Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A
Isomeric Replacement of a Single Aspartic Acid Induces a Marked Change in Protein Function: The Example of Ribonuclease A
复制标题
DOI：
10.1021/acsomega.6b00346
复制
发表时间：
2017-01-01
期刊：
影响因子：
4.1
通讯作者：
Fujii, Noriko
中科院分区：
文献类型：
--
作者：
Sakaue, Hiroaki;Kinouchi, Tadatoshi;Fujii, Noriko
蛋白质中的L α-天冬氨酸（Asp）残基在生理条件下非酶促异构化为异常的L β-、D α-和D β-Asp异构体。Asp残基的这种异构化被认为是蛋白质变性的触发因素，因为它延长了主链或诱导蛋白质结构内侧链的不同取向或两者兼而有之。然而，以前的研究没有发现直接证据表明天冬氨酸异构体对蛋白质功能的影响。因此，需要生产含Asp异构体的蛋白质来验证Asp异构化的影响。在这里，我们描述了使用表达的蛋白质连接来生产含Asp异构体的蛋白质。作为模型蛋白，使用牛胰腺核糖核酸酶A（RNase A，EC 3.1.27.5），其催化RNA中磷酸二酯键的裂解。在这项研究中，RNase A中121位的L α-Asp被L β-、D α-和D β-Asp取代。121位的目标天冬氨酸位于活性位点附近，与RNA切割有关。在121位具有L α-Asp的RNase A显示正常活性。相比之下，含L α-、D α-和D β-Asp的RNase A的催化活性显著降低。这项研究代表了第一次合成和分析的蛋白质含有四种不同的天冬氨酸异构体。
L alpha-Aspartic acid (Asp) residues in proteins are nonenzymatically isomerized to abnormal L beta-, D alpha-, and D beta-Asp isomers under physiological conditions. Such an isomerization of Asp residues is considered to be a trigger of protein denaturation because it either elongates the main chain or induces a different orientation of the side chain within the protein structure or both. However, previous studies have found no direct evidence of the effects of Asp isomers on protein function. Therefore, the production of Asp-isomercontaining proteins is required to verify the effects of Asp isomerization. Here, we describe the production of an Asp-isomer-containing protein using the expressed protein ligation. As a model protein, bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5), which catalyzes the cleavage of phosphodiester bonds in RNA, was used. In this study, L alpha-Asp at position 121 in RNase A was replaced by L beta-, D alpha-, and D beta-Asp. The objective aspartic acid at position 121 is located near the active site and related to RNA cleavage. The RNase A with L alpha-Asp at position 121 showed a normal activity. By contrast, the catalytic activity of L alpha-, D alpha-, and D beta-Asp-containing RNase A was markedly decreased. This study represents the first synthesis and analysis of a protein containing four different Asp isomers.