Apparatus for NMR spectroscopy of encapsulated proteins

描述（由申请人提供）：生物医学研究继续扩大使用详细的原子尺度结构，以详细了解生命和疾病的分子基础。在分子一级确定干预手段的工具至关重要。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，通过基因组序列分析已知的蛋白质的显著部分是标准溶液NMR方法无法获得的。这通常是因为它们太大，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，将需要采取辅助办法，以充分执行以知识为基础的办法，解决人类健康和疾病方面的根本问题。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，以允许应用现有三重共振技术的全部电池，即使没有氘代的好处。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。在第一阶段的进展已经看到的方法主要是在一个专门的实验室环境中实施，迄今为止获得的结果提供了一个诱人的一瞥的方法的潜力。该提案旨在完善和扩展在第一阶段开发的原型设备和方法，以允许采用NMR光谱学的学术和非学术结构生物学家使用该方法，从而获得不适合标准NMR方法或晶体学的蛋白质。我们相信，在这里充分开发的方法可能会在基于结构的生物医学研究的各种重要领域提供突破性技术。生物医学研究继续扩大使用详细的原子尺度结构，以详细了解生命和疾病的分子基础。在分子一级确定干预手段的工具至关重要。该提案旨在继续开发一种通过核磁共振确定结构的新方法。如果成功，这项技术可以作为一个强大的平台，用于合理设计治疗一系列人类疾病的药物。

DESCRIPTION (provided by applicant): Biomedical research continues to expand the use of detailed atomic-scale structure in developing a detailed understanding of the molecular basis for life and for disease. Tools for the identification of means for intervention at the molecular level are of paramount importance. 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 standard solution NMR methods. This is often 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 to fully implement a knowledge-based approach to fundamental problems in human health and disease. 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 can, 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. 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. Progress during Phase I has seen the approach be largely implemented in a specialized laboratory setting and the results obtained thus far provide a tantalizing glimpse of the method's potential. This proposal seeks to refine and extend prototype apparatus and methods developed during Phase I 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. We believe that the method to be fully developed here may provide a break-out technology in a variety of important arenas in structure-based biomedical research. Biomedical research continues to expand the use of detailed atomic-scale structure in developing a detailed understanding of the molecular basis for life and for disease. Tools for the identification of means for intervention at the molecular level are of paramount importance. This proposal seeks to continue the development of a novel approach to structure determination by nuclear magnetic resonance. If successful, this technology could serve as a powerful platform for the rational design of pharmaceuticals for the treatment of an array of human diseases.