The Manipulation of the Internal Hydrophobicity of FraC Nanopores Augments Peptide Capture and Recognition
The detection of analytes and the sequencing of DNA using biological nanopores have seen major advances over recent years. The analysis of proteins and peptides with nanopores, however, is complicated by the complex physicochemical structure of polypeptides and the lack of understanding of the mechanism of capture and recognition of polypeptides by nanopores. In this work, we show that introducing aromatic amino acids at precise positions within the lumen of α-helical fragaceatoxin C (FraC) nanopores increased the capture frequency of peptides and largely improved the discrimination among peptides of similar size. Molecular dynamics simulations determined the sensing region of the nanopore, elucidated the microscopic mechanism enabling accurate characterization of the peptides via ionic current blockades in FraC, and characterized the effect of the pore modification on peptide discrimination. This work provides insights to improve the recognition and to augment the capture of peptides by nanopores, which is important for developing a real-time and single-molecule size analyzer for peptide recognition and identification.
SMD simulation of Ang-I peptide translocation through FraC-Wt-T1 pore. The movie shows an 80 ns MD trajectory where an Ang-I peptide (DRVYIHPFHL) was driven through the FraC WT type-I pore using the SMD protocol. The pore is shown as a grey, cutaway molecular surface; the DPhPC lipid bilayer is shown in cyan (head groups) and blue (lipid tails); the backbone of the peptide is shown in green; from the bottom, the first residue (D) is shown in orange, the second residue (R) is shown in yellow, the next 5 residues (VYIHPF) are shown in red, followed by the last two residues (HL) in cyan. The sensing region defined in Fig. 6D is shown as a translucent yellow region.
SMD simulation of Ang-I peptide translocation through FraC-G13F-T1 pore. The movie shows an 80 ns MD trajectory where an Ang-I peptide (DRVYIHPFHL) was driven through the FraC G13F type-I pore using the SMD protocol. The pore is shown as a grey, cutaway molecular surface; the DPhPC lipid bilayer is shown in cyan (head groups) and blue (lipid tails); the backbone of the peptide is shown in green; from the bottom, the first residue (D) is shown in orange, the second residue (R) is shown in yellow, the next 5 residues (VYIHPF) are shown in red, followed by the last two residues (HL) in cyan. The sensing region defined in Fig. 6E is shown as a translucent yellow region.
SMD simulation of Ang-I peptide translocation through FraC-Wt-T2 pore. The movie shows an 80 ns MD trajectory where an Ang-I peptide (DRVYIHPFHL) was driven through the FraC WT type-II pore using the SMD protocol. The pore is shown as a grey, cutaway molecular surface; the DPhPC lipid bilayer is shown in cyan (head groups) and blue (lipid tails); the backbone of the peptide is shown in green; from the bottom, the first residue (D) is shown in orange, the second residue (R) is shown in yellow, the next 5 residues (VYIHPF) are shown in red, followed by the last two residues (HL) in cyan. The sensing region defined in Fig. 6F is shown as a translucent yellow region.