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The gram-negative bacterium Escherichia coli exhibits a so-called outer membrane, which acts as a barrier against the cell environment. Here, the trimeric matrix porin OmpF is located. The porin forms a β-barrel like structure and is restricted to 7 x 11 Å at the eyelet due to loop region L3. This protein was used as a template for generating hybrid ion-channels. Two different modification strategies were employed, and the resulting pore properties were characterised by single channel measurements via the Black Lipid Membrane method (BLM) and crystallographic analysis.
The first modification strategy is based on splitting the protein into an N-terminal fragment, which is synthesized by Solid Phase Peptide Synthesis (SPPS), whereas the C-terminal polypeptide is produced by recombinant means. The synthesis of the N-terminal peptide allows the incorporation of a non-natural amino acid comprising an alkyne moiety. By using [3+2]-Click-Chemistry, the fluorophore dansylazide was coupled to the alkyne modified peptide. Subsequent ligation via Native Chemical Ligation (NCL) under 8 M urea yielded full-length OmpF.
To perform the second strategy several cysteine residues were introduced into OmpF by site-directed mutagenesis. Two different iodoacetamide-activated modulators, the above mentioned dansyl-compound and a dibenzo-18-crown-6-derivative, were coupled to OmpF via an S-alkylation reaction. Hybrid OmpF pores generated by both strategies were refolded by rapid dilution into a vesicle containing suspension.
Single channel measurements of the refolded reference OmpF proteins showed clearly that their conductance is comparable to wild type OmpF (0.99±0.02 nS) extracted from cell envelopes. This demonstrates that both strategies are valid routes for obtaining nature-like hybrid ion-channels.
It was found, that, beside the properties of the modulator and the attachment site in the pore, the way of generating the hybrids was crucial for modulating the ion current. The produced hybrid ion-channels via S-alkylation exhibited a strong distribution of measured conductance levels, which corresponds to a reduction by 16-19% compared to the wild type. Surprisingly, the dansylhybrid derived by [3+2]-Click-Chemistry showed no significant alteration of its conductance. The dansyl-modulator was also introduced in a site-dependent fashion, and a distance of only ~5 Å was sufficient to change the effect of conductance levels dramatically.
The crystallisation of the crown-ether hybrid yielded a novel crystal form. X-ray analysis with a resolution of up to 3.2 Å revealed an inward-oriented crown-ether conformation. This orientation is strongly stabilized by electrostatic contacts between the ethylene oxygen atoms and several acidic and basic side chains of the inner porin wall, which causes partial blockage of the pore.