The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.
The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.
复制标题
DOI：
10.1021/bi500654y
复制
发表时间：
2014-09-30
期刊：
影响因子：
2.9
通讯作者：
Crofts, Antony R.
中科院分区：
文献类型：
--
作者：
Hong, Sangjin;de Almeida, Wagner B.;Taguchi, Alexander T.;Samoilova, Rimma I.;Gennis, Robert B.;O'Malley, Patrick J.;Dikanov, Sergei A.;Crofts, Antony R.
在残基或取代基水平上的特定同位素标记将不同光谱方法的范围扩展到原子水平。在本文中，我们用~(13)C标记的~(13)C标记的侧链甲基和甲硫氨酸，通过在球形红杆菌BC17中添加L-蛋氨酸构建了甲硫氨酸营养缺陷体，从而实现了泛醌的甲基和甲氧基环取代基的~(13)C标记。用二维电子自旋回波包络调制(HYSCORE)方法研究了Bc1络合物在膜环境中原位生成的半醌分子中13C甲基和甲氧基的超精细偶联.根据13C超精细张量的密度泛函理论计算，表征了BC1络合物(蛋白质数据库条目1PP9)的几何优化X射线结构中的未配对自旋密度分布和甲氧基取代基的构象。与其他蛋白质的比较表明，在每个特定的情况下，甲氧基的个别取向总是与在冷冻乙醇溶液中制备的阴离子自由基的甲氧基构象不同。在蛋氨酸营养缺陷症的产生中使用的方案更普遍地适用，并且因为它引入了使用自杀质粒的基因缺失，所以可以重复应用。
Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly.
登录
查看更多内容
影响因子：
4.8
作者：
Esser, Lothar;Elberry, Maria;Xia, Di
通讯作者：
Xia, Di
影响因子：
2.9
作者：
Gao, XG;Wen, XL;Xia, D
通讯作者：
Xia, D
影响因子：
5.6
作者：
Huang, LS;Cobessi, D;Berry, EA
通讯作者：
Berry, EA
影响因子：
4.8
作者：
DikanovO, Sergei A.;Hollan, J. Todd;Crofts, Antony R.
通讯作者：
Crofts, Antony R.
影响因子：
2.9
作者：
DONOHUE, TJ;MCEWAN, AG;KAPLAN, S
通讯作者：
KAPLAN, S