Efficient scouting instrumentation for the determination of reverse micelle encap

用于测定反胶束包封的高效侦察仪器

• 批准号: 8251081
• 负责人: Ronald William Peterson
• 金额: $ 15.7万
• 依托单位: DAEDALUS INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2013-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8251081
• 关键词: Address; Adoption; Algorithms; Arts; Biology; Biomedical Research; Buffers; Characteristics; Complex; Computer software; Conceptions; Consumption; Development; Devices; Disease; Encapsulated; Ethane; Fluorescence; Gases; Goals; Grant; Health; Human; Human Resources; Integral Membrane Protein; Intervention; Knowledge; Life; Liquid substance; Maintenance; Marketing; Membrane Proteins; Methods; Micelles; Modeling; Molecular; NMR Spectroscopy; Nuclear Magnetic Resonance; Nucleic Acids; Performance; Pharmacologic Substance; Phase; Physics; Preparation; Property; Protein Dynamics; Proteins; Reagent; Relaxation; Reproducibility; Research Personnel; Role; Sampling; Solutions; Solvents; Spectrum Analysis; Structural Biologist; Structural Models; Structure; Surveys; System; Techniques; Technology; Testing; Time; Tube; Viscosity; Water; aqueous; base; design; experience; human disease; innovation; instrument; instrumentation; interest; knowledge base; macromolecule; novel strategies; operation; particle; pressure; protein aggregation; protein structure; prototype; research study; surfactant; tool

DESCRIPTION (provided by applicant): Detailed knowledge of the structures is a vital component of our understanding of the molecular basis of life. From a practical point of view, the atomic-scale structure of the protein can potentially greatly facilitate the design of effective pharmaceuticals. 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. Ongoing advances in experimental techniques continues to push the size limits accessible by NMR and clever sample preparation methods has opened the door for the study of otherwise recalcitrant proteins such as integral membrane proteins. However, progress continues to be largely incremental, and it is clear that a radical shift in approach will likely be necessary to fully implement a knowledge-based approach to fundamental problems in human health and disease. The reverse micelle technology was originally devised to address the slow tumbling problem presented by large soluble proteins to solution NMR methods. From that initial conception it has been shown to be useful for studying a wide array of traditionally intractable proteins such as integral and anchored membrane proteins, aggregation prone proteins, and marginally stable proteins. The basic idea is to take the protein of interest and encapsulate it within the protective aqueous core of a reverse micelle particle and dissolve the entire assembly in a low viscosity fluid such as liquid ethane. In the low viscosity fluid, the reverse micelle particle tumbles faster than the protein dissolved in bulk water. This provides a significant improvement in the NMR relaxation properties governing the efficiency of the modern "triple resonance" experiments. By using this method protein constructs as large as 150 kDa can be studied without benefit of deuteration or the TROSY effect and thus more comprehensive structural and dynamical information can be obtained. To maximize this effect reverse micelle samples must be prepared in liquid ethane, which requires the preparation of samples under significant pressure and maintenance of the pressurized sample within an NMR sample tube. Daedalus Innovations has overcome the initial barrier to the implementation of this approach by developing hardware solutions for researchers to produce such samples in a safe and reproducible manner without the need for any previous experience with high-pressure applications. In this proposal we seek to develop an instrument that overcomes the current critical limitation to regular use, which is the seeming daunting task of finding encapsulation conditions for new proteins. Currently, the conditions for encapsulation (surfactant mixture; sample buffer; etc.) is optimized manually often in a material intensive manner. This is unacceptable for most non-academic applications and is certainly non-ideal in general. A more streamlined and less personnel and material intensive approach is needed. We propose to develop an instrument that will allow relatively automated examination of an array of encapsulation conditions and will identify optimum combinations using a variety of spectroscopic probes, and do so with minimal consumption or reagents. The instrument will build upon Daedalus Innovations' proven technology. The goal is to provide researchers having no intimate knowledge of the art of protein encapsulation to make use of this powerful technology. The proposed instrument will complete the suite of instruments offered by Daedalus Innovations that is designed to provide a turn-key solution for structural studies of macromolecules using the reverse micelle encapsulation strategy. PUBLIC HEALTH RELEVANCE: 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）：结构的详细知识是我们理解生命分子基础的重要组成部分。从实用的角度来看，蛋白质的原子尺度结构可以极大地促进有效药物的设计。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。实验技术的不断进步不断突破核磁共振的尺寸限制，巧妙的样品制备方法为研究其他顽固蛋白质（如整合膜蛋白）打开了大门。然而，进展仍然在很大程度上是渐进的，而且很明显，为了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，可能需要彻底改变方法。反胶束技术最初是为了解决大型可溶性蛋白质在溶液 NMR 方法中出现的缓慢翻滚问题而设计的。从最初的构想开始，它已被证明可用于研究各种传统上难以处理的蛋白质，例如整合膜蛋白和锚定膜蛋白、易于聚集的蛋白质和边缘稳定的蛋白质。基本思想是获取感兴趣的蛋白质并将其封装在反胶束颗粒的保护性水性核心内，并将整个组件溶解在低粘度流体（例如液体乙烷）中。在低粘度流体中，反胶束颗粒比溶解在散装水中的蛋白质翻滚得更快。这显着改善了控制现代“三重共振”实验效率的核磁共振弛豫特性。通过使用这种方法，可以研究大至 150 kDa 的蛋白质构建体，而无需借助氘化或 TROSY 效应，从而可以获得更全面的结构和动力学信息。为了最大限度地提高这种效果，必须在液体乙烷中制备反胶束样品，这需要在很大的压力下制备样品，并在 NMR 样品管内保持加压样品。 Daedalus Innovations 克服了实施这种方法的最初障碍，为研究人员开发了硬件解决方案，以安全且可重复的方式生产此类样品，而无需任何高压应用经验。在本提案中，我们寻求开发一种仪器来克服当前常规使用的关键限制，这是寻找新蛋白质的封装条件看似艰巨的任务。目前，封装条件（表面活性剂混合物；样品缓冲液等）通常以材料密集型方式手动优化。这对于大多数非学术应用来说是不可接受的，而且通常来说也是不理想的。需要一种更精简、更少人员和材料密集的方法。我们建议开发一种仪器，可以相对自动化地检查一系列封装条件，并使用各种光谱探针识别最佳组合，并且以最少的消耗或试剂来实现这一点。该仪器将建立在 Daedalus Innovations 成熟的技术之上。目标是让对蛋白质封装技术没有深入了解的研究人员能够利用这项强大的技术。拟议的仪器将完善 Daedalus Innovations 提供的仪器套件，该套件旨在为使用反胶束封装策略的大分子结构研究提供交钥匙解决方案。 公共卫生相关性：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。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。