Comparison between single-molecule and X-ray crystallography data on yeast F1-ATPase.
Comparison between single-molecule and X-ray crystallography data on yeast F1-ATPase.
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DOI：
10.1038/srep08773
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发表时间：
2015-03-10
影响因子：
4.6
通讯作者：
Berry RM
中科院分区：
文献类型：
--
作者：
Steel BC;Nord AL;Wang Y;Pagadala V;Mueller DM;Berry RM
近几十年来的单分子研究已经阐明了F1-ATP酶的完整化学机械循环，主要是基于嗜热细菌的F1。相比之下，高分辨率晶体结构仅适用于线粒体F1。在这里，我们提出了高分辨率的单分子旋转数据F1从酿酒酵母，获得使用新的高通量检测和分析工具。旋转数据的野生型线粒体酶，一个“肝”亚型，和六个突变形式的酵母F1，以前已被证明是效率较低或部分解偶联。野生型和“肝”亚型显示出与来自大肠杆菌和嗜热菌的F1相同的定性特征。突变体形式的分析揭示了催化停留的延迟和Vmax的相关降低，其幅度与晶体结构中观察到的破坏水平一致。至少一种突变形式显示出先前未观察到的ATP结合角停留，这可能归因于ADP释放减缓。我们讨论了晶体结构和单分子结果之间的相关性。
Single molecule studies in recent decades have elucidated the full chemo-mechanical cycle of F1-ATPase, mostly based on F1 from thermophilic bacteria. In contrast, high-resolution crystal structures are only available for mitochondrial F1. Here we present high resolution single molecule rotational data on F1 from Saccharomyces cerevisiae, obtained using new high throughput detection and analysis tools. Rotational data are presented for the wild type mitochondrial enzyme, a “liver” isoform, and six mutant forms of yeast F1 that have previously been demonstrated to be less efficient or partially uncoupled. The wild-type and “liver” isoforms show the same qualitative features as F1 from Escherichia coli and thermophilic bacteria. The analysis of the mutant forms revealed a delay at the catalytic dwell and associated decrease in Vmax, with magnitudes consistent with the level of disruption seen in the crystal structures. At least one of the mutant forms shows a previously un-observed dwell at the ATP binding angle, potentially attributable to slowed release of ADP. We discuss the correlation between crystal structures and single molecule results.