Acceleration of disulfide-coupled protein folding using glutathione derivatives
Acceleration of disulfide-coupled protein folding using glutathione derivatives
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DOI：
10.1111/j.1742-4658.2011.08039.x
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发表时间：
2011-04-01
期刊：
影响因子：
5.4
通讯作者：
Hidaka, Yuji
中科院分区：
文献类型：
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作者：
Okumura, Masaki;Saiki, Masatoshi;Hidaka, Yuji
蛋白质折叠与二硫键形成同时发生。一般来说，含有二硫键的蛋白质的体外折叠是在氧化还原试剂（如谷胱甘肽）存在的情况下进行的，以允许天然二硫配对发生。众所周知，二硫键的形成和目标蛋白的正确三级结构受到所使用的氧化还原试剂的强烈影响。然而，关于氧化还原试剂中每个氨基酸残基的作用，如谷胱甘肽，所知甚少。因此，我们制备了谷胱甘肽衍生物谷氨酰半胱氨酸精氨酸（ECR）和精氨酸半胱氨酸甘氨酸（RCG），并以溶菌酶和proroguanylin作为模型蛋白，研究了它们促进蛋白质折叠的能力。当使用还原和氧化形式的RCG时，折叠回收率大于典型的谷胱甘肽氧化还原系统。当使用高浓度蛋白质时尤其如此，而使用ECR的折叠回收与谷胱甘肽氧化还原系统相似。氧化折叠动力学分析表明，还原RCG/氧化RCG的折叠速度（K-RCG = 3.69 × 10-3 s-1）比还原谷胱甘肽/氧化谷胱甘肽的折叠速度约高3倍。此外，仅使用RCG或谷胱甘肽氧化形式的折叠实验表明，与谷胱甘肽相比，RCG更有效地将prouroguanylin转化为天然构象。研究结果表明，带正电的氧化还原分子更倾向于加速二硫交换反应，RCG系统有效地介导蛋白质中天然二硫键的形成。
Protein folding occurs simultaneously with disulfide bond formation. In general, the in vitro folding of proteins containing disulfide bond(s) is carried out in the presence of redox reagents, such as glutathione, to permit native disulfide pairing to occur. It is well known that the formation of a disulfide bond and the correct tertiary structure of a target protein are strongly affected by the redox reagent used. However, little is known concerning the role of each amino acid residue of the redox reagent, such as glutathione. Therefore, we prepared glutathione derivatives - glutamyl-cysteinyl-arginine (ECR) and arginyl-cysteinyl-glycine (RCG) - and examined their ability to facilitate protein folding using lysozyme and prouroguanylin as model proteins. When the reduced and oxidized forms of RCG were used, folding recovery was greater than that for a typical glutathione redox system. This was particularly true when high protein concentrations were employed, whereas folding recovery using ECR was similar to that of the glutathione redox system. Kinetic analyses of the oxidative folding of prouroguanylin revealed that the folding velocity (K-RCG = 3.69 x 10-3 s-1) using reduced RCG/oxidized RCG was approximately threefold higher than that using reduced glutathione/oxidized glutathione. In addition, folding experiments using only the oxidized form of RCG or glutathione indicated that prouroguanylin was converted to the native conformation more efficiently in the case of RCG, compared with glutathione. The findings indicate that a positively charged redox molecule is preferred to accelerate disulfide-exchange reactions and that the RCG system is effective in mediating the formation of native disulfide bonds in proteins.