Altenbach C, Marti T, Khorana H G, Hubbell W L
Jules Stein Eye Institute, University of California, Los Angeles 90024-7008.
Science. 1990 Jun 1;248(4959):1088-92. doi: 10.1126/science.2160734.
Transmembrane proteins serve important biological functions, yet precise information on their secondary and tertiary structure is very limited. The boundaries and structures of membrane-embedded domains in integral membrane proteins can be determined by a method based on a combination of site-specific mutagenesis and nitroxide spin labeling. The application to one polypeptide segment in bacteriorhodopsin, a transmembrane chromoprotein that functions as a light-driven proton pump is described. Single cysteine residues were introduced at 18 consecutive positions (residues 125 to 142). Each mutant was reacted with a specific spin label and reconstituted into vesicles that were shown to be functional. The relative collision frequency of each spin label with freely diffusing oxygen and membrane-impermeant chromium oxalate was estimated with power saturation EPR (electron paramagnetic resonance) spectroscopy. The results indicate that residues 129 to 131 form a short water-exposed loop, while residues 132 to 142 are membrane-embedded. The oxygen accessibility for positions 131 to 138 varies with a periodicity of 3.6 residues, thereby providing a striking demonstration of an alpha helix. The orientation of this helical segment with respect to the remainder of the protein was determined.
跨膜蛋白具有重要的生物学功能，然而关于其二级和三级结构的确切信息却非常有限。整合膜蛋白中膜嵌入结构域的边界和结构可以通过一种基于位点特异性诱变和氮氧化物自旋标记相结合的方法来确定。本文描述了该方法在细菌视紫红质（一种作为光驱动质子泵的跨膜色素蛋白）的一个多肽片段上的应用。在18个连续位置（第125至142位残基）引入了单个半胱氨酸残基。每个突变体与特定的自旋标记反应，并重新组装成具有功能的囊泡。利用功率饱和电子顺磁共振（EPR）光谱法估算了每个自旋标记与自由扩散的氧气和膜不通透的草酸铬的相对碰撞频率。结果表明，第129至131位残基形成一个短的水暴露环，而第132至142位残基是膜嵌入的。第131至138位的氧气可及性以3.6个残基的周期变化，从而显著证明了一个α螺旋。确定了该螺旋片段相对于蛋白质其余部分的方向。
