title: Distance Measurement on an Endogenous Membrane Transporter in E. coli Cells and Native Membranes Using EPR Spectroscopy.
abstract: Membrane proteins may be influenced by the environment, and they may be unstable in detergents or fail to crystallize. As a result, approaches to characterize structures in a native environment are highly desirable. Here, we report a novel general strategy for precise distance measurements on outer membrane proteins in whole Escherichia coli cells and isolated outer membranes. The cobalamin transporter BtuB was overexpressed and spin-labeled in whole cells and outer membranes and interspin distances were measured to a spin-labeled cobalamin using pulse EPR spectroscopy. A comparative analysis of the data reveals a similar interspin distance between whole cells, outer membranes, and synthetic vesicles. This approach provides an elegant way to study conformational changes or protein-protein/ligand interactions at surface-exposed sites of membrane protein complexes in whole cells and native membranes, and provides a method to validate outer membrane protein structures in their native environment.
authors.affiliation: Institut für physikalische und theoretische Chemie und Biomolekulares Magnetresonanz Zentrum, Universität Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany) http://www.prisner.de.
authors.forename: Benesh
authors.initials: B
authors.lastname: Joseph
authors.affiliation: Department of Chemistry, University of Virginia, McCormick Road, Charlottesville VA22904-4319 (USA).
authors.forename: Arthur
authors.initials: A
authors.lastname: Sikora
authors.affiliation: Department of Physics, Free University of Berlin, Arnimallee 14, 14195 Berlin (Germany).
authors.forename: Enrica
authors.initials: E
authors.lastname: Bordignon
authors.affiliation: Laboratory for Physical Chemistry, ETH Zurich, Vladimir-Prelog-Weg 2, 8093 Zürich (Switzerland).
authors.forename: Gunnar
authors.initials: G
authors.lastname: Jeschke
authors.affiliation: Department of Chemistry, University of Virginia, McCormick Road, Charlottesville VA22904-4319 (USA). cafiso@virginia.edu.
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
authors.affiliation: Institut für physikalische und theoretische Chemie und Biomolekulares Magnetresonanz Zentrum, Universität Frankfurt, Max-von-Laue-Strasse 7, 60438 Frankfurt am Main (Germany) http://www.prisner.de. prisner@prisner.de.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Prisner
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008958:Models, Molecular
chemical_list: D001425:Bacterial Outer Membrane Proteins; C044592:BtuB protein, E coli; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins
keywords: EPR spectroscopy; in-cell spectroscopy; membrane proteins; spin labeling; vitamin B12 transporter
references.citation: J Bacteriol. 1973 Aug;115(2):506-13
references.pmid: 4579869
references.citation: Biochemistry. 2008 Jan 15;47(2):670-9
references.pmid: 18092811
references.citation: Angew Chem Int Ed Engl. 2014 Sep 22;53(39):10300-14
references.pmid: 25070284
references.citation: Angew Chem Int Ed Engl. 2011 May 23;50(22):5070-4
references.pmid: 21506223
references.citation: J Bacteriol. 1973 Sep;115(3):717-22
references.pmid: 4580564
references.citation: Nat Struct Biol. 2003 May;10(5):394-401
references.pmid: 12652322
references.citation: Q Rev Biophys. 2007 Feb;40(1):1-53
references.pmid: 17565764
references.citation: Nat Struct Biol. 2000 Mar;7(3):205-9
references.pmid: 10700278
references.citation: FEBS Lett. 2010 May 3;584(9):1721-7
references.pmid: 19836392
references.citation: Curr Opin Struct Biol. 2011 Aug;21(4):523-31
references.pmid: 21719274
references.citation: Nat Protoc. 2013 Jan;8(1):131-47
references.pmid: 23257982
references.citation: J Am Chem Soc. 2014 Jan 29;136(4):1238-41
references.pmid: 24428347
references.citation: J Am Chem Soc. 2014 Sep 24;136(38):13458-65
references.pmid: 25163412
references.citation: J Am Chem Soc. 2014 Oct 29;136(43):15366-78
references.pmid: 25325832
references.citation: Phys Chem Chem Phys. 2011 Feb 14;13(6):2356-66
references.pmid: 21116569
references.citation: Annu Rev Phys Chem. 2012;63:419-46
references.pmid: 22404592
references.citation: J Biol Chem. 1986 Feb 25;261(6):2520-3
references.pmid: 3512539
references.citation: J Am Chem Soc. 2010 Jun 23;132(24):8228-9
references.pmid: 20513154
references.citation: Science. 1997 May 23;276(5316):1261-4
references.pmid: 9157886
references.citation: Gene. 1988 Apr 15;64(1):147-53
references.pmid: 3294107
references.citation: Structure. 2011 Nov 9;19(11):1549-61
references.pmid: 22078555
references.citation: Science. 2006 Jun 2;312(5778):1396-9
references.pmid: 16741124
references.citation: Proc Natl Acad Sci U S A. 2009 Dec 22;106(51):21637-42
references.pmid: 19995976
references.citation: Free Radic Res. 2015 Jan;49(1):78-85
references.pmid: 25348344