Donaire A, Zhou Z H, Adams M M, La Mar G N
Department of Chemistry, University of California, Davis 95616, USA.
J Biomol NMR. 1996 Jan;7(1):35-47. doi: 10.1007/BF00190455.
The solution molecular structure of the four-iron ferredoxin (Fd) from the hyperthermophilic archaeon Thermococcus litoralis (Tl) has been investigated by 1H 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 in Tl Fd are a three-stranded antiparallel beta-strand involving the termini of the protein, a double beta-strand (also antiparallel), two alpha-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 of Tl Fd is very similar to that of the 3Fe ferredoxin from the mesophilic bacterium Desulfovibrio 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.
利用¹H NMR光谱研究了嗜热古菌嗜热栖热菌（Tl）的四铁铁氧化还原蛋白（Fd）的溶液分子结构。在H₂O中进行的TOCSY和NOESY实验，旨在检测弱弛豫和强弛豫共振，以及H₂O和D₂O中的稳态NOE，从而鉴定出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，与顺磁簇相连的所有四个半胱氨酸的接触位移和强弛豫信号，可以通过标准的主链连接性进行归属，而无需对三级结构做任何假设。在Tl Fd中鉴定出的二级结构元件包括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。还提出了未连接的半胱氨酸之间存在二硫键。在NOESY图谱中以及通过连接的半胱氨酸与“抗磁性”蛋白质基质之间的稳态NOE观察到的偶极接触表明，Tl Fd的整体折叠模式与嗜温细菌巨大脱硫弧菌的3Fe铁氧化还原蛋白非常相似[Kissinger等人（1991年）《分子生物学杂志》，219，693 - 723]。如其他单簇铁氧化还原蛋白同源质子的晶体结构所预测，簇的顺磁性对簇附近未连接残基以及连接的半胱氨酸的质子信号弛豫特性的影响，与它们到簇铁的距离密切相关。最后，讨论了各种已鉴定的结构因素对该蛋白质热稳定性的潜在作用。
