The affinity purification and characterization of ATP synthase complexes from mitochondria.
The affinity purification and characterization of ATP synthase complexes from mitochondria.
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DOI：
10.1098/rsob.120160
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发表时间：
2013-02-13
期刊：
影响因子：
5.8
通讯作者：
Walker JE
中科院分区：
文献类型：
--
作者：
Runswick MJ;Bason JV;Montgomery MG;Robinson GC;Fearnley IM;Walker JE
线粒体F1-ATP酶抑制剂蛋白IF 1抑制F-ATP合酶的水解活性，但不抑制其合成活性，并且需要ATP的水解以形成被抑制的复合物。在这个复合物中，结合的IF 1的α-螺旋抑制区域占据了酶的三个催化界面之一的深裂缝。它的N-末端区域渗透到酶的中央水腔中，并与酶转子中的γ-亚基相互作用。形成这种复合物的复杂性和抑制剂的结合模式赋予IF 1高特异性。这种性质已被利用在一个单一的色谱步骤中，通过使用具有C-末端亲和标签的抑制剂蛋白质，从线粒体中纯化完整的F-ATP合酶复合物的高选择性亲和程序的发展。用牛IF 1的残基1-60与C-末端绿色荧光蛋白随后His-标签回收抑制复合物，并且用具有C-末端谷胱甘肽-S-转移酶结构域的相同抑制剂回收活性酶。通过从牛、羊、猪和酵母线粒体中纯化酶复合物，证明了该方法的广泛适用性。这些复合物的亚基组合物已被表征。对牛酶的催化性能进行了详细的研究。其水解活性对寡霉素的抑制敏感，并且该酶能够在囊泡中合成ATP，其中质子动力由细菌视紫红质从光产生。偶联酶与亲和层析法制备的非偶联酶进行了比较。在解偶联酶中，酶定子的亚基比偶联酶中降解得更快，表明解偶联涉及定子区域中的显著结构变化。
The mitochondrial F1-ATPase inhibitor protein, IF1, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF1 occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme's rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF1 with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF1 with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme's stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.
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影响因子：
5.6
作者：
Bason JV;Runswick MJ;Fearnley IM;Walker JE
通讯作者：
Walker JE
影响因子：
2.9
作者：
Carroll, Joe;Fearnley, Ian M.;Walker, John E.
通讯作者：
Walker, John E.
影响因子：
11.4
作者：
Kabaleeswaran, Venkataraman;Puri, Neeti;Mueller, David M.
通讯作者：
Mueller, David M.
DOI：
10.1038/nsb966
发表时间：
2003-09-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Cabezón, E;Montgomery, MG;Walker, JE
通讯作者：
Walker, JE
影响因子：
3.5
作者：
NAGLEY, P;HALL, RM;OOI, PG
通讯作者：
OOI, PG