Helix packing in subunit a of the Escherichia coli ATP synthase as determined by chemical labeling and proteolysis of the cysteine-substituted protein.
Helix packing in subunit a of the Escherichia coli ATP synthase as determined by chemical labeling and proteolysis of the cysteine-substituted protein.
复制标题
通过半胱氨酸取代蛋白的化学标记和蛋白水解测定，大肠杆菌 ATP 合酶 a 亚基中的螺旋堆积。
DOI：
10.1021/bi026649t
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发表时间：
2003
期刊：
影响因子：
2.9
通讯作者：
Vik,StevenB
中科院分区：
文献类型：
--
作者：
Zhang,Di;Vik,StevenB
大肠杆菌ATP合酶的亚基被认为在质子驱动的ATP合成过程中控制质子进入C亚基环。在这项研究中，已使用3-N-马来酰亚胺-丙酰生物胞素标记的细胞透化的多粘菌素B九肽的周质表面暴露的亚单位进行了检查，并在周质表面的螺旋包装已探测金属螯合物介导的蛋白水解。将119至146之间的18个残基单独改变为半胱氨酸并测试可及性。标记的位置包括D124和D146，表明至少23个氨基酸的周质环。用5-（α-溴乙酰氨基）-1，10-菲咯啉-铜对跨膜跨膜末端附近的残基进行化学蛋白水解试验。只有残基W241 C和D44 C，以及在较小程度上I43 C，导致氧化后的蛋白水解片段。通过与由工程化到亚基中的因子Xa位点产生的分子量标准品比较来确定片段的大小。片段通过免疫印迹使用在亚基的羧基末端的工程化HA表位检测。结果表明，跨膜跨度5（W241）和跨膜跨度1（D44）都接近跨膜跨度2。
Subunitaof theEscherichia coliATP synthase is thought to control access of protons to the ring ofcsubunits during proton-driven ATP synthesis. In this study, the surface exposure of subunitain the periplasm has been examined using 3-N-maleimidyl-propionyl biocytin labeling in cells permeabilized by polymyxin B nonapeptide, and the helix packing at the periplasmic surface has been probed by metal-chelate mediated proteolysis. Eighteen residues between 119 and 146 were changed individually to cysteine and tested for accessibility. Positions labeled included D124 and D146, indicating a periplasmic loop of at least 23 amino acids. Residues near the ends of the transmembrane spans were tested with 5-(α-bromoacetamido)-1,10-phenanthroline-copper for chemical proteolysis. Only residues W241C and D44C, and to a lesser extent I43C, led to proteolytic fragments after oxidation. The fragments were sized by comparison with molecular weight standards generated by Factor Xa sites engineered into subunita. Fragments were detected by immunoblotting using an engineered HA epitope at the carboxyl-terminal end of subunita. The results indicated that both transmembrane span 5 (W241) and transmembrane span 1 (D44) are close to transmembrane span 2.