Evidence of Unfolded Protein Translocation through a Protein Nanopore

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• 作者：Manuela Pastoriza-Gallego ; Marie-France Breton ; Fran莽oise Discala ; Lo茂c Auvray ; Jean-Michel Betton ; Juan Pelta
• 刊名：ACS Nano
• 出版年：2014
• 出版时间：November 25, 2014
• 年：2014
• 卷：8
• 期：11
• 页码：11350-11360
• 全文大小：758K
• ISSN：1936-086X

文摘

Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein鈥搇igand 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.

Keywords:

nanopore; aerolysin; translocation; qRT-PCR; maltose binding protein; single molecule; protein folding