DETECTION OF ACH-MEDIATED CONFORMATIONAL CHANGES OF ACHBP

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者的研究机构。 乙酰胆碱受体（acetylcholine receptor，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闭合状态改变为开放状态来触发通道开放。在这里，我们要研究乙酰胆碱介导的构象变化，AChBP，一种可溶性蛋白，是同源的配体结合结构域的乙酰胆碱受体。我们的分子动力学模拟（JBC出版）表明乙酰胆碱触发的崩溃的周边环区域称为C环的结合位点。在C环的顶端，两个相邻的半胱氨酸具有显著的构象变化。我们仅对蛋白质中的半胱氨酸残基（U-13 C3，15 N）进行同位素标记，并计划观察添加ACh后核磁共振光谱中化学位移的变化。由于蛋白质中存在其他半胱氨酸，因此将比较具有突变为丝氨酸的邻位半胱氨酸的突变蛋白质的光谱。

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.