Apparatus for NMR spectroscopy of encapsulated proteins

描述（由申请人提供）：现代核磁共振（NMR）波谱学仍然是表征蛋白质、核酸及其复合物的结构和动力学的核心技术。然而，通过基因组序列分析已知的蛋白质的很大一部分是无法通过溶液核磁共振方法获得的。这主要是因为它们太大，因此翻滚太慢，无法获得最佳的NMR性能。此外，通过x射线晶体学或标准核磁共振波谱法进行的高通量解决结构策略的初步测试表明，绝大多数蛋白质根本无法产生适当的样品。显然，辅助方法将是必需的。本提案旨在继续发展一种使用核磁共振为基础的方法的新方法。主要的想法是简单地安排大的蛋白质分子翻滚成一个小得多的蛋白质。这是通过将蛋白质封装在一个反向胶束系统中，并将整个组件溶解在低粘度流体（如液态乙烷）中来实现的。原则上，200 kDa大小的蛋白质组装可以在足够短的相关时间内翻滚，即使没有氘化的好处，也可以应用现有三重共振技术的全部电池。这种方法已经在可溶性的、性能相当良好的蛋白质上得到了证明。此外，倾向于聚集甚至形成不溶性沉淀物的蛋白质已被成功包裹，而相对不稳定因此在体外折叠不完全的蛋白质已被迫在反向胶束的有限空间中折叠。尽管这些成功的应用，该方法还没有被核磁共振界普遍采用。这样做的原因很清楚：用于常规和安全制备和操作所需的高压和易燃样品的设备在商业上是不可用的。本提案旨在开发必要的设备，允许使用核磁共振波谱的学术和非学术结构生物学家使用该方法，从而获得无法适用于标准核磁共振方法或晶体学的蛋白质。

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 primarily because they are too large and therefore tumble too slowly for optimal NMR performance. In addition, initial tests of a high through-put strategy for solving structures by X-ray crystallography or by standard NMR spectroscopy indicate that the vast majority of proteins will simply fail to result in appropriate samples. Clearly, ancillary approaches are going to be required. This proposal seeks to continue the development of a novel approach to using an NMR-based method. The primary idea is to simply arrange for the large 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 such as liquid ethane. Protein assemblies as large as 200 kDa could, in principle, 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 approach has been demonstrated with soluble and reasonably well-behaved proteins. Furthermore, proteins that tend to aggregate or even form insoluble precipitates have been successfully encapsulated and proteins that are relatively unstable and therefore incompletely folded in vitro have been forced to fold in the confined space of the reverse micelle. Despite these successful applications, the method has not been generally adopted by the NMR community. The reasons for this are clear: The apparatus necessary for the routine and safe preparation and manipulation of the highly pressurized and flammable samples that are necessary is not commercially available. This proposal seeks to develop the apparatus necessary to allow academic and non-academic structural biologists employing NMR spectroscopy to use the approach and thereby gain access to proteins that are not amenable to standard NMR methods or to crystallography.