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Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level

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Abstract

Single channel electrophysiological studies have been carried out to elucidate the underlying interactions during the translocation of polypeptides through protein channels. For this we used OmpF from the outer cell membrane of E. coli and arginine-based peptides of different charges, lengths and covalently linked polyethylene glycol as a model system. In order to reveal the fast kinetics of peptide binding, we performed a temperature scan. Together with the voltage-dependent single-channel conductance, we quantify peptide binding and translocation.

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References

  • Ahting U, Thieffry M, Engelhardt H, Hegerl R, Neupert W, Nussberger S (2001) Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria. J Cell Biol 153:1151–1160

    Article  PubMed  CAS  Google Scholar 

  • Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Watler P (2008) Molecular biology of the cell. Garland Science, New York

    Google Scholar 

  • Apetrei A, Asandei A, Park Y, Hahm KS, Winterhalter M, Luchian T (2010) Unimolecular study of the interaction between the outer membrane protein OmpF from E. coli and an analogue of the HP(2–20) antimicrobial peptide. J Bioenerg Biomembr 42:173–180

    Article  PubMed  CAS  Google Scholar 

  • Benz R, Bauer K (1998) Permeation of hydrophilic molecules through the outer membrane of Gram-negative bacteria. Eur J Biochem 176:1–19

    Article  Google Scholar 

  • Bikwemu R, Wolfe AJ, Xing X, Movileanu L (2010) Facilitated translocation of polypeptides through a single nanopore. J Phys: Condens Matter 22:454117

    Article  Google Scholar 

  • Chacinska A, Koehler CM, Milenkovic D, Lithgow T, Pfanner N (2009) Importing mitochondrial proteins: machineries and mechanisms. Cell 2009(138):628–644

    Article  Google Scholar 

  • Chimerel C, Movileanu L, Pezeshki S, Winterhalter M, Kleinekathöfer U (2008) Transport at the nanoscale: temperature dependence of ion conductance. Eur Biophys J 38:121–125

    Article  PubMed  CAS  Google Scholar 

  • Cowan SW, Schirmer T, Rummel G, Steiert M, Ghosh R, Pauptit RA, Jansonius JN, Rosenbusch JP (1992) Crystal structures explain functional properties of two Escherichia coli porins. Nature 358:727–733

    Article  PubMed  CAS  Google Scholar 

  • Cowan SW, Garavito RM, Jansonius JN, Jenkins JA, Karlsson R, Konig N, Pai EF, Pauptit RA, Rizkallah PJ, Rosenbusch JP, Rummel G, Schirmer T (1995) The structure of OmpF porin in a tetragonal crystal form. Structure 3:1041–1050

    Article  PubMed  CAS  Google Scholar 

  • Danelon C, Brando T, Winterhalter M (2003) Probing the orientation of reconstituted maltoporin channels at the single-protein level. J Biol Chem 278:35542–35551

    Article  PubMed  CAS  Google Scholar 

  • Garavito RM, Rosenbusch JP (1986) Isolation and crystallization of bacterial porin. Meth Enzymol 125:309–328

    Article  PubMed  CAS  Google Scholar 

  • Hancock REW, Brinkman FSL (2002) Function of Pseudomonas porins in uptake and efflux. Annu Rev Microbiol 56:17–38

    Article  PubMed  CAS  Google Scholar 

  • Harsman A, Krüger V, Bartsch P, Honigmann A, Schmidt O, Rao S, Meisinger C, Wagner R (2010) Protein conducting nanopores. J Phys Condens Matter 22:454102

    Article  PubMed  Google Scholar 

  • Harsman A, Bartsch P, Hemmis B, Krüger V, Wagner R (2011) Exploring protein import pores of cellular organelles at the single molecule level using the planar lipid bilayer technique. Eur J Cell Biol 90:721–730

    Article  PubMed  CAS  Google Scholar 

  • Hill K, Model K, Ryan MT, Dietmeier K, Martin F, Wagner R, Pfanner N (1998) Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature 395:516–521

    Article  PubMed  CAS  Google Scholar 

  • Lithgow T (2000) Targeting of proteins to mitochondria. FEBS Lett 476:22–26

    Article  PubMed  CAS  Google Scholar 

  • Lugtenberg B, Van Alphen L (1983) Molecular architecture and functioning of the outer membrane of Escherichia coli and other gram-negative bacteria. Biochim Biophys Acta 737:51–115

    Article  PubMed  CAS  Google Scholar 

  • Mahendran KR, Chimerel C, Mach T, Winterhalter M (2009) Antibiotic translocation through membrane channels: temperature-dependent ion current fluctuation for catching the fast events. Eur Biophys J 38:1141–1145

    Article  PubMed  CAS  Google Scholar 

  • Mahendran KR, Hajjar E, Mach T, Lovelle M, Kumar A, Sousa I, Spiga E, Weingart H, Gameiro P, Winterhalter M, Ceccarelli M (2010) Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli—when binding does not imply translocation. J Phys Chem B 114:5170–5179

