Single-molecule nanopore sensing of proline cis/trans amide isomers

Abstract

Molecules known as stereoisomers possess identical numbers and types of atoms, which are oriented differently in space. Cis–trans 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 cis–trans 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 cis and trans 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 cis/trans conformers' equilibrium compared to the natural proline: for 4-(R)-fluoroproline, the trans amide isomer is more favored, while the opposite shift was observed for 4-(S)-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.