DETECTION OF ACH-MEDIATED CONFORMATIONAL CHANGES OF ACHBP

ACH介导的ACHBP构象变化的检测

• 批准号: 7598788
• 负责人: Steven M Sine
• 金额: $ 0.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7598788
• 关键词: Agonist; Binding Sites; Chemicals; Cholinergic Receptors; Computer Retrieval of Information on Scientific Projects Database; Cysteine; Detection; Family; Funding; Gated Ion Channel; Grant; Institution; Label; Ligand Binding Domain; Ligands; Mediating; Molecular Conformation; Mutate; Peripheral; Proteins; Research; Research Personnel; Resources; Serine; Source; United States National Institutes of Health; member; molecular dynamics; mutant

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Acetylcholine receptor (AChR) is a member of ligand-gated ion channel family. Small agonists like ACh trigger the channel opening by changing its conformation from the closed state to the open state. Here we want to study ACh-mediated conformational changes to AChBP, a soluble protein that is homologous to the ligand binding domain of AChR. Our molecular dynamics simulation (JBC in press) indicates ACh triggers the collapse of a peripheral loop region called C-loop on the binding site. At the tip of the C-loop, two vicinal cysteines have dramatic conformational changes. We isotopically-labeled only cysteine residues (U-13C3, 15N) in the protein and plan to see change of chemical shift in NMR spectra following addition of ACh. Since there are other cysteines in the protein, the spectra of a mutant protein with the vicinal cyteines mutated to Serine will be compared.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 乙酰胆碱受体（AChR）是配体门控离子通道家族的成员。像 ACh 这样的小激动剂通过将其构象从关闭状态改变为打开状态来触发通道打开。在这里，我们想要研究 ACh 介导的 AChBP 构象变化，AChBP 是一种与 AChR 配体结合域同源的可溶性蛋白。我们的分子动力学模拟（JBC 正在出版）表明 ACh 触发了结合位点上称为 C 环的外围环区域的崩溃。在 C 环的顶端，两个邻近的半胱氨酸发生了显着的构象变化。我们仅对蛋白质中的半胱氨酸残基（U-13C3、15N）进行同位素标记，并计划在添加 ACh 后观察 NMR 光谱中化学位移的变化。由于蛋白质中还有其他半胱氨酸，因此将比较邻位半胱氨酸突变为丝氨酸的突变蛋白的光谱。