Abstract
Single-channel planar lipid bilayer (PLB) recording of bacterial porins has revealed molecular details of transport across the outer membrane of Gram-negative bacteria, including antibiotic permeation and protein translocation. To explore directional transport processes across cellular membranes, the orientation of porins or other pore-forming proteins must be established in a lipid bilayer prior to experimentation. Here, we describe a direct method for determining the orientation of porins in a PLB—with a focus on E. coli OmpF—by using targeted covalent modification of cysteine mutants. Each of the two possible orientations can be correlated with the porin conductance asymmetry, such that thereafter an I–V curve taken at the start of an experiment will suffice to establish orientation.
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References
Pagès JM, James CE, Winterhalter M (2008) The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol 6:893–903
Qing Y, Pulcu GS, Bell NAW, Bayley H (2018) Bioorthogonal cycloadditions with sub-millisecond intermediates. Angew Chem Int Ed 57:1218–1221
Kullman L, Winterhalter M, Bezrukov SM (2002) Transport of maltodextrins through maltoporin: a single-channel study. Biophys J 82:803–812
Udho E, Jakes KS, Buchanan SK, James KJ, Jiang X, Klebba PE, Finkelstein A (2009) Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes. Proc Natl Acad Sci U S A 22:21990–21995
Housden NG, Hopper JTS, Lukoyanova N, Rodriguez-Larrea D, Wojdyla JA, Klein A, Kaminska R, Bayley H, Saibil HR, Robinson CV, Kleanthous C (2013) Intrinsically disordered protein threads through the bacterial outer-membrane porin OmpF. Science 340:1570–1574
Bayley H, Cremer PS (2001) Stochastic sensors inspired by biology. Nature 413:226–230
Harrington L, Cheley S, Alexander LT, Knapp S, Bayley H (2013) Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate. Proc Natl Acad Sci U S A 110:E4417–E4426
Ramsay WJ, Bayley H (2018) Single-molecule determination of the isomers of d-glucose and d-fructose that bind to boronic acids. Angew Chem Int Ed 57:2841–2845
Song L, Hobaugh MR, Shustak C, Cheley S, Bayley H, Gouaux JE (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274:1859–1866
Hoenger A, Pagès JM, Fourel D, Engel A (1993) The orientation of porin OmpF in the outer membrane of Escherichia coli. J Mol Biol 233:400–413
Brauser A, Schroeder I, Gutsmann T, Cosentino C, Moroni A, Hansen UP, Winterhalter M (2012) Modulation of enrofloxacin binding in OmpF by Mg2+ as revealed by the analysis of fast flickering single-porin current. J Gen Physiol 140:69–82
Ionescu SA, Lee S, Housden NG, Kaminska R, Kleanthous C, Bayley H (2017) Orientation of the OmpF porin in planar lipid bilayers. Chembiochem 18:554–562
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
Chen M, Li QH, Bayley H (2008) Orientation of the monomeric porin OmpG in planar lipid bilayers. Chembiochem 9:3029–3036
Marques EJ, Carneiro CM, Silva AS, Krasilnikov OV (2004) Does VDAC insert into membranes in random orientation? Biochim Biophys Acta 1661:68–77
Gutsmann T, Heimburg T, Keyser U, Mahendran KR, Winterhalter M (2015) Protein reconstitution into freestanding planar lipid membranes for electrophysiological characterization. Nat Protoc 10:188–198
Zakharian E (2013) Recording of ion channel activity in planar lipid bilayer experiments. Methods Mol Biol 998:109–118
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Ionescu, S.A., Lee, S., Bayley, H. (2021). Determining the Orientation of Porins in Planar Lipid Bilayers. In: Fahie, M.A. (eds) Nanopore Technology. Methods in Molecular Biology, vol 2186. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0806-7_5
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DOI: https://doi.org/10.1007/978-1-0716-0806-7_5
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