Apparatus for NMR spectroscopy of encapsulated proteins

封装蛋白质的核磁共振波谱仪

• 批准号: 7937172
• 负责人: Ronald William Peterson
• 金额: $ 13.69万
• 依托单位: DAEDALUS INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7937172
• 关键词: Address; Adopted; Applications Grants; Area; Biomedical Research; Characteristics; Communities; Complex; Computer Assisted; Computers; Confined Spaces; Crystallography; Custom; Development; Devices; Disease; Elements; Encapsulated; Enzymes; Ethane; Evaluation; Feedback; Fluorescence Spectroscopy; Genomics; Government; Health; Human; In Vitro; Intervention; Knowledge; Laboratories; Libraries; Life; Ligands; Liquid substance; Literature; Marketing; Membrane Proteins; Methodology; Methods; Micelles; Modification; Molecular; Monitor; Motion; NMR Spectroscopy; Nature; Noise; Nuclear Magnetic Resonance; Nucleic Acids; Performance; Persons; Pharmacologic Substance; Phase; Preparation; Protein Dynamics; Proteins; Reading; Research; Research Design; Resolution; Rest; Safety; Sampling; Signal Transduction; Solutions; Solvents; Specialist; Structural Biologist; Structure; Survey Methodology; System; Techniques; Technology; Testing; Time; Tube; Viscosity; Water; Work; X-Ray Crystallography; aqueous; base; cost; design; efficacy testing; evaporation; experience; fly; human disease; improved; innovation; insight; interest; knowledge base; member; nanoscale; novel; novel strategies; particle; pressure; protein aggregation; prototype; research study; surfactant; tool

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.

描述（由申请人提供）：生物医学研究不断扩大详细原子尺度结构的使用，以详细了解生命和疾病的分子基础。识别分子水平干预手段的工具至关重要。现代核磁共振 (NMR) 波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很大一部分是标准溶液核磁共振方法无法获得的。这通常是因为它们太大，因此翻滚太慢，无法达到最佳 NMR 性能。此外，通过 X 射线晶体学或标准 NMR 光谱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产生合适的样品。显然，需要采取辅助方法来全面实施基于知识的方法来解决人类健康和疾病的基本问题。该提案旨在继续开发一种使用基于 NMR 的方法的新方法。主要想法是简单地安排大蛋白质分子作为更小的蛋白质分子翻滚。这是通过将蛋白质封装在反胶束系统中并将整个组件溶解在低粘度流体（例如液体乙烷）中来实现的。原则上，可以使大至 200 kDa 的蛋白质组装体以足够短的相关时间进行翻滚，以允许应用现有三重共振技术的全部电池，即使没有氘化的好处。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封装，而相对不稳定并因此在体外折叠不完全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，但该方法尚未被 NMR 界普遍采用。其原因很明显：常规且安全地制备和操作所需的高压易燃样品所需的设备在市场上无法买到。第一阶段的进展表明该方法主要在专门的实验室环境中实施，迄今为止获得的结果让人们对该方法的潜力有了诱人的了解。该提案旨在完善和扩展第一阶段开发的原型设备和方法，以允许学术和非学术结构生物学家使用核磁共振波谱学来使用该方法，从而获得不适合标准核磁共振方法或晶体学的蛋白质。我们相信，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个重要领域提供突破性技术。生物医学研究不断扩大详细原子尺度结构的使用，以深入了解生命和疾病的分子基础。识别分子水平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，这项技术可以成为合理设计治疗一系列人类疾病的药物的强大平台。