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首页> 外文期刊>Biochemistry >Solution structure and dynamics of the human-Escherichia coli thioredoxin chimera: insights into thermodynamic stability.
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Solution structure and dynamics of the human-Escherichia coli thioredoxin chimera: insights into thermodynamic stability.

机译：人类大肠杆菌硫氧还蛋白嵌合体的溶液结构和动力学：对热力学稳定性的见解。

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摘要

We have determined the high-resolution solution structure of the oxidized form of a chimeric human and Escherichia coli thioredoxin (TRX(HE)) by NMR. The overall structure is well-defined with a rms difference for the backbone atoms of 0.27 +/- 0.06 A. The topology of the protein is identical to those of the human and E. coli parent proteins, consisting of a central five-stranded beta-sheet surrounded by four alpha-helices. Analysis of the interfaces between the two domains derived from the human and E. coli sequences reveals that the general hydrophobic packing is unaltered and only subtle changes in the details of side chain interactions are observed. The packing of helix alpha(4) with helix alpha(2) across the hybrid interface is less optimal than in the parent molecules, and electrostatic interactions between polar side chains are missing. In particular, lysine-glutamate salt bridges between residues on helices alpha(2) and alpha(4), which were observed in both human and E. coli proteins, are not present in the chimeric protein. The origin of the known reduced thermodynamic stability of TRX(HE) was probed by mutagenesis on the basis of these structural findings. Two mutants of TRX(HE), S44D and S44E, were created, and their thermal and chemical stabilities were examined. Improved stability toward chaotropic agents was observed for both mutants, but no increase in the denaturation temperature was seen compared to that of TRX(HE). In addition to the structural analysis, the backbone dynamics of TRX(HE) were investigated by (15)N NMR relaxation measurements. Analysis using the model free approach reveals that the protein is fairly rigid with an average S(2) of 0.88. Increased mobility is primarily present in two external loop regions comprising residues 72-74 and 92-94 that contain glycine and proline residues.
机译：我们已经通过NMR确定了嵌合人和大肠杆菌硫氧还蛋白（TRX（HE））的氧化形式的高分辨率溶液结构。整个结构定义明确，骨架原子的均方根差为0.27 +/- 0.06A。该蛋白质的拓扑结构与人和大肠杆菌亲本蛋白的拓扑结构相同，由中央五链β组成页被四个alpha螺旋包围。对源自人和大肠杆菌序列的两个结构域之间的界面的分析表明，一般的疏水性填充没有改变，并且仅观察到侧链相互作用细节上的细微变化。螺旋α（4）与螺旋α（2）在杂化界面上的堆积比在母体分子中的填充效果不佳，并且极性侧链之间的静电相互作用也消失了。特别地，在人和大肠杆菌蛋白质中都观察到的螺旋α（2）和α（4）残基之间的赖氨酸-谷氨酸盐桥不存在于嵌合蛋白中。基于这些结构发现，通过诱变探测了TRX（HE）已知降低的热力学稳定性。创建了TRX（HE）的两个突变体S44D和S44E，并检查了它们的热稳定性和化学稳定性。观察到两个突变体对离液剂的稳定性都有改善，但是与TRX（HE）相比，变性温度没有升高。除结构分析外，还通过（15）N NMR弛豫测量研究了TRX（HE）的骨架动力学。使用无模型方法进行的分析表明，该蛋白质相当坚硬，平均S（2）为0.88。增加的迁移率主要存在于两个外部环区域中，所述两个外部环区域包含含有甘氨酸和脯氨酸残基的残基72-74和92-94。

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