Functional Analysis of Butyrolactone Autoregulator Receptor from Streptomyces virginiae

弗吉尼亚链霉菌丁内酯自动调节受体的功能分析

• 批准号: 07680684
• 负责人: NIHIRA Takuya
• 金额: $ 1.47万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07680684/
• 关键词: butyrolactone autoregulator; receptor; Streptomyces; virginiae butanolide; 放線菌; autoregulator; リセプター; 大量発現

To roughly determine the essential regions on the butyrolactone autoregulator receptor, we at first prepared several deletion mutants by means of PCR.However, the one lacking N-terminal 47 amino acid residues formed high-molecular weight aggregates, while the ones lacking either C-terminal 49 or 96 amino acid resildues formed inclusion bodies when expressed in E.coli. Fusion proteins with thioredoxin also showed the same phenomena, indicating that both the N-terminal and C-terminal 50 amino acid residues are important in maintaining the higher structure of the receptor protein.Next, to identify the essential amino acid residues, we modified the receptor by chemical reagents. DTNB which can specifically react with Cys residues could modify two Cys residues of the receptor. However, the resulting receptor retained the full ligand binding activity and also showed the intact dimeric structure as revealed by gel-filtration experiments, indicating that Cys residues did not play any important roles in the receptor. Dye-sensitized photooxidation resulted in the loss of ligand binding activity, but the modified receptor was present as high-molecular weight aggregates, indicating that the modified residues participate in structure stability.

为了初步确定丁内酯自动调节受体的功能区，我们首先用PCR方法制备了几种缺失突变体，但当在大肠杆菌中表达时，缺失N端47个氨基酸残基的突变体形成了高分子量的聚集体，而缺失C端49或96个氨基酸残基的突变体形成了包涵体。与硫氧还蛋白的融合蛋白也表现出同样的现象，表明N端和C端的50个氨基酸残基对维持受体蛋白的高级结构都很重要。DTNB能特异性地与Cys残基反应，修饰受体的两个Cys残基。然而，得到的受体保留了完整的配体结合活性，也显示了完整的二聚体结构，如凝胶过滤实验所示，表明Cys残基在受体中没有发挥任何重要作用。染料敏化的光氧化导致配体结合活性的损失，但改性的受体是作为高分子量的聚集体，表明改性的残基参与结构稳定性。