title: Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances.
abstract: The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.
authors.affiliation: Department of Physics, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: Devin T
authors.initials: DT
authors.lastname: Edwards
authors.affiliation: Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huber
authors.affiliation: Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: Sunyia
authors.initials: S
authors.lastname: Hussain
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Stone
authors.affiliation: College of Creative Studies, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: Maia
authors.initials: M
authors.lastname: Kinnebrew
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel.
authors.forename: Ilia
authors.initials: I
authors.lastname: Kaminker
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel.
authors.forename: Erez
authors.initials: E
authors.lastname: Matalon
authors.affiliation: Department of Physics, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; Institute of Terahertz Science and Technology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Sherwin
authors.affiliation: Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address: daniella.goldfarb@weizmann.ac.il.
authors.forename: Daniella
authors.initials: D
authors.lastname: Goldfarb
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA; Institute of Terahertz Science and Technology, University of California, Santa Barbara, Santa Barbara, CA 93106, USA. Electronic address: songi@chem.ucsb.edu.
authors.forename: Songi
authors.initials: S
authors.lastname: Han
mesh_terms: D003198:Computer Simulation; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D005682:Gadolinium / Q000737:chemistry*; D008958:Models, Molecular; D009154:Mutation; D009589:Nitrogen Oxides / Q000737:chemistry*; D011487:Protein Conformation; D020560:Proteobacteria / Q000378:metabolism*; D025604:Rhodopsins, Microbial / Q000737:chemistry* / Q000235:genetics; D013113:Spin Labels
chemical_list: D009589:Nitrogen Oxides; D025604:Rhodopsins, Microbial; D013113:Spin Labels; C413548:proteorhodopsin; D005682:Gadolinium