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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 众所周知，饱和水磁化的方法对那些与水交换的共振，即蛋白质中的酰胺基团，会产生较低的灵敏度。因此，为了达到最大的灵敏度，涉及酰胺基团的实验，如NOESY 15N-HSQC，被设计成保持水的磁化强度。我们报告了一种用于同时进行NOESY 13C，15N？HSQC实验的改进方法，该方法基于NOESY 15N-FAST HSQC脉冲序列，其特点是具有用于抑制和保存水的水门元件。结果是一个同步的NOESY实验，提供了更好的水抑制，更好的光谱质量和灵敏度，可与母体获得的15N-NOESY谱相媲美，但与13C-和先前报道的13C，15N同时方法相比有所改善。我们方法的唯一缺点是水抑制方案消除了水附近的共振，最明显的是来自13C的共振？组。正因为如此，我们优化了13C光谱窗口，以获得来自亚甲基，特别是甲基的最大信号和分辨率。考虑到这些特点，我们的同步实验非常适合于收集水中的NOESY光谱，这些样品是U-2H，U-15N和甲基13C，1H标记的中到大尺寸蛋白质的样品。对于U-15N和U-13C标记的蛋白质样品，必须进行第二次3D实验，以从13C？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，其中碳窗针对13C？组。该方法具有良好的抑水性和良好的13C？与常规的全脂碳NOESY实验相比，可以在更短的时间内获得数据。

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.