Subunit epsilon of the Escherichia coli ATP synthase: novel insights into structure and function by analysis of thirteen mutant forms.
Subunit epsilon of the Escherichia coli ATP synthase: novel insights into structure and function by analysis of thirteen mutant forms.
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大肠杆菌 ATP 合酶的亚基 epsilon：通过分析 13 种突变形式对结构和功能的新见解。
作者：
Xiong,H;Zhang,D;Vik,SB
最近，核磁共振技术已经确定了大肠杆菌三磷酸腺苷合成酶ε亚基的结构模型。(1995)NAT。结构。Biol.2,961Uhlin 967]和X射线结晶学[−等人]。(1997)Structire5,1219−1230]，揭示了一个双域蛋白。本研究构建并分析了6个新的ε突变体： Y63A、D81A、T82A和3个截短突变体tr80(S)、tr94(LAS)和tr117(As)。对先前构建的7个突变体进行了分析： E31A、E59A、S65A、E70A、T77A、R58A和D81A/R85A。通过等电聚焦从过量生产这13个突变体的细胞提取液中纯化亚基。用固定化镍亲和层析法制备了缺失ε亚基的F1。三个突变体E70A、S65A和E31A表现出比野生型更高的亲和力和抑制程度。三个突变体T82A、R85A和tr94(LAS)在测试的浓度范围内表现出较低的亲和力和抑制程度。D81a和tr80(S)两株无抑制作用。T77A、Y63A、E59A和tr117(AS)的亲和力低于野生型，但抑制程度接近正常。结果表明，ε亚基的C末端结构域有助于抑制三磷酸腺苷的水解，但对三磷酸腺苷驱动的质子转运并不是必需的。与γ亚基的相互作用可能涉及含有S65、E70、T77、D81和T82残基的表面，而R85和Y63残基可能在ε亚基的构象中起重要作用。
Structural models of subunit ε of the ATP synthase fromEscherichiacolihave been determined recently by NMR [Wilkens et al. (1995)Nat. Struct. Biol.2, 961−967] and by X-ray crystallography [Uhlin et al. (1997)Structure5, 1219−1230], revealing a two-domain protein. In this study, six new ε mutants were constructed and analyzed: Y63A, D81A, T82A, and three truncated mutants, tr80(S), tr94(LAS), and tr117(AS). Seven mutants constructed previously were also analyzed: E31A, E59A, S65A, E70A, T77A, R58A, and D81A/R85A. Subunits were purified by isoelectric focusing from extracts of cells that overproduced these 13 mutants. F1was prepared lacking subunit ε by immobilized-Ni affinity chromatography. Three mutants, E70A, S65A, and E31A, showed somewhat higher affinities and extents of inhibition than the wild type. Three mutants, T82A, R85A, and tr94(LAS), showed both lower affinities and extents of inhibition, over the concentration range tested. Two showed no inhibition, D81A and tr80(S). The others, T77A, Y63A, E59A, and tr117(AS), showed lower affinities than wild type, but the extents of inhibition were nearly normal. Results indicate that the C-terminal domain of subunit ε contributes to inhibition of ATP hydrolysis, but it is not necessary for ATP-driven proton translocation. Interactions with subunit γ are likely to involve a surface containing residues S65, E70, T77, D81, and T82, while residues R85 and Y63 are likely to be important in the conformation of subunit ε.