NMR spectroscopy of proteins in low viscosity fluids

描述（申请人提供）：现代核磁共振（NMR） 光谱学仍然是一个核心技术的特点， 蛋白质、核酸及其复合物的结构和动力学。 尽管如此，通过蛋白质组学已知的蛋白质中有很大一部分， 基因组序列的分析是溶液NMR方法无法实现的。这 是因为它们本身太大，或者因为它们需要 与脂质的大集合体结合，因此翻滚得太慢， 实现最佳NMR性能。这项建议旨在继续发展 一种新的方法来渲染大蛋白质的NMR弛豫特性 适用于现代三重的全面和有效的应用 共振和相关的溶液NMR技术。基本的方法是 使蛋白质分子以更小的蛋白质形式翻滚。这是 通过将蛋白质包封在反胶束系统中来实现， 将整个组件溶解在低粘度流体中。我们计算蛋白质 在足够短的时间内，可以使高达100 kDa的组装体翻滚 相关时间，以允许现有的三重共振的完整电池 即使没有氘化的好处，也要应用技术。基本 已经用小的模型蛋白泛素证明了这种方法。使用 一组特征良好的蛋白质，我们将确定必要的条件 以充分包裹大蛋白质。这种方法也可能 适用于膜蛋白，我们也将调整技术， 目的并将其应用于几种膜蛋白。一些技术 将探索一些进展，包括使用低温探针技术， 制定一种局部调整方法， 用于结构约束的偶极耦合。如果这一总体战略证明， 如果成功，它将为使用高分辨率 溶液NMR技术表征大小高达100 kDa的蛋白质。

Description (provided by applicant): Modern nuclear magnetic resonance (NMR) spectroscopy continues to be a central technique in the characterization of the structure and dynamics of proteins, nucleic acids and their complexes. Nevertheless, a significant fraction of the proteins that are known through the analysis of the genomic sequence are inaccessible to solution NMR methods. This is because they are too large, either by themselves or because they require association with large assemblies of lipids, and therefore tumble too slowly for optimal NMR performance. This proposal seeks to continue the development of a novel approach to rendering the NMR relaxation properties of large proteins amenable to the comprehensive and efficient application of modern triple resonance and related solution NMR techniques. The basic approach is to simply arrange for the protein molecule to tumble as a much smaller protein. This is achieved by encapsulating the protein in a reverse micelle system and dissolving the entire assembly in a low viscosity fluid. We calculate protein assemblies as large as 100 kDa could be made to tumble with sufficiently short correlation times to allow the full battery of existing triple resonance techniques to be applied, even without benefit of deuteration. The basic approach has been demonstrated with a small model protein, ubiquitin. Using a set of well-characterized proteins, we will determine the conditions necessary to adequately encapsulate large proteins. This method is also potentially applicable to membrane proteins and we will also adapt the technology for this purpose and apply it to several membrane proteins. A number of technical advances will be explored and include the use of cryogenic probe technology and development of a method of partial alignment to allow access to residual dipolar couplings for structural restraints. Should this general strategy prove successful, it would provide a powerful approach to using high-resolution solution NMR techniques to characterize proteins up to 100 kDa in size.