Edwards Devin T, Huber Thomas, Hussain Sunyia, Stone Katherine M, Kinnebrew Maia, Kaminker Ilia, Matalon Erez, Sherwin Mark S, Goldfarb Daniella, Han Songi
Department of Physics, University of California, Santa Barbara, Santa Barbara, CA 93106, USA.
Research School of Chemistry, The Australian National University, Canberra, ACT 0200, Australia.
Structure. 2014 Nov 4;22(11):1677-86. doi: 10.1016/j.str.2014.09.008. Epub 2014 Oct 30.
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.
利用传统的氮氧化物自旋标记和最近开发的基于Gd3+的自旋标记，通过双电子-电子共振（DEER）光谱获得了具有功能相关性的绿色吸收型视紫红质（G-PR）六聚体寡聚体的结构组织。通过对Trp58和Thr177残基进行半胱氨酸突变，制备了带有氮氧化物或Gd3+标记的G-PR。通过将G-PR六聚体中多个蛋白质间距离的可靠测量结果与计算机建模相结合，我们获得了一个与同源蓝色吸收型PR（B-PR）六聚体的最新晶体结构相符的结构模型。这些DEER结果提供了在膜模拟环境中以及晶体结构中未解析的环区域内的特定距离信息。此外，使用氮氧化物自旋标记进行的X波段DEER测量受到多自旋效应的影响，这种效应有时会影响对次近邻距离的检测。使用Gd3+自旋标记在高磁场下进行测量大大提高了灵敏度，并减轻了多自旋相互作用带来的困难。
