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: 6320909
• 负责人: STANLEY J OPELLA
• 金额: $ 8.46万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6320909
• 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， 一种高度专业化和严格管制的联系， 神经细胞和另一个可兴奋细胞。 神经递质门控 通道是介导 这对细胞之间的沟通;然而，很少有 通过X射线晶体学或溶液学 因为它们是膜蛋白。 我们已经开始 研究功能性25个残基肽的结构细节 对应于膜的离子通道的成孔元件， 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，电鳐乙酰胆碱受体的模型具有 一个由五个组成的小孔螺旋，每个蛋白质一个 由M2形成的亚基。 已经表明，一个五聚体束， M2螺旋足以显示M2的几种功能特性。 通过重组纯化的胆碱能受体， 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 在细菌中作为融合蛋白的肽， 用于胶束和固态溶液NMR研究的样品 多维溶液NMR光谱可用于确定 基于多个短程距离的三维结构 测量. 相反，在固态NMR光谱中， 定向样品，它是定向的测定， 单个肽平面，用于表征 结构. 这两种方法提供了到 生物聚合物的结构，这是重要的，以验证 结果的新的固态NMR方法对这些系统。 直接 胶束样品溶液NMR研究结果的比较 和固态核磁共振研究的乙酰胆碱M2双层样品 肽产生几乎相同的结构。 所有的核磁共振数据 可以用肽的模型来概括，其中单体M2 肽在双层中自组装以发挥与它们在膜中一样的功能。 完整的蛋白质 下一步是确定 膜结构域的显著更大的区段， 神经递质门控通道，以找到更大的作用， 多肽环境对肽的结构的影响。 我们有 最近获得的初步光谱从一个样品的设计 钾通道蛋白，120个残基。