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

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。次级项目的主要支助 子项目的主要研究者可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 表示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，饱和水磁化的方法将对与水交换的那些共振（即蛋白质中的酰胺基团）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 15 N-HSQC，已经被设计为保持水的磁化。我们在这里报告的同时NOESY 13 C，15 N？HSQC实验，基于NOESY 15 N快速HSQC脉冲序列，具有用于水抑制和保存的WATERGATE元件。其结果是一个同时NOESY实验，提供更好的水抑制，更好的光谱质量和灵敏度可比的父母，15 N-NOESY光谱独立获得，但改善相比，13 C-和以前报道的13 C，15 N同时的方法。我们的方法的唯一缺点是，水抑制计划消灭了附近的水，最显着的是那些从13 C？组正因为如此，我们优化了13 C光谱窗口，以实现最大的信号和分辨率从亚甲基，特别是甲基。鉴于这些特点，我们的同时实验非常适合于收集水中中到大尺寸的蛋白质样品的NOESY谱，这些样品是U-2 H，U-15 N和甲基13 C，1H标记的。组，为此，我们建议使用13 C-NOESY实验的基础上的灵敏度增强13 C-HSQC脉冲序列，与碳窗口优化13 C？组这种方法产生良好的水抑制和良好的S/N为13 C？组，并且可以在比常规的全脂肪族碳NOESY实验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.