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.

Type 1 inositol 1, 4, 5-trisphosphate receptor (IP, R1), an inositol 1, 4, 5-trisphosphate (IP, R1), an inositol 1, 4, 5-trisphosphate (IP, D23E D2-gated Caiy D12+E D1 release channel binds IPiy D23E D2 within the N-terminal ligand-binding region. Here we report an改进的大肠杆菌表达系统，其中大量的IPIY D23E D2结合位点可以有效地作为固体活性蛋白产生，我们发现IPII D23E D2的结构在大肠杆菌中表达的结合构建体表达了大肠杆菌的结合构建体。总体分数。 IPI D23 D23 D2的分子识别。纯化的小鼠小脑1型肌醇的依赖性，1、4、5-三磷酸受体（IPI D23 D2R1）/CAI D22+IE D22+IE D2通道在细胞质CAI D12+IE D1 d1 d1 d1 d1 d1 d1 d1 d1 d1 d1 d1 d1 d1。与粗系统中的通道相反，纯化的IPII D23I D2R1重构为平面脂质双拉剂，并未显示对CAII D12+E D1的钟形依赖性。它通过增加CAII D12+E D1的增加而被激活，即使没有抑制至2000μm也没有抑制。在较高的CAII D12+E D1浓度下，向细胞质侧添加钙调蛋白抑制了通道。钙调蛋白拮抗剂逆转了小脑微粒体中天然通道的CAII D12+E D1依赖性失活。这些结果表明，对细胞质CAI D12+IE D1的钟形依赖性不是IPI D23的内在特性，而CAI D12+IE D1？依赖性失活是由钙调蛋白直接介导的。