Computational nanopore redesign for the sensing of chiral peptide isomers

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n the broad portfolio of nanopore sensing applications, the identification of chiral peptide isoforms is an important one, relevant for example to disease diagnostics and the elucidation of the enantiomeric purity of peptide therapeutics. Differentiating enantiomeric isoforms of peptides using standard bioanalytical and chromatographic techniques is both complicated, expensive, and time-consuming. Nanopore sensing offers the possibility of a portable, inexpensive, and rapid detection of chiral peptides. Early studies of nanopore sensing of chiral molecules relied on the introduction of chiral moieties to bias the detection of one chiral form over the other. More recent work on peptides has shown that differences in the peptide-nanopore interactions, and consequently in the affinities and kinetics of peptide isoforms, can give rise to characteristic current signals. Based on these findings, we hypothesize that for a given nanopore one could design a set of mutations that can bind the D and L isoforms of a peptide with distinct conformations and affinities – such a mutant nanopore should in principle generate different currents for the two isoforms. To arrive at such a nanopore design, we propose to develop a computational nanopore redesign pipeline that combines the Rosetta protein redesign algorithms with a fast analytical method for estimating the open pore and blockade currents to generate nanopore designs that are computationally predicted to be stable and produce a distinct signal for the peptide isoforms. Within this protocol, the computational design algorithm would serve to simultaneously optimize the nanopore-peptide interactions of both peptide isoforms to enable the binding of the peptides with different affinities and conformations. For the estimation of the open pore and blockage currents, we plan to employ the recently developed steric exclusion model enabling the screening for promising designs after successive rounds of computational design optimization. In our previous work, we had investigated the OmpF nanopore for the detection of the enantiomeric forms of a pentapeptide, demonstrating the potential of the pore for chirality sensing. In the proposed project, we plan to investigate the same pore (and its homologues) together with similar peptides, with the goal of improving the ionic current discrimination of the peptides through the application of the proposed computational pipeline, enabling a straightforward comparison with the available experimental data. A major part of the proposed work would focus on the development and testing of the design pipeline and the current estimation procedure with comparisons to data from all-atom MD simulations where appropriate. In conclusion, we expect to arrive at a set of potential nanopore mutants that allow a clear discrimination of different isoforms and that can be used as base for the experimental testing of such nanopores.