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）：生物医学研究继续扩大详细的原子尺度结构的使用，以发展对生命和疾病的分子基础的详细理解。在分子水平上识别干预手段的工具是至关重要的。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无法通过标准溶液核磁共振方法获得的。这通常是因为它们太大，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，为了全面实施以知识为基础的方法来解决人类健康和疾病的基本问题，将需要辅助方法。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大小的蛋白质组件可以在足够短的相关时间内翻转，即使没有氘化的好处，也可以应用现有三重共振技术的完整电池。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。在第一阶段的进展中，该方法主要在专门的实验室环境中实施，到目前为止获得的结果为该方法的潜力提供了诱人的一瞥。本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。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的技术。生物医学研究继续扩大对详细的原子尺度结构的使用，以发展对生命和疾病的分子基础的详细了解。在分子水平上识别干预手段的工具是至关重要的。本提案旨在继续发展核磁共振结构测定的新方法。如果成功，这项技术可以作为一个强大的平台，为合理设计药物治疗一系列人类疾病。