Apparatus for NMR spectroscopy of encapsulated proteins

描述（由申请人提供）：现代核磁共振（NMR）光谱仍然是表征蛋白质、核酸及其复合物的结构和动力学的核心技术。然而，通过分析基因组序列已知的蛋白质的显著部分是溶液NMR方法无法获得的。这主要是因为它们太大，因此对于最佳NMR性能来说翻滚太慢。此外，通过X射线晶体学或标准NMR光谱学解析结构的高通量策略的初步测试表明，绝大多数蛋白质根本无法得到合适的样品。显然，需要采取辅助措施。该提案旨在继续开发使用基于NMR的方法的新方法。主要的想法是简单地安排大蛋白质分子作为一个小得多的蛋白质翻滚。这是通过将蛋白质包封在反胶束系统中并将整个组装体溶解在低粘度流体如液体乙烷中来实现的。大到200 kDa的蛋白质组装体原则上可以在足够短的相关时间内翻滚，以允许应用现有的三重共振技术的全部电池，即使没有氘代的好处。该方法已被证明与可溶性和合理的良好行为的蛋白质。此外，倾向于聚集或甚至形成不溶性沉淀物的蛋白质已被成功地包封，并且相对不稳定并因此在体外不完全折叠的蛋白质已被迫在反胶束的有限空间中折叠。尽管有这些成功的应用，该方法尚未被NMR界普遍采用。其原因很清楚：常规和安全制备和操作高压和易燃样品所需的设备无法在市场上买到。该提案旨在开发必要的设备，允许学术和非学术结构生物学家采用NMR光谱学使用的方法，从而获得不适合标准NMR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.