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正在被开发作为一种额外的方法， 确定膜蛋白复合物的结构。 的 固态NMR实验的实施将几个 对系统的要求。 蛋白质必须均匀为15 N/2 H/13 C 在细菌表达系统中标记。 蛋白质必须配合 以提供单轴取向。 这就留下了 分辨率和光谱分配。 一种系统方法 用于对均匀标记的 蛋白质必须是强大的，独立于二级 结构要素 结构计算可以在 可以测量和分配一组完整的谐振频率。 强的和弱的均质偶极耦合都提供了 用于识别空间接近的核对的机制， 其提供了一种用于进行顺序谐振分配的方法 特别是在阿尔法等规则二级结构区域 膜蛋白中的螺旋 共振现在完全解决了 在均匀15 N标记的多维固态NMR谱中， proteins. 模型中邻近~ 1H核间的丰富自旋交换 肽和稀释自旋交换之间的15 N网站在这两个模型 系统已经实现。 最近，三维稀自旋 交换实验已经被用来分配大量的 共振在一个统一的15 N标记的M2通道肽在定向 双层。 13 C/15 N均匀标记样品的制备 使我们能够开发三重共振分配策略， 蛋白质，以及给予充分的访问所有的骨干， 侧链网站 研制了三重共振脉冲序列 其影响磁化沿着肽骨架的转移 从15 N到13 C。 实验关联了~（13）C化学位移 各向异性与15 N-1H偶极耦合和15 N/13 C相关。 通过 控制13 C和15 N的相关的定时， 可以选择磁化从酰胺氮转移到 直接键合的碳邻居或连续残基中的碳。 该脉冲序列已在双15 N/13 C标记的 乙酰化甘氨酸的晶体。 收购这三家公司的潜力 15 N/13 C标记蛋白质在定向脂质中的空间相关性 双层承诺对位点进行完全顺序分配 在肽骨架中。