Nanopore Analysis of Peptides and Proteins

Nanopore sensing is a single-molecule technique capable of detecting peptide and protein molecules by monitoring the change in current generated by their interaction with protein or solid-state nanopores in an applied electric field. The interaction of a small globular protein HPr and two of its mutants, with the aerolysin nanopore were analyzed and compared with earlier results obtained with the α-hemolysin nanopore. The HPr molecules interact differently with the two nanopores while the anatomy and net charge of the pores affect their translocation parameters. Cleavage of insulin’s disulfide bonds with the reducing agent TCEP and the release of the component polypeptides could also be detected by nanopore analysis. An alternating current field superimposed on the direct current field inhibited the translocation of a peptide with a permanent dipole moment, while another peptide with no dipole moment was less affected. The detection of conformational changes in peptides and small proteins caused by metal ion binding also proved possible. A Zn-finger protein was able to translocate the α-hemolysin pore in the absence of Zn(II), while mostly bumping events were observed when Zn(II) was added. By comparison, the FSD-1 protein, which folds into a Zn-finger motif by hydrophobic interactions alone, was not able to translocate. The metal binding ability of three prion peptides was studied with an α-hemolysin pore. The results clearly indicated that Cu(II) and Zn(II) bound to all three peptides and caused conformational changes reflected in their interaction parameters with the α-hemolysin pore. The interaction of HPr, calmodulin and maltose binding protein with 7 nm and 5 nm diameter silicon nitride (SixNy) pores indicated that protein molecules with dimensions comparable to, or larger than the pore diameter do not translocate. However, smaller proteins are able to translocate in a folded conformation. Finally, the formation of prion/antibody complexes was successfully detected with an 11 nm SixNy pore but not with a 19 nm pore. The results underline the importance of choosing a pore with a suitable diameter in relation to the size of the analytes