Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.
Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.
复制标题
使用设计的金属离子结合位点和定点自旋标记确定蛋白质中的距离：应用于大肠杆菌的乳糖通透酶。
DOI：
10.1073/pnas.92.26.12300
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发表时间：
1995
影响因子：
11.1
通讯作者：
Kaback,HR
中科院分区：
文献类型：
--
作者：
Voss,J;Hubbell,WL;Kaback,HR
如随附论文所示，可以利用定点金属-硝基氧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。为了评估这种潜在的强大方法的通用性，特别是对于难以结晶的膜蛋白，在大肠杆菌乳糖通透酶（多胞膜蛋白的范例）的选定位置引入了顺磁性金属离子结合位点和硝基氧侧链。因此，将三个单独的半胱氨酸残基引入缺乏天然半胱氨酸残基的乳糖通透酶突变体的假定螺旋IV中，该突变体含有螺旋III和IV之间的周质环中的六个连续组氨酸残基形式的高亲和力二价金属离子结合位点。此外，该构建体在中部细胞质环中含有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，用纯化的蛋白质获得电子顺磁共振光谱。结果表明位置 103、111 和 121 距金属结合位点 8、14 和 > 23 A。这些数据与通透酶跨膜结构域 IV 的 α 螺旋构象一致。讨论了该技术在乳糖通透酶中测定螺旋堆积的应用。
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 A 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 packing in lactose permease is discussed.