3D Domain Swapping of Azurin from Alcaligenes xylosoxidans
3D Domain Swapping of Azurin from Alcaligenes xylosoxidans
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来自木糖氧化产碱菌的天青蛋白的 3D 结构域交换
DOI：
10.1039/c9mt00255c
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发表时间：
2020
期刊：
影响因子：
3.4
通讯作者：
Shun Hirota
中科院分区：
文献类型：
--
作者：
Robby Noor Cahyono;Masaru Yamanaka;Satoshi Nagao;Naoki Shibata;Yoshiki Higuchi;Shun Hirota
蛋白质低聚物因其在细胞中的知识增加和在未来材料中的应用前景而受到人们的关注。各种蛋白质的3D结构域交换已经被证明，但还没有关于蓝铜蛋白的结构域交换的报道。在此，我们发现产碱杆菌的天青素在pH为5.0时通过2，2，2-三氟乙醇与铜(I)-天青素反应生成齐聚物，然后在pH为7.0时进行冷冻干燥和溶解，而使用铜(II)-天青素时，天青素发生轻微的齐聚。在类似的溶解过程中，随着铜(II)离子的加入，高阶齐聚物的数量增加，表明铜(II)离子促进了天青齐聚反应。天青二聚体在460 nm处的吸光度与∼620 nm处的吸光度之比(Abs460/Abs618=0.113)高于单体的吸光度(Abs460/Abs622=0.067)，而二聚体的‖值(5.85mT)略小于单体(5.95mT)，表明二聚体的铜配位更具方向性。天青二聚体的氧化还原电位为342±5 mVs.NHE，比单体高50 mV。根据X-射线晶体分析，天青二聚体呈现结构域交换结构，其中含有三条β链的N-末端区域在原型之间交换。天青二聚体的铜配位结构发生了四面体扭曲，与单体相似，但二聚体的铜-氧(Gly45)键长较长(单体为2.46-2.59；二聚体为2.98-3.25)。这些结果为通过结构域交换设计蓝铜蛋白低聚物打开了大门。
Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to 3D domain swap, but there has been no domain swapping report on a blue copper protein. Here, we found that azurin fromAlcaligenes xylosoxidansoligomerizes by the procedure of 2,2,2-trifluoroethanol addition to Cu(i)-azurin at pH 5.0, lyophilization, and dissolution at pH 7.0, whereas it slightly oligomerizes when using Cu(ii)-azurin. The amount of high order oligomers increased with the addition of Cu(ii) ions to the dissolution process of a similar procedure for apoazurin, indicating that Cu(ii) ions enhance azurin oligomerization. The ratio of the absorbance at 460 nm to that at ∼620 nm of the azurin dimer (Abs460/Abs618= 0.113) was higher than that of the monomer (Abs460/Abs622= 0.067) and the EPRA‖value of the dimer (5.85 mT) was slightly smaller than that of the monomer (5.95 mT), indicating a slightly more rhombic copper coordination for the dimer. The redox potential of the azurin dimer was 342 ± 5 mVvs.NHE, which was 50 mV higher than that of the monomer. According to X-ray crystal analysis, the azurin dimer exhibited a domain-swapped structure, where the N-terminal region containing three β-strands was exchanged between protomers. The copper coordination structure was tetrahedrally distorted in the azurin dimer, similar to that in the monomer; however, the Cu–O(Gly45) bond length was longer for the dimer (monomer, 2.46–2.59 Å; dimer, 2.98–3.25 Å). These results open the door for designing oligomers of blue copper proteins by domain swapping.