title: Ligand Induced Conformational Changes of a Membrane Transporter in E. coli Cells Observed with DEER/PELDOR.
abstract: An unrealized goal in structural biology is the determination of structure and conformational change at high resolution for membrane proteins within the cellular environment. Pulsed electron-electron double resonance (PELDOR) is a well-established technique to follow conformational changes in purified membrane protein complexes. Here we demonstrate the first proof of concept for the use of PELDOR to observe conformational changes in a membrane protein in intact cells. We exploit the fact that outer membrane proteins usually lack reactive cysteines and that paramagnetic spin labels entering the periplasm are selectively reduced to achieve specific labeling of the cobalamin transporter BtuB in Escherichia coli. We characterize conformational changes in the second extracellular loop of BtuB upon ligand binding and compare the PELDOR data with high-resolution crystal structures. Our approach avoids detergent extraction, purification, and reconstitution usually required for these systems. With this approach, structure, function, conformational changes, and molecular interactions of outer membrane proteins can be studied at high resolution in the cellular environment.
authors.affiliation: Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, University of Frankfurt , Max-von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany.
authors.forename: Benesh
authors.initials: B
authors.lastname: Joseph
authors.affiliation: Department of Chemistry and Center for Membrane Biology, University of Virginia , McCormick Road, Charlottesville, Virginia 22904-4319, United States.
authors.forename: Arthur
authors.initials: A
authors.lastname: Sikora
authors.affiliation: Department of Chemistry and Center for Membrane Biology, University of Virginia , McCormick Road, Charlottesville, Virginia 22904-4319, United States.
authors.forename: David S
authors.initials: DS
authors.lastname: Cafiso
mesh_terms: D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry*; D011487:Protein Conformation
chemical_list: D029968:Escherichia coli Proteins; D008024:Ligands; D026901:Membrane Transport Proteins