1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis.
1H NMR investigation of the secondary structure, tertiary contacts and cluster environment of the four-iron ferredoxin from the hyperthermophilic archaeon Thermococcus litoralis.
复制标题
1H NMR 研究来自超嗜热古细菌 Thermococcus litoralis 的四铁铁氧还蛋白的二级结构、三级接触和簇环境。
DOI：
10.1007/bf00190455
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发表时间：
1996
影响因子：
2.7
通讯作者：
LaMar,GN
中科院分区：
文献类型：
--
作者：
Donaire,A;Zhou,ZH;Adams,MM;LaMar,GN
用~1H核磁共振波谱研究了嗜热古细菌(TL)的四铁铁还蛋白(FD)的溶液分子结构。TOCSY和NOESY在H2O中的实验是为检测弱弛豫和强弛豫共振而量身定做的，加上H2O和D2O中的稳态NO，允许识别59个残基中的58个，顺磁中心附近的一个残基未被检测到。结果表明，连接到顺磁团簇上的所有四个半胱氨酸的接触位移和强弛豫信号都可以由标准主链连接性来指定，而不需要任何关于三级结构的假设。在TlFd中发现的二级结构元件是一个涉及蛋白质末端的三链反平行β链，一个双β链(也是反平行)，两个α螺旋和四个转角。还提出了在未连接的半胱氨酸之间存在二硫键的观点。在NOESY图谱和连接的半胱氨酸与‘抗磁性’蛋白质基质之间的稳态NOE中观察到的偶极接触表明，T1Fd的总体折叠模式与来自中嗜菌Desulfovibrio gias的3Fe铁氧还蛋白非常相似[Kisinger等人。(1991)J.Mol.生物，219,693-723]。团簇的顺磁性对团簇附近未连接残基的质子信号的松弛性质以及连接的半胱氨酸的影响与团簇铁的接近程度(S)有很好的相关性，正如从其他单簇铁还蛋白的同源质子结构预测的那样。最后，讨论了各种已确定的结构因素在该蛋白质的超热稳定性中的潜在作用。
The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeonThermococcus litoralis (Tl)has been investigated by1H NMR spectroscopy. TOCSY and NOESY experiments in H2O, tailored to detect both weakly and strongly relaxed resonances, together with steady-state NOEs in both H2O and D2O, allowed the identification of 58 of the 59 residues, with one residue near the paramagnetic center undetected. It is shown that the contact shifted and strongly relaxed signals for all four cysteines ligated to the paramagnetic cluster can be assigned by standard backbone connectivities that do not require any assumptions about the tertiary structure. Secondary structural elements identified inTlFd are a three-stranded antiparallel β-strand involving the termini of the protein, a double β-strand (also antiparallel), two α-helices and four turns. The existence of a disulfide bridge between the nonligated cysteines is also proposed. Dipolar contacts observed in the NOESY maps and by steady-state NOEs between the ligated cysteines and the ‘diamagnetic’ protein matrix indicate that the overall folding pattern ofTlFd is very similar to that of the 3Fe ferredoxin from the mesophilic bacteriumDesulfovibrio gigas[Kissinger et al. (1991)J. Mol. Biol.,219, 693–723]. The influence of the paramagnetism of the cluster on the relaxation properties of the proton signals of nonligated residues near the cluster, as well as on the ligated cysteines, correlates well with the proximity to the cluster iron(s), as predicted from the crystal structures for homologous protons of other single-cluster ferredoxins. Finally, the potential role of the various identified structural factors in contributing to the hyperthermostability of this protein is discussed.