Harnisch U, Weiss H, Sebald W
Eur J Biochem. 1985 May 15;149(1):95-9. doi: 10.1111/j.1432-1033.1985.tb08898.x.
The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora mitochondria was determined by cDNA and genomic DNA sequencing. A first cDNA was identified from a cDNA bank cloned in Escherichia coli by hybridization selection of mRNA, cell-free protein synthesis and immunoadsorption. Further cDNA and geonomic DNA were identified by colony filter hybridization. The N-terminal sequence of the mature protein was determined by automated Edman degradation. From the sequence a molecular mass of 24749 Da results for the precursor protein and of 21556 Da for the mature protein. The presequence consists of 32 amino acids with four arginines as the only charged residues. The mature protein consists of 199 amino acids. It is characterized by a small N-terminal hydrophilic part of 29 residues, a hydrophobic stretch of 25 residues and a large C-terminal hydrophilic domain of 145 residues. The only four cysteines of the protein, which are assumed to bind the 2 Fe-2S cluster, are located in a moderate hydrophobic region of this large domain. Cysteines 3 and 4 are unusually arranged in that they are separated by only one proline. From sequence data the arrangement of the subunit in the membrane is deduced.
通过cDNA和基因组DNA测序确定了粗糙脉孢菌线粒体泛醇-细胞色素c还原酶铁硫亚基的一级结构。通过mRNA的杂交选择、无细胞蛋白质合成和免疫吸附，从克隆于大肠杆菌的cDNA文库中鉴定出第一个cDNA。通过菌落滤膜杂交鉴定出更多的cDNA和基因组DNA。通过自动Edman降解确定成熟蛋白的N端序列。根据该序列，前体蛋白的分子量为24749 Da，成熟蛋白的分子量为21556 Da。前导序列由32个氨基酸组成，其中四个精氨酸是唯一的带电荷残基。成熟蛋白由199个氨基酸组成。其特征在于有一个由29个残基组成的小的N端亲水部分、一个由25个残基组成的疏水延伸段和一个由145个残基组成的大的C端亲水结构域。该蛋白仅有的四个半胱氨酸被认为与2Fe-2S簇结合，它们位于这个大结构域的一个中等疏水区。半胱氨酸3和4的排列方式不同寻常，它们之间仅间隔一个脯氨酸。根据序列数据推断该亚基在膜中的排列方式。
