Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

• Published in: Structure (London) 2014-11, Vol.22 (11), p.1677-1686
• Main Authors: Edwards, Devin T., Huber, Thomas, Hussain, Sunyia, Stone, Katherine M., Kinnebrew, Maia, Kaminker, Ilia, Matalon, Erez, Sherwin, Mark S., Goldfarb, Daniella, Han, Songi
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2014.09.008

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. [Display omitted] •Gd3+ DEER techniques are introduced to study membrane protein oligomer structures•Gd3+ measurements in green-absorbing proteorhodopsin hexamers show two distances•This oligomeric structure is similar to that of blue-absorbing proteorhodopsin•Distances to multiple neighbors resolved better with Gd3+ than with nitroxide labels The structure of transmembrane protein oligomers remains difficult to study. Edwards et al. use EPR measurements of interprotein distances to verify a model of the proteorhodopsin hexamer. Gd3+-based spin labels are shown to improve measurements in oligomers by reporting multiple distances better than traditional labels.