The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S.
The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S.
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镓取代的腐氧还蛋白突变体的溶液结构：GaPdx C85S。
DOI：
10.1023/a:1008354113765
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发表时间：
1998
影响因子：
2.7
通讯作者：
Kazanis,S
中科院分区：
文献类型：
--
作者：
Pochapsky,TC;Kuti,M;Kazanis,S
铁氧还蛋白putidaredoxin （Pdx）的fe2s2簇可以被单个镓离子取代，从而产生一种无色、抗磁性的蛋白，其中除了金属结合位点外，天然铁氧还蛋白的主要结构特征是保守的。用均匀的15n、13C标记的C85S GaPdx样品，通过多层核磁共振（NMR）方法测定了g85s GaPdx的溶液结构，其中非配体半胱氨酸被丝氨酸取代。利用13c标记样品获得的13c、1H HSQC光谱对亮氨酸和valine甲基共振进行了立体定向配位，并利用二维j调制15n、1H HSQC和三维（HN）CO(CO)NH实验的组合获得了主二面角约束。计算中共使用了1117个noe导出的距离约束，包括454个短程（），456个远程（i - j≥4）互残约束和207个非琐屑残留约束。97 φ和55 χ1角约束也包括在使用组合距离几何模拟退火方案的20个结构族的计算中。在计算中，蛋白质的大多数区域都被很好地定义，主链原子的RMSD为0.525 Å，不包括金属结合环（残基34-48）和最后三个c端残基（残基103-106）。在有可能进行比较的地方，这些区域的动态行为比天然蛋白有所增加，含有残基74-76的环也是如此。讨论了天然Pdx和GaPdx的结构和动力学差异与金属结合位点的电荷和包装有关。
The Fe2S2cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly15N,13C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using13C,1H HSQC spectra obtained with fractionally13C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated15N,1H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range (), 456 long range (i - j ≥ 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 φ and 55 χ1angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 Å for backbone atoms excluding the metal binding loop (residues 34–48) and the last three C-terminal residues (residues 103–106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74–76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.
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