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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >DISTANCE DETERMINATION IN PROTEINS USING DESIGNED METAL ION BINDING SITES AND SITE-DIRECTED SPIN LABELING - APPLICATION TO THE LACTOSE PERMEASE OF ESCHERICHIA COLI
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DISTANCE DETERMINATION IN PROTEINS USING DESIGNED METAL ION BINDING SITES AND SITE-DIRECTED SPIN LABELING - APPLICATION TO THE LACTOSE PERMEASE OF ESCHERICHIA COLI

机译：使用设计的金属离子结合位点和定向的自旋标签测定蛋白质中的距离-在大肠埃希氏菌的乳糖通透中的应用

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摘要

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure, To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV, In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification, After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II), The results demonstrate that positions 103, 111, and 121 are 8, 14, and >23 Angstrom from the metal binding site, These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease, Application of the technique to determine helix parking in lactose permease is discussed.
机译：如随附文件所示，可以利用定点金属-氮氧化物对之间的磁偶极相互作用来测量T4溶菌酶（一种已知结构的蛋白质）中的距离，以评估这种可能有效的通用方法，尤其是对于膜蛋白，由于难于结晶，因此顺磁性金属离子结合位点和一氧化氮侧链均被引入到大肠杆菌的乳糖通透酶中的选定位置，这是多位膜蛋白的范例。因此，将三个单独的半胱氨酸残基引入乳糖通透酶突变体的推定螺旋IV中，该突变体不含天然半胱氨酸残基，该天然半胱氨酸残基在螺旋III和IV之间的周质环中以六个连续的组氨酸残基的形式包含高亲和力的二价金属离子结合位点，此外，该构建体在中间细胞质环中包含一个生物素受体结构域，以促进纯化。纯化和自旋标记后，在不存在和存在Cu（II）的情况下，纯化的蛋白质均获得了电子顺磁共振波谱，结果表明：位置103、111和121与金属结合位点分别为8、14和> 23埃，这些数据与通透酶的跨膜结构域IV的α螺旋构象一致，该技术在确定乳糖中的螺旋沉积中的应用渗透酶进行了讨论。

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