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: 6465910
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-06-19 至 2002-05-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6465910
• 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.

固态核磁共振正在开发作为一种额外的方法 确定膜蛋白复合物的结构。 这 固体核磁共振实验的实施有几个地方 对系统的要求。 蛋白质必须统一为 15N/2H/13C 在细菌表达系统中进行标记。 蛋白质必须合作 提供单轴方向。 这留下了负担 分辨率和光谱分配。 系统的方法 用于对统一标记的序列进行共振分配 必须开发出稳健且独立于次级的蛋白质 结构元素。 一旦结构计算可以进行 可以测量和分配完整的共振频率集。 强和弱同核偶极耦合都提供了 识别空间接近的核对的机制， 它提供了一种进行顺序共振分配的方法 特别是在规则二级结构区域，如α 膜蛋白中的螺旋。 共振现已完全解决 均匀 15N 标记的多维固态 NMR 谱 蛋白质。 模型中附近 1H 原子核之间存在丰富的自旋交换 两个模型中 15N 位点之间的肽和稀释自旋交换 系统已经实现。 最近，三维稀旋 交换实验已被用来分配大量的 均匀 15N 标记的 M2 通道肽中的共振 双层。 13C/15N统一标记样品的制备 使我们能够为这些开发三重共振分配策略 蛋白质，以及充分访问所有主链和 侧链站点。 已开发出三重共振脉冲序列 影响磁化强度沿着肽主链的转移 从 15N 到 13C。 该实验关联 13C 化学位移 具有 15N-1H 偶极耦合和 15N/13C 相关性的各向异性。 经过 控制13C和15N相关的时序，序列 可以选择磁化强度从酰胺氮转移到 直接键合相邻的碳或连续残基中的碳。 该脉冲序列已在双 15N/13C 标记的 乙酰化甘氨酸晶体。 获得这三者的潜力 定向脂质中 15N/13C 标记蛋白的维度相关性 双层有望实现位点的完全顺序分配 在肽骨架中。