title: Relative Orientation of POTRA Domains from Cyanobacterial Omp85 Studied by Pulsed EPR Spectroscopy.
abstract: Many proteins of the outer membrane of Gram-negative bacteria and of the outer envelope of the endosymbiotically derived organelles mitochondria and plastids have a β-barrel fold. Their insertion is assisted by membrane proteins of the Omp85-TpsB superfamily. These proteins are composed of a C-terminal β-barrel and a different number of N-terminal POTRA domains, three in the case of cyanobacterial Omp85. Based on structural studies of Omp85 proteins, including the five POTRA-domain-containing BamA protein of Escherichia coli, it is predicted that anaP2 and anaP3 bear a fixed orientation, whereas anaP1 and anaP2 are connected via a flexible hinge. We challenged this proposal by investigating the conformational space of the N-terminal POTRA domains of Omp85 from the cyanobacterium Anabaena sp. PCC 7120 using pulsed electron-electron double resonance (PELDOR, or DEER) spectroscopy. The pronounced dipolar oscillations observed for most of the double spin-labeled positions indicate a rather rigid orientation of the POTRA domains in frozen liquid solution. Based on the PELDOR distance data, structure refinement of the POTRA domains was performed taking two different approaches: 1) treating the individual POTRA domains as rigid bodies; and 2) using an all-atom refinement of the structure. Both refinement approaches yielded ensembles of model structures that are more restricted compared to the conformational ensemble obtained by molecular dynamics simulations, with only a slightly different orientation of N-terminal POTRA domains anaP1 and anaP2 compared with the x-ray structure. The results are discussed in the context of the native environment of the POTRA domains in the periplasm.
authors.affiliation: Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany; Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany.
authors.forename: Reza
authors.initials: R
authors.lastname: Dastvan
authors.affiliation: Molecular Cell Biology of Plants, Goethe University Frankfurt, Frankfurt am Main, Germany.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Brouwer
authors.affiliation: Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany; Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Schuetz
authors.affiliation: Molecular Cell Biology of Plants, Goethe University Frankfurt, Frankfurt am Main, Germany.
authors.forename: Oliver
authors.initials: O
authors.lastname: Mirus
authors.affiliation: Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany; Molecular Cell Biology of Plants, Goethe University Frankfurt, Frankfurt am Main, Germany; Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany. Electronic address: schleiff@bio.uni-frankfurt.de.
authors.forename: Enrico
authors.initials: E
authors.lastname: Schleiff
authors.affiliation: Institute of Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University Frankfurt, Frankfurt am Main, Germany; Cluster of Excellence Macromolecular Complexes, Goethe University Frankfurt, Frankfurt am Main, Germany. Electronic address: prisner@chemie.uni-frankfurt.de.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Prisner
mesh_terms: D017033:Anabaena / Q000737:chemistry*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005615:Freezing; D056004:Molecular Dynamics Simulation; D000072417:Protein Domains; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary
chemical_list: D001425:Bacterial Outer Membrane Proteins