Functional Analysis of Butyrolactone Autoregulator Receptor from Streptomyces virginiae

为了大致确定丁内酯自调节受体上的必要区域，我们首先用PCR方法制备了几个缺失突变体。然而，缺少n端47个氨基酸残基的氨基酸在大肠杆菌中表达时形成了高分子量的聚集体，而缺少c端49或96个氨基酸残基的氨基酸在大肠杆菌中表达时形成了包涵体。与硫氧还蛋白融合的蛋白也表现出相同的现象，这表明n端和c端50个氨基酸残基对维持受体蛋白的高级结构都很重要。接下来，为了鉴定必需氨基酸残基，我们用化学试剂修饰受体。DTNB能与Cys残基发生特异性反应，能修饰受体的两个Cys残基。然而，通过凝胶过滤实验，得到的受体保留了完全的配体结合活性，并且显示出完整的二聚体结构，这表明Cys残基在受体中没有发挥任何重要作用。染料敏化光氧化导致配体结合活性丧失，但修饰后的受体以高分子量聚集体的形式存在，表明修饰后的残基参与了结构稳定性。

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.