    Article  PubMed  CAS  Google Scholar 

  • Mahendran KR, Romero-Ruiz M, Schlösinger A, Winterhalter M, Nussberger S (2012) Protein translocation through Tom40: kinetics of peptide release. Biophys J 102:39–47

    Article  PubMed  CAS  Google Scholar 

  • Modi N, Singh P, Mahendran K, Schulz R, Winterhalter M, Kleinekathoefer U (2011) Probing the transport of ionic liquids in aqueous solution through nanopores. J Phys Chem Lett 2:2331–2336

    Article  CAS  Google Scholar 

  • Mohammad MM, Movileanu L (2008) Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology. Eur Biophys J 37:913–925

    Article  PubMed  CAS  Google Scholar 

  • Movileanu L, Bayley H (2001) Partitioning of a polymer into a nanoscopic protein pore obeys a simple scaling law. Proc Natl Acad Sci USA 98:10137–10141

    Article  PubMed  CAS  Google Scholar 

  • Movileanu L, Schmittschmitt JP, Scholtz JM, Bayley H (2005) Interactions of peptides with a protein pore. Biophys J 89:1030–1045

    Article  PubMed  CAS  Google Scholar 

  • Nestorovich EM, Danelon C, Winterhalter M, Bezrukov SM (2002) Designed to penetrate. Time resolved interaction of single antibiotic molecules with bacterial pores. Proc Natl Acad Sci USA 99:9789

    Article  PubMed  CAS  Google Scholar 

  • Neupert W, Herrmann JM (2007) Translocation of proteins into mitochondria. Annu Rev Biochem 76:723–749

    Article  PubMed  CAS  Google Scholar 

  • Orlik F, Andersen C, Danelon C, Winterhalter M, Pajatsch M, Böck A, Benz R (2003) CymA of Klebsiella oxytoca outer membrane. Binding of cyclodextrins and study of the current noise of open channels. Biophys J 85:876–885

    Article  PubMed  CAS  Google Scholar 

  • Oukhaled G, Mathé J, Biance AL, Bacari L, Betton JM, Lairez D, Pelta J, Auvray L (2007) Unfolding of proteins and long transient conformations detected by single nanopore recording. Phys Rev Lett 98:158101–158104

    Article  PubMed  CAS  Google Scholar 

  • Pastoriza-Gallego M, Rabah L, Gibrat G, Thiebot B, Van der Goot FG, Auvray L, Betton JM, Pelta J (2010) Dynamics of unfolded protein transport through an aerolysin pore. J Am Chem Soc 133:2923–2931

    Article  Google Scholar 

  • Pezeshki S, Chimerel C, Bessonov AN, Winterhalter M, Kleinekathöfer U (2009) Understanding ion conductance on a molecular level: an all-atom modeling of the bacterial porin OmpF. Biophys J 97:1898–1906

    Article  PubMed  CAS  Google Scholar 

  • Prilipov A, Phale PS, Van Gelder P, Rosenbusch JP, Koebnik R (1998) Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. FEMS Microbiol Lett 163:65–72

    Article  PubMed  CAS  Google Scholar 

  • Romero-Ruiz M, Mahendran KR, Eckert R, Winterhalter M, Nussberger S (2010) Interactions of mitochondrial presequence peptides with the mitochondrial outer membrane preprotein translocase TOM. Biophys J 99:774–781

    Article  PubMed  CAS  Google Scholar 

  • Rosenbusch JP (1974) Characterization of the major envelope protein from Escherichia coli. Regular arrangement on the peptidoglycan and unusual dodecyl sulfate binding. J Biol Chem 249:8019–8029

    PubMed  CAS  Google Scholar 

  • Rostovtseva TK, Nestorovich EM, Bezrukov SM (2002) Partitioning of differently sized poly (ethylene glycol)s into OmpF porin. Biophys J 82:160–169

    Article  PubMed  CAS  Google Scholar 

  • Schatz G, Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271:1519–1526

    Article  PubMed  CAS  Google Scholar 

  • Singh PR, Ceccarelli M, Lovelle M, Winterhalter M, Mahendran KR (2012) Antibiotic permeation across the OmpF channel: modulation of the affinity site in the presence of magnesium. J Phys Chem B 116:4433–4438

    Article  PubMed  Google Scholar 

  • Walther DM, Rapaport D (2009) Biogenesis of mitochondrial outer membrane proteins. Biochim Biophys Acta 1793:42–51

    Article  PubMed  CAS  Google Scholar 

  • Wolfe AJ, Mohammad MM, Cheley S, Bayley H, Movileanu L (2007) Catalyzing the translocation of polypeptides through attractive interactions. J Am Chem Soc 129:14034–14041

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge the DFG for financial support through Wi2278/18-1. Special thanks to Niraj Modi for providing the structural schema of OmpF.

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Correspondence to Mathias Winterhalter.

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Lamichhane, U., Islam, T., Prasad, S. et al. Peptide translocation through the mesoscopic channel: binding kinetics at the single molecule level. Eur Biophys J 42, 363–369 (2013). https://doi.org/10.1007/s00249-012-0885-6

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  • DOI: https://doi.org/10.1007/s00249-012-0885-6

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