Oligomeric protein nanopores with rigid structures have been engineered for the purpose of sensing a wide range of analytes including small molecules and biological species such as proteins and DNA. We chose a monomeric β-barrel porin, OmpG, as the platform from which to derive the nanopore sensor. OmpG is decorated with seven flexible loops that move dynamically to create a distinct gating pattern when ionic current passes through the pore. Biotin was chemically tethered to the most flexible one of these loops. The gating characteristic of the loop’s movement in and out of the porin was substantially altered by analyte protein binding. The gating characteristics of the pore with bound targets were remarkably sensitive to molecular identity – even providing the ability to distinguish between homologues within an antibody mixture. A total of five gating parameters were analyzed for each analyte to create a unique fingerprint for each biotin binding protein. Our exploitation of gating noise as a molecular identifier may allow more sophisticated sensor design while OmpG’s monomeric structure greatly simplifies nanopore production.

Background: Cytolysin A (ClyA) is an ␣-pore-forming toxin secreted from pathogenic E. coli. Results: ClyA monomer assembles to an oligomeric pre-pore structure independently of lipid membrane and detergent. Conclusion: Our results support a model that ClyA proteins may oligomerize to a prepore within outer membrane vesicles before arriving on the target cell membrane. Significance: The proposed model for ClyA represents a non-classical pathway to attack eukaryotic host cells.

The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded β-barrel membrane protein functioning as a non-specific porin for the uptake of oligosaccharides in E. coli. Two different crystal structures of OmpG obtained at different values of pH suggest a pH-gated pore opening mechanism. In these structures, extracellular loop 6 extends away from the barrel wall at neutral pH, but is folded back into the pore lumen at low pH, blocking transport through the pore. Loop 6 was invisible in a previously published solution NMR structure of OmpG in DPC micelles, presumably due to conformational exchange on an intermediate NMR time-scale. Here we present an NMR paramagnetic relaxation enhancement (PRE)-based approach to visualize the conformational dynamics of loop 6 and to calculate conformational ensembles that explain the pH-gated opening and closing of the OmpG channel. The different loop conformers detected by the PRE ensemble calculations were validated by disulfide cross-linking of strategically engineered cysteines and electrophysiological single channel recordings. The results indicate a more dynamically regulated channel opening and closing than previously thought and reveal additional membrane-associated conformational ensembles at pH 6.3 and 7.0. We anticipate this approach to be generally applicable to detect and characterize functionally important conformational ensembles of membrane proteins.

Outer membrane protein G (OmpG) from Escherichia coli has exhibited pH-dependent gating that can be employed by bacteria to alter the permeability of their outer membranes in response to environmental changes. We developed a computational model, Protein Topology of Zoetic Loops (Pretzel), to investigate the roles of OmpG extracellular loops implicated in gating. The key interactions predicted by our model were verified by single-channel recording data. Our results indicate that the gating equilibrium is primarily controlled by an electrostatic interaction network formed between the gating loop and charged residues on the lumen. The results shed light on the mechanism of OmpG gating and will provide a fundamental basis for the engineering of OmpG as a nanopore sensor. Our computational Pretzel model could be applied to other outer membrane proteins that contain intricate dynamic loops that are functionally important.

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