The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship
The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship
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DOI：
10.1074/jbc.m109.002980
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发表时间：
2009-06-12
影响因子：
4.8
通讯作者：
Cain, Brian D.
中科院分区：
文献类型：
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作者：
Claggett, Shane B.;Plancher, Mac O'Neil;Cain, Brian D.
F1 F0 ATP合酶的外周柄对于F-1与F-0的结合以及酶的两个部分之间的能量适当转移是必需的。大肠杆菌的外周柄由相同的B亚基的二聚体组成。相反，光合生物表达两个b样基因，形成异二聚体的外周柄。先前我们产生了嵌合外周柄，其中E. coli的B亚基被来自细长热聚球藻的B和B'亚基（Claggett，S. B.，Grabar，T. B.，Dunn，S. D、和该隐，B。D.等人（2007）J. Bacteriol. 189，5463-5471）。嵌合B和B'亚基，缩写为Tb和Tb'的空间排列，已通过Cu 2+介导的二硫键交联形成进行了研究。研究了可溶性模型多肽和完整功能F1 F0 ATP合酶复合物内全长亚基之间的二硫键形成。在这两种情况下，二硫键优先在Tb-A83 C和Tb '（A90 C）之间形成，表明两个嵌合亚基的螺旋之间存在交错关系。即使在严格的条件下，也会在这些位置之间快速形成二硫化物。重要的是，这种交联的形成对ATP驱动的质子泵没有可检测的影响，表明交错构象与正常的酶活性相容。在不太严格的反应条件下，也可能检测到通过相同位置交联的B亚基，这表明也可能存在配准、非交错平行构象。
The peripheral stalk of F1F0 ATP synthase is essential for the binding of F-1 to F-0 and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b' subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463-5471). The spatial arrangement of the chimeric b and b' subunits, abbreviated Tb and Tb', has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between Tb-A83C and Tb'(A90C), indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, non-staggered parallel conformation may also exist.