TRIPLE RESONANCE SOLID STATE NMR EXPERIMENTS TO ASSIGN PEPTIDE BACKBONE SITES

神经系统的主要功能单位是突触， 高度专业化和严格监管的连接 神经细胞和另一个可兴奋的细胞。神经递质门控 通道是起中介作用的关键分子成分 这对细胞之间的通信；然而，几乎没有 从x射线结晶学或溶液中了解它们的结构 核磁共振波谱，因为它们是膜蛋白。我们已经开始 25个功能残基多肽的结构研究 对应于离子通道的成孔元件 乙酰胆碱受体和NMDA受体。序列中的 第二个跨膜片段M2在所有基因中高度保守 神经递质门控通道超家族的成员。 值得注意的是，鱼雷乙酰胆碱受体的模型具有 由五个螺旋组成的毛孔，每个蛋白质一个 由M2形成的亚基。已经证明，五聚体中的一束 M2螺旋足以显示的几个功能性质 重组纯化后的胆碱能受体分析 用单通道法测定平面脂双层中肽的含量 在电压钳位条件下的记录。我们已经表达了这些 多肽作为融合蛋白在细菌中并获得均一标记 胶束和固体中的溶液核磁共振研究样品 多维溶液核磁共振波谱可以用来确定 基于多近程距离的三维结构 测量。相比之下，在固态核磁共振波谱中 定向样本它是确定的取向 单个多肽平面用于表征 结构。这两种方法提供了通往 生物聚合物的结构，这对于验证 新的固体核磁共振方法在这些体系上的结果。一位直接的 胶束样品的溶液核磁共振研究结果比较 乙酰胆碱M2双层样品的固体核磁共振研究 多肽产生的结构几乎完全相同。所有的核磁共振数据都可以 用单体M2所在的多肽模型进行总结 多肽在双层中自组装，其功能与它们在细胞内的作用相似。 完整的蛋白质。下一步是确定 膜结构域的大得多的片段 神经递质门控通道的发现作用更大 多肽环境对多肽结构的影响。我们有 最近从一个设计的样品中获得了初步的光谱 钾通道蛋白，含120个残基。

The primary functional unit of the nervous system is the synapse, a highly specialized and tightly regulated connection between one nerve cell and another excitable cell. Neurotransmitter-gated channels are the pivotal molecular components that mediate communication between the pair of cells; however, little has been learned about their structures from x-ray crystallography or solution NMR spectroscopy because they are membrane proteins. We have begun to study the structural details of the functional 25 residue peptides corresponding to the pore forming elements of the ion-channels of the acetylcholine receptor and the NMDA receptor. The sequences of the second transmembrane segment, M2, are highly conserved among all members of the superfamily of neurotransmitter-gated channels. Significantly, the model of the Torpedo acetylcholine receptor has a pore formed by a bundle of five ?-helices, one from each protein subunit formed from M2. It has been shown that a pentameric bundle of M2 helices is sufficient to display several functional properties of the cholinergic receptor as analyzed by reconstitution of purified peptides in planar lipid bilayers, measured by single-channel recordings under voltage-clamp conditions. We have expressed these peptides as fusion proteins in bacteria and obtained uniformly labeled samples for solution NMR studies in micelles and solid-state multidimensional solution NMR spectroscopy can be used to determine three-dimensional structures based on many short-range distance measurements. In contrast, in solid state NMR spectroscopy of oriented samples it is the determinations of the orientations of individual peptide planes that are used to characterize the structures. The two approaches provide independent paths to the structure of biopolymers, which is important in order to verify the results of the new solid-state NMR method on these systems. A direct comparison of the results from solution NMR studies of micelle samples and solid-state NMR studies of bilayer samples of the acetylcholine M2 peptide yield virtually identical structures. All of the NMR data can be summarized with the model of the peptide where the monomeric M2 peptides self-assemble in bilayers to function much as they do in the intact proteins. The next step is to determine the structures of substantially larger segments of the membrane domains of the neurotransmitter-gated channels to find the role of the larger polypeptide environment on the structure of the peptides. We have recently obtained preliminary spectra from a sample of a designed potassium channel protein with 120 residues.