STRUCT OF ACETYLCHOLINE BINDING REGION FROM NICOTINIC ACETYLCHOLINE RECEPTOR

烟碱乙酰胆碱受体乙酰胆碱结合区的结构

• 批准号: 6120246
• 负责人: VLADIMIR J BASUS
• 金额: $ 0.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120246
• 关键词: biological products; biomaterials; biomedical resource; biotechnology; spectrometry; structural biology

The nicotinic acetylcholine receptor is found at neuromuscular junctions, where it functions to depolarize the postsynaptic membrane, triggering muscle contraction. Depolarization occurs by channel opening which is induced by the binding of two acetylcholine molecules. Neurotoxins inhibit the binding of acetylcholine by binding to a large portion of the receptor's a-subunit, blocking at least in part the acetylcholine binding site. The best structure for the receptor is at a low resolution level of 9 A, using a combination of electron microscopy and x-ray diffraction. NMR spectroscopy for this large membrane bound protein complex is clearly beyond current technological capabilities. However, peptides corresponding to the portion of the receptor near the acetylcholine binding site have been found to bind a-bungarotoxin with about the same affinity as the intact a-subunit. We propose here (1) to determine the rates of exchange and activation energies between R- and S-disulfide bond rearrangement and between a cis-trans amide bond interconversion in an eight-membered cysteinyl-cysteine ring, C192-C193 found in the active sight of the a-subunit; (2) to determine the active conformation of the cysteinyl-cysteine ring in peptide fragments derived from the AChR's a-subunit; (3) to determine the structure of the N-terminal extracellular region of the a-subunit that binds acetylcholine, agonists and antagonists in its free, agonist-bound and antagonist-bound states.Some of the peptides for this study have already been synthesized. We shall need to make a variety of different peptides whose syntheses have not been completely worked out. Products and reactions will have to be verified by mass spectrometry, which can be performed locally at UCSF. Once the desired products are made we shall pursue the structures and dynamics of these peptides in free and bound states by multi-dimensional multinuclear magnetic resonance techniques. The objective of this research is to determine whether or not the rare structural motif of a cysteinyl-cysteine ring may act as a molecular switch in opening and closing the ion channel of the AChR. From the molecular details of AChR ligand interactions with their binding site as well as the structural changes of the receptor binding site, we hope to shed light on AChR's highly specific recognition, mechanism and control.

烟碱乙酰胆碱受体存在于神经肌肉 连接，在那里它的功能是去突触后膜， 引发肌肉收缩 去极化按通道发生 由两个乙酰胆碱结合引起的开放 分子。 神经毒素抑制乙酰胆碱的结合， 与受体α亚基的大部分结合， 至少部分是乙酰胆碱结合位点。 最好的结构， 该受体处于9 A低分辨率水平，使用组合 电子显微镜和X射线衍射。 核磁共振波谱 这种大的膜结合蛋白复合物显然超出了目前的水平， 技术能力。 然而，对应于所述多肽的肽 乙酰胆碱结合位点附近的受体部分已经被 发现与α-银环蛇毒素结合的亲和力与 完整的α-亚单位。 我们在这里建议（1）确定 R-和S-二硫键之间的交换能和活化能 重排和顺-反酰胺键之间的相互转化， 活性物质中发现的八元半胱氨酰半胱氨酸环，C192-C193 a亚基的视线;（2）确定活性构象 衍生自半胱氨酸的肽片段中的半胱氨酸-半胱氨酸环 AChR的α-亚基;（3）确定N-末端的结构 结合乙酰胆碱的α-亚单位的胞外区， 激动剂和拮抗剂以其游离的、激动剂结合的和 拮抗剂结合状态。本研究的一些肽具有 已经合成了。 我们需要做各种各样的 不同的肽，其合成尚未完全完成 出去 产物和反应将必须通过质量来验证 光谱，这可以在UCSF本地进行。 一旦 我们将追求结构和动力学 这些肽在自由和结合状态的多维 多核磁共振技术。 的目的 研究的目的是确定一种罕见的结构基序是否 半胱氨酰-半胱氨酸环可以作为打开的分子开关， 关闭AChR的离子通道。 从分子的细节来看， AChR配体与其结合位点的相互作用以及 受体结合位点的结构变化，我们希望能够揭示 AChR的高度特异性识别，机制和控制。