title: Expression, purification and spin labelling of the ferrous iron transporter FeoB from Escherichia coli BL21 for EPR studies.
abstract: The ferrous iron transporter FeoB is an important factor in the iron metabolism of various bacteria. As a membrane bound GTPase it also represents an interesting evolutionary link between prokaryotic and eukaryotic membrane signalling pathways. To date, structural information for FeoB is limited to the cytosolic GTPase domain and structural features such as the oligomeric state of the transporter in the membrane, and thereby the nature of the transport pore are a matter of constant debate. Recently, EPR distance measurements have become an important tool to investigate such questions in frozen solution. As a prerequisite for these experiments, we designed protocols to express and purify both the cytosolic domain of FeoB (NFeoB) and full-length FeoB from Escherichia coli BL21 in purity, quantity and quality needed for EPR studies. Since FeoB from E. coli contains 12 native cysteines, we incorporated the unnatural amino acid para-acetylphenylalanine (pAcF) into the protein. We spin labelled the mutant protein using the HO4120 spin label and performed preliminary EPR experiments using cw-X-band EPR spectroscopy. Our results provide new insights concerning the oligomeric state of full-length FeoB.
authors.affiliation: Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany. Electronic address: hagelueken@pc.uni-bonn.de.
authors.forename: Gregor
authors.initials: G
authors.lastname: Hagelueken
authors.affiliation: Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany.
authors.forename: Fraser G
authors.initials: FG
authors.lastname: Duthie
authors.affiliation: Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Florin
authors.affiliation: Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Schubert
authors.affiliation: Institute for Physical and Theoretical Chemistry, University of Bonn, Wegelerstr. 12, 53115 Bonn, Germany.
authors.forename: Olav
authors.initials: O
authors.lastname: Schiemann
mesh_terms: D027682:Cation Transport Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D029968:Escherichia coli Proteins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008958:Models, Molecular; D013113:Spin Labels
chemical_list: D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; C470580:FeoB protein, E coli; D013113:Spin Labels
keywords: EPR; FeoB; Membrane protein; Spin label; Unnatural amino acid