Single-molecule nanopore sensing of proline / amide isomers.

Molecules known as stereoisomers possess identical numbers and types of atoms, which are oriented differently in space. - isomerization of proline, a distinctive case of stereoisomerism in peptides and proteins, includes the rearrangement of chemical groups around an acyl-proline amide bond that bears the partial double bond character. Many cellular processes are affected by - proline isomerization and associated conformational protein interconversions. This work explored the conformer ratio of natural and chemically modified prolines using the aerolysin pore as a nanosensor. Despite the well-known involvement of proline in protein folding, stability, and aggregation, the highly demanding discrimination of and isomers of the Xaa-Pro peptide bond has not so far been reported at a single-molecule level using an electrical detection with a nanopore. For a proline-rich 19 amino acid residue fragment of the Dynamin 2 protein, one of the subfamilies of GTP-binding proteins, the third proline in the sequence was substituted by two stereoisomeric 4-fluoroprolines. The nanopore experiments were able to sense the influence of fluorination in shifting the / conformers' equilibrium compared to the natural proline: for 4-()-fluoroproline, the amide isomer is more favored, while the opposite shift was observed for 4-()-fluoroproline. NMR spectroscopy was used to validate the nanopore results. Overall, our findings demonstrate the high sensitivity of single-molecule nanopore sensing as an analytical tool for stereoisomer identification within peptides.