NMR Studies of Alpha-Bungarotoxin-Acetylcholine Receptor Peptide Complexes

烟碱型乙酰胆碱受体位于神经肌肉交界处，其功能是使突触后膜上的肌肉细胞去极化，引发肌肉收缩。去极化是由乙酰胆碱结合引起的通道开放，以允许小阳离子通过。神经毒素通过结合很大一部分乙酰胆碱受体亚单位，阻断至少部分乙酰胆碱结合部位，从而抑制乙酰胆碱的结合。结合电子显微镜和X射线衍射数据，该受体的最佳结构是在22A的低分辨率水平。这种大型膜结合复合体的核磁共振波谱显然超出了当前的技术能力。然而，与乙酰胆碱结合位点附近受体部分相对应的多肽与银环蛇毒素结合的亲和力与整个亚基结合这种神经毒素的亲和力大致相同。本文建议使用二维和三维核磁共振波谱，包括具有选择性同位素标记的异核技术，来确定这些与银环蛇毒素结合的多肽的结构。这将首次确定含有神经毒素结合位点主要决定因素的部分乙酰胆碱受体与至少部分乙酰胆碱结合位点之间相互作用的细节。通过研究不同多肽形成的复合体，将阐明银环蛇毒素与乙酰胆碱受体之间特异性结合作用的相对重要性。这将有助于了解蛋白质之间非共价相互作用的一般知识。

The nicotinic acetylcholine receptor is found at neuromuscular junctions, where its function is to depolarize muscle cells at the postsynaptic membrane, triggering muscle contraction. Depolarization occurs by channel opening, induced by the binding of acetylcholine, to allow the passage of small cations. Neurotoxins inhibit the binding of acetylcholine, by binding a large portion of the acetylcholine receptor -subunit, blocking at least part of the acetylcholine binding site. The best structure for the receptor is at the low resolution level of 22 A, using a combination of electron microscopy and x-ray diffraction data. NMR spectroscopy for this large membrane bound 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 - bungarotoxin with about the same affinity as the entire - subunit binds this neurotoxin. It is proposed here to determine the structure of these peptides bound to -bungarotoxin, using two and three-dimensional NMR spectroscopy, including heteronuclear techniques with selective isotopic labeling. This will allow for the first time the details of the interactions between a portion of the acetylcholine receptor containing the major determinant of the neurotoxin binding site, and at least part of the acetylcholine binding site to be determined. By studying complexes formed with different peptides the relative importance of specific binding interactions between - bungarotoxin and the acetylcholine receptor, will be clarified. This will contribute to the general knowledge of non-covalent interactions between proteins.