期刊
STRUCTURE
卷 22, 期 11, 页码 1677-1686出版社
CELL PRESS
DOI: 10.1016/j.str.2014.09.008
关键词
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资金
- NSF [CHE-0821589, MCB-1244651]
- W.M. Keck Foundation [SB-080017]
- Institute for Collaborative Biotechnologies from the U.S. Army Research Office [W911NF-09-0001]
- Israel-USA BSF Science Foundation
- Australian Research Council [DP120100561, FT09917009]
- Direct For Biological Sciences [1244651] Funding Source: National Science Foundation
- Div Of Molecular and Cellular Bioscience [1244651] Funding Source: National Science Foundation
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 multi-spin 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.
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