Unidirectional single-file transport of full-length proteins through a nanopore.

Autor: Yu L; Department of Physics, Northeastern University, Boston, MA, USA., Kang X; Department of Bioengineering, Northeastern University, Boston, MA, USA., Li F; Department of Chemistry, University of Massachusetts at Amherst, Amherst, MA, USA., Mehrafrooz B; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA., Makhamreh A; Department of Bioengineering, Northeastern University, Boston, MA, USA., Fallahi A; Department of Bioengineering, Northeastern University, Boston, MA, USA., Foster JC; Molecular and Cellular Biology Program, University of Massachusetts at Amherst, Amherst, MA, USA., Aksimentiev A; Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL, USA.; Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA.; Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, IL, USA., Chen M; Department of Chemistry, University of Massachusetts at Amherst, Amherst, MA, USA.; Molecular and Cellular Biology Program, University of Massachusetts at Amherst, Amherst, MA, USA., Wanunu M; Department of Physics, Northeastern University, Boston, MA, USA. wanunu@neu.edu.; Department of Bioengineering, Northeastern University, Boston, MA, USA. wanunu@neu.edu.; Chemistry and Chemical Biology, Northeastern University, Boston, MA, USA. wanunu@neu.edu.
Jazyk: angličtina
Zdroj: Nature biotechnology [Nat Biotechnol] 2023 Aug; Vol. 41 (8), pp. 1130-1139. Date of Electronic Publication: 2023 Jan 09.
DOI: 10.1038/s41587-022-01598-3
Abstrakt: The electrical current blockade of a peptide or protein threading through a nanopore can be used as a fingerprint of the molecule in biosensor applications. However, threading of full-length proteins has only been achieved using enzymatic unfolding and translocation. Here we describe an enzyme-free approach for unidirectional, slow transport of full-length proteins through nanopores. We show that the combination of a chemically resistant biological nanopore, α-hemolysin (narrowest part is ~1.4 nm in diameter), and a high concentration guanidinium chloride buffer enables unidirectional, single-file protein transport propelled by an electroosmotic effect. We show that the mean protein translocation velocity depends linearly on the applied voltage and translocation times depend linearly on length, resembling the translocation dynamics of ssDNA. Using a supervised machine-learning classifier, we demonstrate that single-translocation events contain sufficient information to distinguish their threading orientation and identity with accuracies larger than 90%. Capture rates of protein are increased substantially when either a genetically encoded charged peptide tail or a DNA tag is added to a protein.
(© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)
Databáze: MEDLINE