Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances

Devin T Edwards; Thomas Huber; Sunyia Hussain; Katherine M Stone; Maia Kinnebrew; Ilia Kaminker; Erez Matalon; Mark S Sherwin; Daniella Goldfarb; Songi Han

doi:10.1016/j.str.2014.09.008

Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances

Structure. 2014 Nov 4;22(11):1677-86. doi: 10.1016/j.str.2014.09.008. Epub 2014 Oct 30.

Authors

Affiliations

¹ Department of Physics, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
² Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia.
³ Department of Chemical Engineering, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
⁴ Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
⁵ College of Creative Studies, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
⁶ Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel.
⁷ 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.
⁸ Department of Chemical Physics, Weizmann Institute of Science, Rehovot, 7610001, Israel. Electronic address: daniella.goldfarb@weizmann.ac.il.
⁹ 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.

PMID: 25438671
DOI: 10.1016/j.str.2014.09.008

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Computer Simulation
Dimerization
Electron Spin Resonance Spectroscopy
Gadolinium / chemistry*
Models, Molecular
Mutation
Nitrogen Oxides / chemistry*
Protein Conformation
Proteobacteria / metabolism*
Rhodopsins, Microbial / chemistry*
Rhodopsins, Microbial / genetics
Spin Labels

Substances

Nitrogen Oxides
Rhodopsins, Microbial
Spin Labels
proteorhodopsin
Gadolinium