Real-Time Nanopore-Based Recognition of Protein Translocation Success
David Paul Hoogerheide, Philip A. Gurnev, Tatiana K. Rostovtseva, Sergey M. Bezrukov
A growing number of new technologies are supported by a single- or multi-nanopore architecture for capture, sensing, and delivery of polymeric biomolecules.1-6 Nanopore-based single-molecule DNA sequencing, in which engineered nanopores7 are augmented with molecular machinery to control the motion of the DNA strand, 8,9 is the premier example. 10-12 This method relies on the uniform linear charge density of DNA, so that each DNA strand is overwhelmingly likely to "translocate", i.e. pass through the nanopore and across the separating membrane. For disordered peptides, 13 folded proteins, 14 or block copolymers with heterogeneous charge densities, translocation is not assured, and additional strategies to monitor the progress of the polymer molecule through a nanopore are required. Here we demonstrate a single-molecule method for direct, model-free, real-time monitoring of the translocation of a disordered, heterogeneously charged polypeptide through a nanopore. The crucial elements are two "selectivity tags" - regions of different but uniform charge density-at the ends of the polypeptide. These affect the selectivity of the nanopore differently and enable discrimination between polypeptide translocation and retraction. Our results demonstrate demonstrate exquisite sensitivity of polypeptide translocation to applied transmembrane potential and prove the principle that nanopore selectivity reports on biopolymer substructure. We anticipate the selectivity tag technique to be broadly applicable to nanopore-based protein dectection, analysis, and separation technologies. This work opens an avenue to solving long-standing, medically relevant question regarding protein translocation, such as bacterial toxicity mechanisms15,16 and the operation of cellular protein transport machinery. 17
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Real-Time Nanopore-Based Recognition of Protein Translocation Success, Biophysical Journal Biophysical Letter, [online], https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=923362
(Accessed May 31, 2023)