Abstract
Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein-ligand complexes. This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Here, we report the translocation of a chimera molecule through the aerolysin nanopore in the presence of a denaturing agent, guanidium chloride (1.5 M) and KCl (1 M). The chimera molecule is composed of the recombinant MalE protein with a unique cysteine residue at the C-terminal position covalently linked to a single-stranded DNA oligonucleotide. Real-time polymerase chain reaction (PCR) was used to detect the presence of chimera molecules that have been effectively translocated from the cis to trans chamber of the set up. Comparing the electrical signature of the chimera related to the protein or oligonucleotide alone demonstrates that each type of molecule displays different dynamics in term of transport time, event frequency, and current blockade. This original approach provides the possibility to study protein translocation through different biological, artificial, and biomimetic nanopores or nanotubes. New future applications are now conceivable such as protein refolding at the nanopore exit, peptides and protein sequencing, and peptide characterization for diagnostics.
Highlights
Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein ligand complexes
Proteins nanopores are mainly used to study protein translocation,25À27 protein unfolding by denaturing agent,[21,28,29] electrical force,[22] pH,30 or by molecular motor,[14] to study protein traffic in cells,[8] to probe disordered protein folding by divalent cations[31] or by a peptide ligand,[17] to detect native proteins with aptamers covalently attached to the nanopore[32,33] or with directed evolution approach to design and obtain new channels.[34,35]
We have designed and produced a monodisperse chimera molecule composed of a recombinant protein with a unique cysteine residue at the C-terminal position covalently linked to a single-stranded DNA oligonucleotide
Summary
Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein ligand complexes This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Comparing the electrical signature of the chimera related to the protein or oligonucleotide alone demonstrates that each type of molecule displays different dynamics in term of transport time, event frequency, and current blockade This original approach provides the possibility to study protein translocation through different biological, artificial, and biomimetic nanopores or nanotubes. In contrast with other previous results,[50,55] we describe here the first assay to directly detect translocated molecules, and not their activity, using single protein channels
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
