DEVELOPMENT OF NMR EXPERIMENTS FOR THE ANALYSIS OF LARGER 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。 该项目的目标是测试、优化和开发新的核磁共振脉冲程序和方法，用于在配备低温探针的高场仪器上研究大型蛋白质和蛋白质-蛋白质复合体。处理大蛋白质的挑战来自系统的不利松弛特性造成的信号损失，以及制备可长时间稳定的浓缩蛋白质样品的困难。缓解这种弛豫问题的最常见的方法是制备完全氢化的蛋白质，也就是说，所有不可交换的氢原子都被氢原子取代。虽然这种方法确实允许用核磁共振研究更大的蛋白质，但用重氢取代所有不可交换的质子也限制了可以从蛋白质中提取的信息量。因此，最近的蛋白质样品是用替代的氚标记策略制备的，其中脂肪侧链中的一些质子被保留下来。特别是，一个非常有用的方法是保存亮氨酸、异亮氨酸或Valine侧链上的一个甲基质子，从而扩大可以进行的核磁共振实验的类型，从而增加从给定蛋白质样本获得的知识。在NMRFAM，我们正在与CESG合作，评估通过无细胞表达来制备氚代蛋白质的新方法。同时，我们还在不断地测试、优化和进一步发展用于研究重氢蛋白质样品的核磁共振实验。我们的目标是开发适合于研究中给定的蛋白质标记方案的实验，以及确保确实获得了从困难的蛋白质样本中提取的所有可能的信号。在配备了低温探头的高场仪器上，这不是一项微不足道的任务，我们经常会发现，相同的实验可能需要不同的参数才能在不同的光谱仪上以最佳方式工作。 最后，一种研究大系统的新方法是制备完全由高度区域和立体选择性的‘2’H，‘13’C，‘15’N标记氨基酸组成的蛋白质。这些新型标记蛋白质被命名为立体阵列同位素标记(SAIL)蛋白质，已被证明非常适合于核磁共振结构分析。在NMRFAM，我们一直致力于研究SAIL标记蛋白质的核磁共振实验的优化和发展，包括应用我们的快速数据收集方法HIFI。 对于蛋白质复合体的研究，我们正在设计新的方法，通过利用不对称标记方案，允许单个组分的光谱去卷积。然后，我们可以使用单个核磁共振样本来观察和获取关于络合物的每个组分的信息，从而节省资金、测量时间并确保所有信息都是在相同的条件下获得的。此外，使用相同的样本，我们还在开发交叉饱和实验，利用特定的标记方案来研究蛋白质-蛋白质界面。

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. The goal of this project is to test, optimize and develop new NMR pulse programs and methods for studying large proteins and protein-protein complexes on high-field instruments, equipped with cryogenic probes. The challenge of working with large proteins comes from the loss of signal due to the unfavorable relaxation properties of the system, as well as, the difficulty of preparing concentrated protein samples that are stable for extended periods of time. The most common approach taken to alleviate this relaxation problem is to prepare proteins that are fully deuterated, i.e. where all non-exchangeable hydrogens atoms are replaced with deuterium atoms. Although this method does allow for larger proteins to be studied by NMR, replacing all non-exchangeable protons with deuterium also limits the amount of information that can be extracted from a protein. Thus, more recently protein samples have been prepared using alternative deuterium labeling strategies, where some of the protons in the aliphatic side chains are preserved. In particular, a very useful approach is to preserve one of the methyl protons on Leucine, Isoleucine or Valine side chains, thus expanding the type of NMR experiments that can be run and consequently increasing the knowledge obtainable from a given protein sample. At NMRFAM, we are working in collaboration with CESG to assess new methods for preparing deuterated proteins by cell-free expression. At the same time, we are also continuously testing, optimizing and further developing NMR experiments that are used for studying deuterated protein samples. Our goal is to develop experiments that are suited for the given labeling scheme of the protein at study, as well as, to make sure that all possible signal that can be extracted from a difficult protein sample is indeed obtained. This is no trivial task on high-field instruments that are equipped with cryogenic probes, where we often find that the same experiment may require different parameters to work optimally on different spectrometers. Finally, a novel approach to study large systems is to prepare proteins that are fully composed of highly regio- and stereoselectively '2'H, '13'C, '15'N -labeled amino acids. These new type of labeled proteins, which have been named as stereo-array isotope-labeled (SAIL) proteins, have been shown to be exceptionally suitable for NMR structural analyses. At NMRFAM we have been working on the optimization and development of NMR experiments for the studying of SAIL labeled protein, including the application of our fast method for data collection HIFI. For the study of protein complexes, we are devising new methods that, by taking advantage of asymmetric labeling schemes, allow the deconvolution of the spectra for the individual components. We can then observe and obtain information about each component of the complex using a single NMR sample, saving money, measuring time and ensuring that all information is obtained under identical conditions. Furthermore, using the same sample we are also developing cross-saturation experiments that take advantage of the particular labeling scheme to study the protein-protein interface.