Unfolding of β-hairpin and α-helical peptides through a biological nanopore.

Abstract

Nanopore sensing is a useful tool for the rapid detection of analytes at the single-molecule level. Since this technology allows high sensitivity detection based on the ion currents, it is expected to be applied to protein sequencing and folding analysis of proteins. In particular, biological nanopores are 1-4 nm in the size, and proteins pass through the nanopore with unfolding its inside. Therefore, the ion currents will provide insight into the folding process of each secondary structure of proteins, and these analyses may be able to detect specific structures of proteins. Here, we synthesized β-hairpin and α-helix peptides, and analyzed the ion current of the translocation with unfolding. β-hairpin peptides were synthesized using cell-free synthesis, and mass spectrometry characterized each peptide and confirmed secondary structures by fluorescence measurement and circular dichroism spectrum. Channel current measurements identified the behavior of β-hairpin peptides passing through αHL nanopores. As result, signals with three current blocking rates were observed, reflecting the stepwise unfolding of β-hairpin peptides within αHL nanopores. Therefore, we estimated the unfolding behavior of peptides from each blocking state of the ion currents. The estimated results for each level are shown below. Level 1: β-hairpin structure hooked with the upper of nanopore. Level 2: in the pores, the pulling force caused partial unfolding allowing β-hairpin structure to traverse the pore. Level 3: remainder structures unfolded completely and left through the trans entrance. Our experiments with protein denaturants supported our estimations. We are trying to observe the translocation of α-helix peptides, and research the difference in unfolding process of β-hairpin peptides and that of α-helix peptides.