Functional Analysis of Butyrolactone Autoregulator Receptor from Streptomyces virginiae

为了粗略地确定丁内酯自身调节受体的必需区域，我们首先通过PCR的方法制备了几个缺失突变体，但缺乏N-端47个氨基酸残基的突变体在大肠杆菌中表达时形成了高相对分子质量的聚集体，而缺乏C-末端49或96个氨基酸残基的突变体在大肠杆菌中表达时形成了包涵体。与硫氧还蛋白的融合蛋白也表现出相同的现象，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是重要的。DTNB能与Cys残基发生特异性反应，可以修饰受体的两个Cys残基。然而，凝胶过滤实验显示，所得到的受体保留了完整的配体结合活性，并显示出完整的二聚体结构，表明半胱氨酸残基在受体中不起任何重要作用。染料敏化的光氧化导致配体结合活性的丧失，但修饰的受体以高分子量聚集体的形式存在，表明修饰残基参与了结构的稳定。

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.