Pulling peptides across nanochannels: resolving peptide binding and translocation through the hetero-oligomeric channel from Nocardia farcinica.

We investigated translocation of cationic peptides through nanochannels derived from the Gram-positive bacterium Nocardia farcinica at the single-molecule level. The two subunits NfpA and NfpB form a hetero-oligomeric cation selective channel. On the basis of amino acid comparison we performed homology modeling and obtained a channel structurally related to MspA of Mycobacterium smegmatis. The quantitative single-molecule measurements provide an insight into transport processes of solutes through nanochannels. High-resolution ion conductance measurements in the presence of peptides of different charge and length revealed the kinetics of peptide binding. The observed asymmetry in peptide binding kinetics indicated a unidirectional channel insertion in the lipid bilayer. In the case of cationic peptides, the external voltage acts as a driving force that promotes the interaction of the peptide with the channel surface. At low voltage, the peptide just binds to the channel, whereas at higher voltage, the force is strong enough to pull the peptide across the channel. This allows distinguishing quantitatively between peptide binding and translocation through the channel.

[1]  A. Wolfe,et al.  Facilitated translocation of polypeptides through a single nanopore , 2010, Journal of physics. Condensed matter : an Institute of Physics journal.

[2]  H. Bayley,et al.  Catalyzing the translocation of polypeptides through attractive interactions. , 2007, Journal of the American Chemical Society.

[3]  J. M. Scholtz,et al.  Interactions of peptides with a protein pore. , 2005, Biophysical journal.

[4]  S. Bezrukov,et al.  Polymeric nonelectrolytes to probe pore geometry: application to the alpha-toxin transmembrane channel. , 1999, Biophysical journal.

[5]  J. Betton,et al.  Unfolding of proteins and long transient conformations detected by single nanopore recording. , 2007, Physical review letters.

[6]  J. Rosenbusch,et al.  Coupling site-directed mutagenesis with high-level expression: large scale production of mutant porins from E. coli. , 1998, FEMS microbiology letters.

[7]  H. Nikaido,et al.  Diffusion of solutes through channels produced by phage lambda receptor protein of Escherichia coli: inhibition by higher oligosaccharides of maltose series. , 1980, Biochemical and biophysical research communications.

[8]  B. Krasniqi,et al.  Nanopore analysis of tethered peptides , 2010, Journal of peptide science : an official publication of the European Peptide Society.

[9]  R. Benz,et al.  Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior. , 1994, Biophysical journal.

[10]  H. Nikaido,et al.  Porins and specific channels of bacterial outer membranes , 1992, Molecular microbiology.

[11]  H. Nikaido,et al.  The envelope of mycobacteria. , 1995, Annual review of biochemistry.

[12]  M. Winterhalter,et al.  Probing the Orientation of Reconstituted Maltoporin Channels at the Single-protein Level* , 2003, Journal of Biological Chemistry.

[13]  C. Dobson.  Protein folding and misfolding , 2003, Nature.

[14]  L. Movileanu,et al.  Excursion of a single polypeptide into a protein pore: simple physics, but complicated biology , 2008, European Biophysics Journal.

[15]  Eric Hajjar,et al.  Molecular basis of enrofloxacin translocation through OmpF, an outer membrane channel of Escherichia coli--when binding does not imply translocation. , 2010, The journal of physical chemistry. B.

[16]  Sergey M. Bezrukov,et al.  Counting polymers moving through a single ion channel , 1994, Nature.

[17]  C. Andersen,et al.  The channel-tunnel HI1462 of Haemophilus influenzae reveals differences to Escherichia coli TolC. , 2006, Microbiology.

[18]  L. Movileanu,et al.  Substrate Specificity within a Family of Outer Membrane Carboxylate Channels , 2012, PLoS biology.

[19]  G. Patriarche,et al.  Protein transport through a narrow solid-state nanopore at high voltage: experiments and theory. , 2012, ACS nano.

[20]  R. Benz,et al.  Ion selectivity of gram-negative bacterial porins , 1985, Journal of bacteriology.

[21]  H. Bayley,et al.  Identification of epigenetic DNA modifications with a protein nanopore. , 2010, Chemical communications.

[22]  H. Nikaido,et al.  Porin channels in Escherichia coli: studies with liposomes reconstituted from purified proteins , 1983, Journal of bacteriology.

[23]  K. Schulten,et al.  Imaging alpha-hemolysin with molecular dynamics: ionic conductance, osmotic permeability, and the electrostatic potential map. , 2005, Biophysical journal.

[24]  S. Bezrukov,et al.  Functional subconformations in protein folding: evidence from single-channel experiments. , 2005, Physical review letters.

[25]  M Montal,et al.  Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[26]  H. Engelhardt,et al.  MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis , 2001, Molecular microbiology.

[27]  A. Berezhkovskii,et al.  Optimizing transport of metabolites through large channels: molecular sieves with and without binding. , 2005, Biophysical journal.

[28]  F. G. van der Goot,et al.  Dynamics of unfolded protein transport through an aerolysin pore. , 2011, Journal of the American Chemical Society.

[29]  S. Bezrukov,et al.  Ion Channels as Molecular Coulter Counters to Probe Metabolite Transport , 2000, The Journal of Membrane Biology.

[30]  D. O. Rudin,et al.  Induced excitability in reconstituted cell membrane structure. , 1963, Journal of theoretical biology.

[31]  Cees Dekker,et al.  Hybrid pore formation by directed insertion of α-haemolysin into solid-state nanopores. , 2010, Nature nanotechnology.

[32]  R. Benz,et al.  Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli. , 1978, Biochimica et biophysica acta.

[33]  Georg E. Schulz,et al.  The Structure of a Mycobacterial Outer-Membrane Channel , 2004, Science.

[34]  R. Benz,et al.  Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica. , 2011, Biochimica et biophysica acta.

[35]  R. Benz,et al.  The cell wall porin of Nocardia farcinica: biochemical identification of the channel‐forming protein and biophysical characterization of the channel properties , 1998, Molecular microbiology.

[36]  R. Benz,et al.  The cell wall porin of the gram-positive bacterium Nocardia asteroides forms cation-selective channels that exhibit asymmetric voltage dependence , 1999, Archives of Microbiology.

[37]  M. Muthukumar,et al.  Polymer translocation through alpha-hemolysin pore with tunable polymer-pore electrostatic interaction. , 2010, The Journal of chemical physics.

[38]  M. Muthukumar.  Mechanism of DNA transport through pores. , 2007, Annual review of biophysics and biomolecular structure.