title: Unfoldase-mediated protein translocation through an α-hemolysin nanopore.
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.
authors: Nanopore Group, Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, California, USA.
authors: Jeff
authors: J
authors: Nivala
authors: Douglas B
authors: DB
authors: Marks
authors: Mark
authors: M
authors: Akeson
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism*; D001709:Biotechnology; D049071:Endopeptidase Clp / Q000737:chemistry* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry* / Q000378:metabolism*; D058608:Nanopores; D021381:Protein Transport; D058767:Protein Unfolding
chemical_list: D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D018832:Molecular Chaperones; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities
references: Nat Nanotechnol. 2010 Nov;5(11):798-806
references: 20871614
references: J Am Chem Soc. 2008 Mar 26;130(12):4081-8
references: 18321107
references: Nature. 2005 Oct 20;437(7062):1115-20
references: 16237435
references: Cell. 2011 Apr 15;145(2):257-67
references: 21496645
references: Phys Rev Lett. 2007 Apr 13;98(15):158101
references: 17501386
references: EMBO J. 1997 Sep 15;16(18):5509-19
references: 9312010
references: Trends Biotechnol. 2009 Jun;27(6):333-41
references: 19394097
references: Cell. 2003 Aug 22;114(4):511-20
references: 12941278
references: Protein Sci. 2002 Jun;11(6):1482-91
references: 12021447
references: Nat Biotechnol. 2012 Feb 14;30(4):344-8
references: 22334048
references: Cell. 2011 Apr 29;145(3):459-69
references: 21529717
references: ACS Chem Biol. 2012 Apr 20;7(4):652-8
references: 22260417
references: J Pept Sci. 2011 Nov;17(11):726-34
references: 21766390
references: J Am Chem Soc. 2009 Jul 8;131(26):9287-97
references: 19530678
references: Mol Cell. 2000 Apr;5(4):639-48
references: 10882100
references: Cell. 1991 May 3;65(3):371-80
references: 1902142
references: Biochim Biophys Acta. 2012 Jan;1823(1):15-28
references: 21736903
references: Genes Dev. 1998 May 1;12(9):1338-47
references: 9573050
references: Nat Biotechnol. 2012 Mar 25;30(4):349-53
references: 22446694