title: Nanodiscs allow phage display selection for ligands to non-linear epitopes on membrane proteins.
abstract: In this work, we exploited a method that uses polytopic membrane proteins as targets for phage display selections. Membrane proteins represent the largest class of drug targets and drug discovery is mostly based on the identification of ligands binding to target molecules. The screening of a phage display library for ligands against membrane proteins is typically hindered by the requirement of these proteins for a membrane environment, which is necessary to retain correct folding and epitope formation. Especially in proteins with multiple transmembrane domains, epitopes often are non-linear and consist of a combination of loops between transmembrane stretches of the proteins. Here, we have used bacteriorhodopsin (bR) as a model of polytopic membrane protein, assembled into nanoscale phospholipid bilayers, so called nanodiscs, to screen a phage display library for potential ligands. Nanodiscs provide a native-like environment to membrane proteins and thus selection of ligands can take place in a near physiological state. Screening a 12-mer phage display peptide library against bR nanodiscs led to the isolation of phage clones binding specifically to bR. We were further able to identify the binding site of selected phage clones proving that the clones bind to extramembranous, non-linear epitopes of bR. Thus, nanodiscs provide a suitable and general tool that allows screening of a phage display library against membrane proteins in a near native environment.
authors.affiliation: Institute of Complex Systems (ICS-6), Forschungszentrum Jülich, Jülich, Germany ; Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Pavlidou
authors.forename: Karen
authors.initials: K
authors.lastname: Hänel
authors.forename: Luis
authors.initials: L
authors.lastname: Möckel
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
mesh_terms: D000595:Amino Acid Sequence; D001436:Bacteriorhodopsins / Q000737:chemistry*; D001665:Binding Sites; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry*; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry; D008024:Ligands; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D053758:Nanoparticles / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D019151:Peptide Library; D011485:Protein Binding
chemical_list: D000939:Epitopes; D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D001436:Bacteriorhodopsins; D004134:Dimyristoylphosphatidylcholine
references.citation: Biochemistry. 2007 Feb 27;46(8):2059-69
references.pmid: 17263563
references.citation: Curr Opin Struct Biol. 2010 Aug;20(4):471-9
references.pmid: 20570504
references.citation: Biosci Biotechnol Biochem. 2006 Sep;70(9):2035-41
references.pmid: 16960395
references.citation: Chembiochem. 2003 Sep 5;4(9):811-5
references.pmid: 12964153
references.citation: Molecules. 2011 Jan 21;16(1):857-87
references.pmid: 21258295
references.citation: J Huazhong Univ Sci Technolog Med Sci. 2008 Jun;28(3):299-303
references.pmid: 18563328
references.citation: Science. 1985 Jun 14;228(4705):1315-7
references.pmid: 4001944
references.citation: Mol Biosyst. 2010 Jan;6(1):126-33
references.pmid: 20024074
references.citation: Protein Sci. 1998 Apr;7(4):1029-38
references.pmid: 9568909
references.citation: J Struct Biol. 1998 Sep;123(1):37-44
references.pmid: 9774543
references.citation: Arch Biochem Biophys. 1998 May 15;353(2):365-73
references.pmid: 9606971
references.citation: Eur J Biochem. 1996 Feb 1;235(3):526-33
references.pmid: 8654397
references.citation: J Immunol Methods. 2002 Mar 1;261(1-2):37-48
references.pmid: 11861064
references.citation: J Proteome Res. 2008 Aug;7(8):3535-42
references.pmid: 18557639
references.citation: Science. 1996 Mar 29;271(5257):1854-7
references.pmid: 8596952
references.citation: Mol Cell Proteomics. 2008 Nov;7(11):2246-53
references.pmid: 18603642
references.citation: Anal Biochem. 2011 Jan 1;408(1):46-52
references.pmid: 20804721
references.citation: Chembiochem. 2003 Aug 4;4(8):748-53
references.pmid: 12898626
references.citation: Biochemistry. 1983 Mar 15;22(6):1323-34
references.pmid: 6838856
references.citation: J Am Chem Soc. 2004 Mar 24;126(11):3477-87
references.pmid: 15025475
references.citation: Biophys J. 2002 Sep;83(3):1557-66
references.pmid: 12202380
references.citation: Anal Chem. 2008 Aug 15;80(16):6245-52
references.pmid: 18616345
references.citation: Nat Med. 2002 Feb;8(2):121-7
references.pmid: 11821895
references.citation: Arch Biochem Biophys. 2006 Jun 15;450(2):215-22
references.pmid: 16620766
references.citation: Nat Rev Drug Discov. 2006 Dec;5(12):993-6
references.pmid: 17139284
references.citation: FEBS J. 2010 Apr;277(7):1653-65
references.pmid: 20156289
references.citation: Biol Chem. 2009 Aug;390(8):805-14
references.pmid: 19453280
references.citation: Protein Sci. 2003 Nov;12(11):2476-81
references.pmid: 14573860
references.citation: Annu Rev Biochem. 2010;79:737-75
references.pmid: 20307193
references.citation: J Am Chem Soc. 2009 Sep 2;131(34):12060-1
references.pmid: 19663495