of sensing distinct polypeptide conformations corresponds to disordered protein unfolding, suggesting that this pore can find applications in proteomics.

Conformational flexibility driving charge-selective substrate translocation across a bacterial transporter

“…For example, pores showed slightly higher conductance at positive voltages and more frequent gating at negative voltages. Based on this asymmetry in the pore conductance and gating with the direction of the voltage, we presumed the pore orientation, as shown in our previous studies 22,31 (Fig. 1 and S1–S3†).…”

“…17 Previously, we have shown that cationic CDs and peptides bind to the trans side of the pore with high affinity compared to the cis side, which agrees with the charge distribution in the pore, which allowed us to presume pore orientation. 22,31 Thus, adding CymA to the cis side of the preformed bilayer results in pore insertion with the extracellular side (pore entrance and dense charge pattern) exposed at the trans compartment (Fig. 1a and S1†).…”