NMR spectroscopy of proteins in low viscosity fluids

描述(申请人提供)：现代核磁共振(核磁共振) 光谱学仍然是表征生物多样性的核心技术。 蛋白质、核酸及其复合体的结构和动力学。 然而，已知的蛋白质中有相当一部分是通过 用溶液核磁共振方法无法对基因组序列进行分析。这 是因为它们太大了，或者是因为它们本身，或者因为它们需要 与大的脂类聚集在一起，因此翻滚太慢 以获得最佳的核磁共振性能。这项建议旨在继续发展 一种再现大分子蛋白质核磁共振弛豫特性的新方法 适应现代三元复合物流的全面高效应用 共振和相关的溶液核磁共振技术。基本方法是简单地 安排蛋白质分子以小得多的蛋白质形式翻滚。这是 通过将蛋白质包裹在反胶束系统中而实现的 将整个组件溶解在低粘度流体中。我们计算蛋白质 大到100 kDa的组件可以在足够短的情况下翻滚 相关时间允许现有三重谐振的满电池 即使没有重氢的好处，也要应用技术。最基本的 这种方法已经用一种名为泛素的小模型蛋白质进行了演示。使用 一组特性良好的蛋白质，我们将确定必要的条件 以充分包裹大的蛋白质。此方法也有可能 适用于膜蛋白，我们也将对此进行技术改造 目的：将其应用于几种膜蛋白。多项技术支持 将探索进展，包括使用低温探测器技术和 开发一种部分对齐的方法以允许访问残留物 用于结构约束的偶极联轴器。如果这一总体战略被证明 如果成功，它将提供一种使用高分辨率 溶液核磁共振技术用于表征大小达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.