Apparatus for NMR spectroscopy of encapsulated proteins

描述(申请人提供)：现代核磁共振(核磁共振)光谱学仍然是表征蛋白质、核酸及其复合体的结构和动力学的中心技术。然而，通过对基因组序列的分析已知的很大一部分蛋白质是溶液核磁共振方法无法获得的。这主要是因为它们太大，因此翻滚太慢，无法获得最佳的核磁共振性能。此外，通过X射线结晶学或标准核磁共振光谱学对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合适的样品。显然，将需要采取辅助措施。这项提议寻求继续开发一种新的方法来使用基于核磁共振的方法。主要的想法是简单地安排大的蛋白质分子作为小得多的蛋白质翻滚。这是通过将蛋白质包裹在反胶束系统中并将整个组装件溶解在低粘度流体(如液体乙烷)中实现的。原则上，可以使长达200 kDa的蛋白质组件以足够短的关联时间翻滚，从而使现有的三重共振技术得以充分应用，即使没有氚的好处。这种方法已经用可溶的和表现相当良好的蛋白质进行了演示。此外，易于聚集甚至形成不可溶沉淀物的蛋白质已经被成功包裹，而相对不稳定的蛋白质在体外不完全折叠，被迫在反胶束的受限空间中折叠。尽管有这些成功的应用，但该方法并未被核磁共振社区普遍采用。原因很清楚：对高压和易燃样品进行常规和安全的制备和操作所需的设备在商业上是不存在的。这项建议旨在开发必要的仪器，以允许学术和非学术结构生物学家使用核磁共振光谱学方法，从而获得不适用于标准核磁共振方法或结晶学的蛋白质。

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.