Apparatus for NMR spectroscopy of encapsulated proteins

封装蛋白质的核磁共振波谱仪

• 批准号: 7463930
• 负责人: Ronald William Peterson
• 金额: $ 34.5万
• 依托单位: DAEDALUS INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7463930
• 关键词: Address; Adopted; Applications Grants; Area; Biomedical Research; Characteristics; Class; Communities; Complex; Computer Assisted; Computers; Condition; 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; Numbers; Performance; Persons; Pharmacologic Substance; Phase; Preparation; Protein Dynamics; Proteins; Purpose; Reading; Research; Research Design; Resolution; Rest; Safety; Sampling; Signal Transduction; Solutions; Solvents; Specialist; Standards of Weights and Measures; Structural Biologist; Structure; Survey Methodology; System; Techniques; Technology; Testing; Time; Tube; Viscosity; Water; Work; X-Ray Crystallography; aqueous; base; cost; design; desire; 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使用详细的原子级结构，以发展对生命和疾病的分子基础的详细了解。在分子水平上确定干预手段的工具至关重要。现代核磁共振波谱仍然是表征蛋白质、核酸及其络合物结构和动力学的核心技术。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标准的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，要全面实施以知识为基础的方法来解决人类健康和疾病的根本问题，将需要采取辅助方法。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，甚至不需要氚的好处。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。在第一阶段的进展中，该方法在很大程度上在专门的实验室环境中实施，到目前为止所获得的结果提供了对该方法潜力的诱人一瞥。这项提议寻求改进和扩展在第一阶段开发的原型设备和方法，以允许使用核磁共振光谱的学术和非学术结构生物学家使用该方法，从而获得不适用于标准核磁共振方法或结晶学的蛋白质。我们相信，将在这里充分开发的方法可能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级结构，以发展对生命和疾病的分子基础的详细了解。在分子水平上确定干预手段的工具至关重要。这项提议寻求继续开发一种新的核磁共振确定结构的方法。如果成功，这项技术可能成为合理设计治疗一系列人类疾病的药物的强大平台。