Orientation Identification of Peptide Translocation Through an Aerolysin Nanopore

Abstract

Nanopores have been proven to have a wide application in biosensing at the single-molecule level. Nanopore-based protein sequencing is getting more and more attention. Recently it has been reported that 13 of the 20 natural amino acids with a short polycationic carrier can be distinguished by aerolysin nanopore via the current blockades, demonstrated that aerolysin nanopore is a suitable tool for protein sequencing. However, there are still more challenges that are needed to overcome while nanopore-based protein sequencing such as the uncertain orientation of the peptide through the pore and the non-uniform charge of the polypeptidic chain, etc. To determine the direction of the protein translocating through the aerolysin nanopore, a mutant aerolysin nanopore was used to detect the Aβ25-35 and Aβ35-25 fragments modified by specific charged tags at C-terminal or N-terminal in our experiments. The results showed that the duration time of N-terminal modified peptide Aβ25-35 and Aβ35-25 is nearly 6 times and 4 times respectively of the C-terminal modified polypeptides at +100 mV, indicating the N-terminal modified peptide have a stronger interaction with aerolysin nanopore. The difference in the interaction behavior between the N-terminal and C-terminal and aerolysin nanopore provides a new idea for designing the threading orientation of peptide for peptide sequencing.