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; 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展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。在分子水平上识别用于干预的平均值的工具至关重要。现代的核磁共振（NMR）光谱仍然是蛋白质，核酸及其复合物的结构和动力学表征的中心技术。然而，通过基因组序列分析已知的蛋白质的很大一部分无法访问标准溶液NMR方法。这通常是因为它们太大，因此滚动太慢，无法获得最佳的NMR性能。此外，针对通过X射线晶体学或标准NMR光谱求解结构的高贯穿策略的初始测试表明，绝大多数蛋白质只会无法获得适当的样品。显然，将需要采取辅助方法来充分实施基于知识的人类健康和疾病基本问题的方法。该建议旨在继续开发一种新型方法，用于使用基于NMR的方法。主要思想是简单地安排大型蛋白质分子作为小得多的蛋白质滚动。这是通过将蛋白质封装在反向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，可以使蛋白质组件的大小为200 kDa，以便在足够短的相关时间下翻滚，以允许使用现有的三重共振技术的全部电池，即使没有侵犯也没有效益。此外，倾向于聚集甚至形成不溶性沉淀物的蛋白质已成功封装，并且相对不稳定且因此在体外不完全折叠的蛋白质已被迫在反向胶束的狭窄空间中折叠。尽管有这些成功的应用，但该方法通常尚未被NMR社区采用。这样做的原因很明确：无法在商业上获得常规和安全准备和操纵高度加压和易燃样品所必需的设备。第一阶段的进展已经看到该方法在专门的实验室环境中已很大程度上实现，迄今为止获得的结果可欣赏该方法潜力。该提案旨在完善和扩展I阶段期间开发的原型设备和方法，以允许采用NMR光谱法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。我们认为，这里要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展使用详细的原子级结构，以详细了解生命和疾病的分子基础。在分子水平上识别用于干预的平均值的工具至关重要。该提案旨在继续开发一种新颖的方法来通过核磁共振结构确定。如果成功的话，这项技术可以作为制药理性设计的强大平台，用于治疗一系列人类疾病。