Specific heterodimer formation by the cytoplasmic domains of the b and b′ subunits of cyanobacterial ATP synthase
Specific heterodimer formation by the cytoplasmic domains of the b and b′ subunits of cyanobacterial ATP synthase
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DOI：
10.1021/bi001821j
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发表时间：
2001-01-09
期刊：
影响因子：
2.9
通讯作者：
Lill, H
中科院分区：
文献类型：
--
作者：
Dunn, SD;Kellner, E;Lill, H
蓝细菌集胞藻PCC 6803的ATP合酶的B和B'亚基的可溶性结构域与连接到其N-末端的His标签一起表达。纯化后，多肽的特征在于化学交联，分析超离心，和圆二色谱。用化学交联剂处理可溶性B和B'结构域的混合物导致交联二聚体的大量形成，而对任一结构域本身的类似处理仅导致交联物质的痕量形成。通过沉降平衡测量的B和B'结构域在20 ℃溶液中的分子量分别为17 800 +/- 700和16 300 +/- 400，而计算的多肽分子量为16 635和15 422，而B和B'的混合物得到29800 +/-800的分子量。等摩尔混合物的沉降系数为1.73 +/-0.03。单个多肽的圆二色性光谱表明螺旋含量在40-50%的范围内;混合物的光谱揭示了指示卷曲螺旋形成的变化和60%的螺旋含量。结果表明，B和B'亚基的胞质结构域作为单体单独存在，但当它们混合在一起时形成高度延伸的异源二聚体。
The soluble domains of the b and b' subunits of the ATP synthase of the cyanobacterium Synechocystis PCC 6803 were expressed with His tags attached to their N-termini. Following purification, the polypeptides were characterized by chemical cross-linking, analytical ultracentrifugation, and circular dichroism spectroscopy. Treatment of a mixture of the soluble b and b' domains with a chemical crosslinking agent led to substantial formation of cross-linked dimers, whereas similar treatment of either domain by itself resulted in only trace formation of cross-linked species. The molecular weights of the domains of b and b' in solution at 20 degreesC, measured by sedimentation equilibrium, were 17 800 +/- 700 and 16 300 +/- 400, respectively, compared to calculated polypeptide molecular weights of 16 635 and 15 422, whereas a mixture of b and b' gave a molecular weight of 29 800 +/- 800. The sedimentation coefficient of an equimolar mixture was 1.73 +/- 0.03. The circular dichroism spectra of the individual polypeptides indicated helical contents in the range of 40-50%; the spectrum of the mixture revealed changes indicative of coiled-coil formation and a helical content of 60%. The results indicate that the cytosolic domains of the b and b' subunits exist individually as monomers but form a highly extended heterodimer when they are mixed together.