STRUCT OF ACETYLCHOLINE BINDING REGION FROM NICOTINIC ACETYLCHOLINE RECEPTOR

烟碱型乙酰胆碱受体存在于神经肌肉 连接，在那里它起到去极化突触后膜的作用， 引发肌肉收缩。去极化按通道进行 由两个乙酰胆碱结合引起的开放 分子。神经毒素通过以下途径抑制乙酰胆碱的结合 与受体a亚单位的很大一部分结合，阻断在 最少部分是乙酰胆碱结合部位。最好的结构是 接收器处于9A的低分辨率水平，使用组合 电子显微镜和x射线衍射仪。核磁共振波谱的研究 这种大的膜结合蛋白复合体显然超出了目前的范围 技术能力。然而，与之相对应的多肽 受体靠近乙酰胆碱结合部位的部分已经被 发现与银环蛇毒素结合的亲和力与 完整的a亚基。我们建议在此(1)确定 R-和S-二硫键之间的交换和活化能 重排和顺式-反式酰胺键之间的相互转化 八元半胱氨基-半胱氨酸环，C192-C193 (2)测定α-亚基的活性构象 肽片段中的半胱氨基-半胱氨酸环 AchR的α-亚基；(3)确定N-末端的结构 与乙酰胆碱结合的a亚基的胞外区， 激动剂和拮抗剂在其自由、激动剂结合和 拮抗剂结合状态。本研究的一些肽具有 已经被合成了。我们需要生产各种不同的 合成尚未完全完成的不同多肽 出去。产品和反应必须通过质量来验证 光谱分析，可在加州大学旧金山分校就地进行。一旦 我们将追求产品的结构和动力 这些多肽在自由态和结合态的多维 多核磁共振技术。这样做的目的是 研究的目的是确定一个稀有的结构主题是否 半胱氨基-半胱氨酸环可作为分子开关 关闭AChR的离子通道。从分子细节上看 Achr配体与其结合部位的相互作用以及 受体结合部位的结构变化，希望对我们有所启示 对AChR高度特异的识别、机制和控制。

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.