TRIPLE RESONANCE SOLID STATE NMR EXPERIMENTS TO ASSIGN PEPTIDE BACKBONE SITES

用于指定肽骨架位点的三重共振固态核磁共振实验

• 批准号: 6592518
• 负责人: STANLEY J OPELLA
• 金额: $ 17.24万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-31 至 2003-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6592518
• 关键词: biological products; biomedical resource; nervous system; nuclear magnetic resonance spectroscopy; proteins; structural biology; technology /technique

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.

神经系统的主要功能单位是突触， 人与人之间高度专业化且严格监管的联系 神经细胞和另一个兴奋细胞。 神经递质门控 通道是介导的关键分子成分 细胞对之间的通讯；然而，几乎没有 从 X 射线晶体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 研究功能性 25 残基肽的结构细节 对应于离子通道的成孔元素 乙酰胆碱受体和NMDA受体。 的序列 第二个跨膜片段 M2 在所有跨膜片段中高度保守 神经递质门控通道超家族的成员。 值得注意的是，鱼雷乙酰胆碱受体模型具有 由一束五个 β 螺旋形成的孔，每个蛋白质都有一个螺旋 由M2形成的亚基。 研究表明，五聚体束 M2 螺旋足以显示 通过重构纯化的胆碱能受体进行分析 平面脂质双层中的肽，通过单通道测量 电压钳条件下的记录。 我们已经表达了这些 肽作为细菌中的融合蛋白并获得统一标记 用于胶束和固态溶液 NMR 研究的样品 多维溶液核磁共振波谱可用于确定 基于许多短程距离的三维结构 测量。 相反，在固态核磁共振波谱中 定向样本，它是方向的确定 用于表征的单个肽平面 结构。 这两种方法提供了独立的路径 生物聚合物的结构，这对于验证 新固态核磁共振方法在这些系统上的结果。 直接一个 胶束样品溶液核磁共振研究结果的比较 乙酰胆碱 M2 双层样品的固态核磁共振研究 肽产生几乎相同的结构。 所有 NMR 数据均可 可以用肽模型来概括，其中单体 M2 肽在双层中自组装，其功能与它们在双层中的功能一样 完整的蛋白质。 下一步是确定结构 实质上更大的膜域片段 神经递质门控通道寻找作用较大的 多肽环境对肽结构的影响。 我们有 最近从设计的样品中获得了初步光谱 具有 120 个残基的钾通道蛋白。