Altenbach C, Flitsch S L, Khorana H G, Hubbell W L
Jules Stein Eye Institute, University of California, Los Angeles 90024-1771.
Biochemistry. 1989 Sep 19;28(19):7806-12. doi: 10.1021/bi00445a042.
Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.
定点诱变用于产生细菌视紫红质的突变体，其中甘氨酸-72、苏氨酸-90、亮氨酸-92或丝氨酸-169被半胱氨酸取代。然后将两种不同的自旋标记共价连接到这些位点。连接位点的选择涵盖了两个假定的环（72,169）和一个跨膜片段（90,92）。由于空间位阻问题，在位置90处标记的蛋白质无法正确重折叠，但在磷脂囊泡中成功重构的其他突变体的电子顺磁共振光谱提供了关于标记位点附近蛋白质侧链动力学的信息。采用功率饱和方法研究自旋弛豫时间，而自旋弛豫时间又可能受到与顺磁性物质碰撞的影响。利用氧气和水溶性铬配合物对自旋标记突变体功率饱和行为的差异效应，获得膜结合蛋白位点的拓扑信息。结果表明，72位和169位残基位于暴露于水相的结构化环中，92位残基位于膜内部，可能靠近螺旋-螺旋接触区域。
