Structural model for an alkaline form of ferricytochrome c
Structural model for an alkaline form of ferricytochrome c
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DOI：
10.1021/ja027180s
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发表时间：
2003-03-12
影响因子：
15
通讯作者：
Gray, HB
中科院分区：
文献类型：
--
作者：
Assfalg, M;Bertini, I;Gray, HB
通过 NMR 光谱检查了碱性构象的酵母 iso-1-ferricytochrome c 三重变体 (Lys72Ala/Lys79Ala/Cys102Thr) 的 N-15 富集样品。这些突变计划产生具有单一构象异构体的细胞色素 c。尽管光谱收集的条件不理想（即 pH 值接近 11），NMR 仍然是一种能够利用顺磁性的合适的研究技术。 76%的氨基酸和49%的质子被成功分配。该分配部分是通过标准方法实现的，部分是通过识别与 pH 7 下的天然蛋白质中保持相同构象的基团，以及对于其他一些残基，通过对核间距离预测的初步分析来实现的。 Lys73 与 His18 一起被指定为轴向配体。以这种方式，108个氨基酸的838个有意义的NOES、50个主链角度约束和203个假接触位移允许随机生成的结构收敛到主链RMSD为1.5+/-0.2埃的构象异构体家族。大多数天然细胞色素 c 构象在高 pH 条件下得以维持。涉及 His18 的 NOE 模式清楚地表明该蛋白质的近侧（包括 20s 和 40s 环）基本保持完整。结构差异集中在 70-80 环（因为作为轴向配体的 Lys73 取代了 Met80），以及面向该环的 50s 螺旋；因此，血红素基团与溶剂的接触增加。基于几个光谱特征，我们得出结论，折叠的多肽具有高度流动性。
An N-15-enriched sample of the yeast iso-1-ferricytochrome c triple variant (Lys72Ala/Lys79Ala/Cys102Thr) in an alkaline conformation was examined by NMR spectroscopy. The mutations were planned to produce a cytochrome c with a single conformer. Despite suboptimal conditions for the collection of spectra (i.e., pH approximate to 11), NMR remains a suitable investigation technique capable of taking advantage of paramagnetism. 76% of amino acids and 49% of protons were assigned successfully. The assignment was in part achieved through standard methods, in part through the identification of groups maintaining the same conformation as in the native protein at pH 7 and, for a few other residues, through a tentative analysis of internuclear distance predictions. Lys73 was assigned as the axial ligand together with His18. In this manner, 838 meaningful NOES for 108 amino acids, 50 backbone angle constraints, and 203 pseudocontact shifts permitted the convergence of randomly generated structures to a family of conformers with a backbone RMSD of 1.5+/-0.2 Angstrom. Most of the native cytochrome c conformation is maintained at high pH. The NOE pattern that involves His18 clearly indicates that the proximal side of the protein, including the 20s and 40s loops, remains essentially intact. Structural differences are concentrated in the 70-80 loop, because of the replacement of Met80 by Lys73 as an axial ligand, and in the 50s helix facing that loop; as a consequence, there is increased exposure of the heme group to solvent. Based on several spectral features, we conclude that the folded polypeptide is highly fluxional.