Steinhoff H J, Mollaaghababa R, Altenbach C, Khorana H G, Hubbell W L
Institut für Biophysik, Ruhr-Universität Bochum, Germany.
Biophys Chem. 1995 Sep-Oct;56(1-2):89-94. doi: 10.1016/0301-4622(95)00019-t.
Site-directed spin labeling of membrane proteins has been used to determine: (1) the topography of the polypeptide chain with respect to the membrane/solution interface, and (2) the identity and orientation of secondary structure in selected regions. These features are deduced from the collision rates of nitroxide side chains with paramagnetic reagents in solution, and the principles of the method are reviewed with reference to bacteriorhodopsin. The dynamics of the nitroxide side chains relative to the backbone reveal tertiary interactions of the labeled site, and provide a promising means of time-resolving conformational changes. This aspect is illustrated by recent studies of structural changes in bacteriorhodopsin during the photocycle. In these experiments, nitroxide side chains were introduced at residues 72, 101 and 105 after replacement of the original residues by cysteine. Upon flash photolysis, the electron paramagnetic resonance spectrum of a nitroxide at 101, but not those at 72 or 105, is time-dependent. The spectral change develops during the decay of the M-intermediate, and reverses upon return to the ground state. The results suggest a movement of the C-D or E-F interhelical loops during the protonation changes of aspartate 96.
（1）多肽链相对于膜/溶液界面的拓扑结构，以及（2）选定区域中二级结构的特征和取向。这些特征是根据溶液中氮氧侧链与顺磁性试剂的碰撞速率推导出来的，并以细菌视紫红质为例对该方法的原理进行了综述。氮氧侧链相对于主链的动力学揭示了标记位点的三级相互作用，并提供了一种有前景的时间分辨构象变化的方法。细菌视紫红质在光循环过程中的结构变化的最新研究说明了这一方面。在这些实验中，在将原始残基替换为半胱氨酸后，在残基72、101和105处引入了氮氧侧链。闪光光解后，101位氮氧的电子顺磁共振谱随时间变化，而72位或105位的则不然。光谱变化在M中间体衰减过程中发展，并在回到基态时逆转。结果表明，在天冬氨酸96质子化变化过程中，C-D或E-F螺旋间环发生了移动。
