Analysis of the molecular dynamics of intracellular signal transduction by chromophore, assisted inactivatid

发色团辅助灭活细胞内信号转导的分子动力学分析

基本信息

批准号：
10558112
负责人：
MIKOSHIBA Katsuhiko
金额：
$ 8.26万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Type 1 inositol 1, 4, 5-trisphosphate receptor (IP, R1), an inositol 1, 4, 5-trisphosphate (IPィイD23ィエD2-gated CaィイD12+ィエD1 release channel binds IPィイD23ィエD2 within the N-terminal ligand-binding region. Here we report an improved Escherichia coli expression system in which large amounts of the IPィイD23ィエD2 binding sites could be efficiently produced as soluble active proteins. We have found that the structures of IPィイD23ィエD2, binding constructs expressed in E. coli significantly affect their production as soluble protein Residues 1-6O4 (T604), which contain the putative protein folding units, yielded about 4.6% of the total soluble fraction. As a result, soluble active T604 would be 19 mg per liter of culture. The affinity for IPィイD23ィエD2 of T604 (KィイD2dィエD2 = 45nM) is comparable to of the native IPィイD23ィエD2R1, whereas that of an R441Q mutant is much higher (8.1 nM). This system should provide an invaluable and powerful means to unveil the molecular recognition of IPィイD23ィエD2R1 for IPィイD23ィエD2.The dependency of purified mouse cerebellar type 1 inositol, 1, 4, 5-trisphosphate receptor (IPィイD23ィエD2R1)/CaィイD22+ィエD2 channel function on cytoplasmic CaィイD12+ィエD1 was examined. In contrast to the channels in crude systems, the purified IPィイD23ィエD2R1 reconstituted into planar lipid bilaryers did not show the bell-shaped dependence on CaィイD12+ィエD1. It was activated with increasing CaィイD12+ィエD1 sublinearly without inhibition even up to 2000 μM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high CaィイD12+ィエD1 concentrations. Calmodulin antagonists reversed the CaィイD12+ィエD1 -dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic CaィイD12+ィエD1 is not an intrinsic property of the IPィイD23ィエD2R1, and the CaィイD12+ィエD1 ?dependent inactivation is directly mediated by calmodulin.

1 型肌醇 1, 4, 5-三磷酸受体 (IP, R1)，肌醇 1, 4, 5-三磷酸（IP-D23 门控 Ca-D12+D1 释放通道在 N 端配体内结合 IP-D23 D2在这里，我们报告了一种改进的大肠杆菌表达系统，其中大量的IP-D23-D2 结合位点可以作为可溶性活性蛋白有效产生。我们发现，在大肠杆菌中表达的 IP-D23-D2 结合构建体的结构显着影响其作为可溶性蛋白残基 1-6O4 (T604) 的产生。，其中包含推定的蛋白质折叠单位，产生约 4.6% 的总可溶性级分，因此，可溶性活性 T604 为每 19 毫克。 T604 对 IPD23D2 的亲和力 (KD2D2 = 45nM) 与天然 IPD23D2R1 相当，而 R441Q 突变体的亲和力要高得多 (8.1 nM)。 IPD23D2R1 的识别IP-D23-D2。检查了纯化的小鼠小脑1型肌醇、1,4,5-三磷酸受体(IP-D23-D2R1)/Ca-D22+D2通道功能对细胞质Ca-i D12+D1的依赖性。与粗制系统中的通道相比，纯化的 IPD23D2R1 重组为平面脂质胆管没有表现出对CaiD12+D1的钟形依赖性。它随着CaiD12+D1的增加而亚线性地被激活，即使达到2000μM，在细胞质侧添加钙调蛋白也会在高CaiD12+D1浓度下抑制通道。拮抗剂逆转了CaiD12+D1依赖性的天然失活这些结果表明，对细胞质 CaD12+D1 的钟形依赖性并不是 IPD23D2R1 的固有特性，并且 CaD12+D1 依赖性失活是由钙调蛋白直接介导的。