Isolation of yeast complex IV in native lipid nanodiscs.
Isolation of yeast complex IV in native lipid nanodiscs.
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DOI：
10.1016/j.bbamem.2016.09.004
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发表时间：
2016-12
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我们用两亲性苯乙烯马来酸（SMA）共聚物在天然脂质环境中从S.酿酒酵母线粒体。使用亲和色谱法纯化每个盘含有一个CytcO的天然纳米盘。天然纳米盘的最长横截面为11 nm × 14 nm。基于该大小，我们估计每个CytcO被约100个磷脂包围。天然纳米盘含有与线粒体内膜中发现的那些相同的主要磷脂。尽管CytcO在线粒体膜上与细胞色素bc 1形成超复合物，但cyt.在原生纳米盘中未发现bc 1。然而，发现松散结合的呼吸超复合物因子与分离的CytcO相关。天然纳米盘显示出约130电子CytcO−1 s−1的O2还原活性，并且完全还原的CytcO与O2的反应动力学与线粒体膜中观察到的CytcO基本相同。CO-配体结合到CytcO催化位点的动力学在天然纳米盘和线粒体膜中是相似的。我们还发现，过量的SMA可逆地抑制线粒体CytcO的催化活性，推测是通过干扰细胞色素。C绑定。这些数据指出了在提取膜蛋白后去除过量SMA的重要性。总之，我们的数据显示了使用SMA提取的CytcO进行功能和结构研究的高潜力。
We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm × 14 nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc1 in the mitochondrial membrane, cyt. bc1 was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O2-reduction activity of ~130 electrons CytcO−1 s−1 and the kinetics of the reaction of the fully reduced CytcO with O2 was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.
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