Unfoldase-mediated protein translocation through an α-hemolysin nanopore

Nat Biotechnol. 2013 Mar;31(3):247-50. doi: 10.1038/nbt.2503. Epub 2013 Feb 3.

Abstract

Using nanopores to sequence biopolymers was proposed more than a decade ago. Recent advances in enzyme-based control of DNA translocation and in DNA nucleotide resolution using modified biological pores have satisfied two technical requirements of a functional nanopore DNA sequencing device. Nanopore sequencing of proteins was also envisioned. Although proteins have been shown to move through nanopores, a technique to unfold proteins for processive translocation has yet to be demonstrated. Here we describe controlled unfolding and translocation of proteins through the α-hemolysin (α-HL) pore using the AAA+ unfoldase ClpX. Sequence-dependent features of individual engineered proteins were detected during translocation. These results demonstrate that molecular motors can reproducibly drive proteins through a model nanopore--a feature required for protein sequence analysis using this single-molecule technology.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / metabolism*
  • Biotechnology
  • Endopeptidase Clp / chemistry*
  • Endopeptidase Clp / metabolism*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism*
  • Hemolysin Proteins / chemistry*
  • Hemolysin Proteins / metabolism*
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism
  • Models, Molecular
  • Molecular Chaperones / chemistry*
  • Molecular Chaperones / metabolism*
  • Nanopores*
  • Protein Transport
  • Protein Unfolding*

Substances

  • Escherichia coli Proteins
  • Hemolysin Proteins
  • Lipid Bilayers
  • Molecular Chaperones
  • Endopeptidase Clp
  • Adenosine Triphosphatases
  • ClpX protein, E coli
  • ATPases Associated with Diverse Cellular Activities