COMPLETE RESOLUTION OF SPECTRAL PARAMETERS OF COAT PROTEIN OF FD PHAGE

FD噬菌体外壳蛋白光谱参数的完整解析

• 批准号: 6592516
• 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/6592516
• 关键词: biological products; biomedical equipment development; biomedical resource; building /facility design /renovation; nuclear magnetic resonance spectroscopy; proteins; structural biology; technology /technique

Solid state NMR is being developed as an additional method for determining structures of membrane protein complexes. The implementation of the solid state NMR experiments places several requirements on the system. The protein must be uniformly 15N/2H/13C labeled in bacterial expression systems. The protein must cooperate to provide a uniaxis of orientation. This leaves the burden of resolution and assignment to the spectroscopy. A systematic method for making sequential resonance assignments of the uniformly labeled proteins must be developed that is robust and independent of secondary structure elements. Structure calculations can follow once the complete set of resonance frequencies can be measured and assigned. Both the strong and weak homonuclear dipolar couplings provide a mechanism for identifying pairs of nuclei in close spatial proximity, which provides a method for making sequential resonance assignments especially in regions of regular secondary structure like the alpha helices in membrane proteins. The resonances are now fully resolved in multidimensional solid-state NMR spectra of uniformly 15N labeled proteins. Abundant spin-exchange among nearby 1H nuclei in model peptides and dilute spin-exchange among 15N sites in both model systems has been achieved. Recently, three-dimensional dilute spin exchange experiments have been used to assign a significant number of resonances in a uniformly 15N labeled M2 channel peptide in oriented bilayers. The preparation of uniformly 13C/15N labeled samples enables us to develop triple-resonance assignment strategies for these proteins, as well as giving full access to all of the backbone and sidechain sites. A triple resonance pulse sequence has been developed that effects the transfer of magnetization along the peptide backbone from 15N to 13C. The experiment correlates 13C chemical shift anisotropy with 15N-1H dipolar coupling and 15N/13C correlation. By controlling the timing of the correlation of 13C and 15N, the sequence can select the transfer of magnetization from amide nitrogens to directly bonded carbon neighbors or to carbons in sequential residues. This pulse sequence has been demonstrated on a double 15N/13C labeled crystal of acetylated glycine. The potential of acquiring the three dimensional correlation on 15N/13C labeled proteins in oriented lipid bilayers holds the promise of full sequential assignment of the sites in the peptide backbone.

固态NMR被开发为 确定膜蛋白复合物的结构。 这 固态NMR实验的实施放置了几个 系统要求。 蛋白质必须均匀地为15N/2H/13C 在细菌表达系统中标记。 蛋白质必须配合 提供方向的单素。 这留下了 分辨率和分配到光谱法。 一种系统的方法 用于进行均匀标记的顺序共振分配 必须开发蛋白质稳健且独立于次要的蛋白质 结构元素。 结构计算一旦 可以测量和分配完整的共振频率集。 强和弱的同核偶极耦合都提供了 识别核近距离近端的核对的机制， 这提供了一种使顺序共振分配的方法 特别是在常规二级结构（如alpha）的区域 膜蛋白中的螺旋。 共振现在已经完全解决 在均匀15N的多维固态NMR光谱中 蛋白质。 模型中附近1H核之间的大量自旋交换 两种模型中15n位点之间的肽和稀释自旋交换 系统已经实现。 最近，三维稀释自旋 交换实验已用于分配大量 在面向的15N标记的M2通道肽中共鸣 双层。 制备统一的13C/15N标记样品 使我们能够为这些制定三重谐和分配策略 蛋白质，并完全访问所有主链和 Sidechain站点。 已经开发了三重共振脉冲序列 这会影响磁化沿肽主链的转移 从15N到13C。 该实验将13C化学移位相关 各向异性具有15N-1H偶极耦合和15N/13C相关性。 经过 控制13C和15N的相关时间的时机，序列 可以选择从酰胺氮的转移到 直接粘合碳邻居或连续残基中的碳。 该脉冲序列已在标记的双15N/13C上证明 乙酰化甘氨酸的晶体。 获取三个的潜力 在定向脂质中标记为15N/13C标记蛋白的尺寸相关性 BiLayers拥有完全顺序分配站点的承诺 在肽主链中。