IMPROVED TIME SHARED (TS) 3D-NOESY 13C,15N-HSQC FOR LARGE PROTEINS

针对大蛋白质改进的分时 (TS) 3D-NOESY 13C,15N-HSQC

• 批准号: 8361202
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 0.37万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361202
• 关键词: Amides; Biological Preservation; Carbon; Data; Elements; Funding; Grant; Knowledge; Label; NOESY; National Center for Research Resources; Parents; Physiologic pulse; Principal Investigator; Proteins; Reporting; Research; Research Infrastructure; Resolution; Resources; Sampling; Scheme; Signal Transduction; Source; Time; United States National Institutes of Health; Water; base; carbene; cost; design; improved; methyl group; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. It is common knowledge that approaches that saturate water magnetization will yield lower sensitivity for those resonances that exchange with water, namely amide groups in proteins. Thus, to achieve maximum sensitivity, experiments that involve amide groups, such as NOESY 15N-HSQC, have been designed to preserve water magnetization. We report here a modified approach for the simultaneous NOESY 13C,15N?HSQC experiment, based on the NOESY 15N-fast HSQC pulse sequence, that features a WATERGATE element for water suppression and preservation. The result is a simultaneous NOESY experiment that provides better water suppression, better spectral quality and sensitivity comparable to that achieved by the parent the 15N-NOESY spectrum acquired independently, but improved compared to a 13C- and to previously reported 13C,15N simultaneous approaches. The only drawback of our approach is that the water suppression scheme wipes out resonances near the water, most notably those from 13C? groups. Because of this, we optimize the 13C spectral window to achieve maximal signal and resolution from methylene and especially methyl groups. Given these features, our simultaneous experiment is ideally suited for collecting NOESY spectra in water with samples of medium to large size proteins that are U-2H, U-15N and methyl 13C,1H labeled.For U-15N and U-13C labeled protein samples, a second 3D experiment must be collected to get NOESY peaks from 13C? groups, and for this, we propose using a 13C-NOESY experiment based on the sensitivity-enhanced 13C-HSQC pulse sequence, with the carbon window optimized for 13C? groups. This approach yields good water suppression and good S/N for the 13C? groups, and the data can be acquired in less time than for a regular full aliphatic carbon NOESY experiment.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 众所周知，使水磁化饱和的方法会对与水交换的共振（即蛋白质中的酰胺基团）产生较低的灵敏度。因此，为了实现最大灵敏度，涉及酰胺基团（例如 NOESY 15N-HSQC）的实验被设计为保持水磁化。我们在此报告了一种基于 NOESY 15N 快速 HSQC 脉冲序列的同步 NOESY 13C,15N?HSQC 实验的修改方法，该方法具有用于水抑制和保存的 WATERGATE 元件。结果是同步NOESY实验提供了更好的水抑制、更好的光谱质量和灵敏度，与独立获得的15N-NOESY光谱的母体所实现的结果相当，但与13C-和之前报道的13C、15N同时方法相比有所改进。我们方法的唯一缺点是水抑制方案消除了水附近的共振，尤其是来自 13C？组。因此，我们优化了 13C 光谱窗口，以实现亚甲基（尤其是甲基）的最大信号和分辨率。鉴于这些特征，我们的同步实验非常适合收集水中的 NOESY 光谱，其中包含 U-2H、U-15N 和甲基 13C,1H 标记的中型至大尺寸蛋白质样品。对于 U-15N 和 U-13C 标记的蛋白质样品，必须收集第二个 3D 实验才能获得 13C 的 NOESY 峰？为此，我们建议使用基于灵敏度增强的 13C-HSQC 脉冲序列的 13C-NOESY 实验，其中碳窗口针对 13C 进行了优化？组。这种方法对 13C? 具有良好的水抑制和良好的信噪比。组，并且与常规全脂肪族碳 NOESY 实验相比，可以在更短的时间内获得数据。