Efficient scouting instrumentation for the determination of reverse micelle encap

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，蛋白质的原子级结构可能极大地促进有效药物的设计。现代核磁共振（NMR）光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继续推动NMR可达到的尺寸限制，聪明的样品制备方法为研究其他不稳定的蛋白质（如整合膜蛋白）打开了大门。然而，进展在很大程度上仍然是渐进的，显然，要充分实施以知识为基础的办法来解决人类健康和疾病的根本问题，可能需要彻底改变方法。反胶束技术最初是为了解决大的可溶性蛋白质溶液NMR方法的缓慢翻滚问题而设计的。从最初的概念，它已被证明是有用的，用于研究广泛的传统上棘手的蛋白质，如整合和锚定的膜蛋白，聚集倾向的蛋白质，和边缘稳定的蛋白质。其基本思想是将目标蛋白质封装在反胶束颗粒的保护性水性核心内，并将整个组装体溶解在低粘度流体如液体乙烷中。在低粘度流体中，反胶束颗粒翻滚得比溶解在本体水中的蛋白质快。这提供了一个显着的改善NMR弛豫性能的现代“三重共振”实验的效率。通过使用这种方法，可以研究大至150 kDa的蛋白质构建体，而无需受益于氘代或TROSY效应，因此可以获得更全面的结构和动力学信息。为了最大化该效果，反胶束样品必须在液体乙烷中制备，这需要在显著压力下制备样品并将加压样品保持在NMR样品管内。Daedalus Innovations已经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，以安全和可重复的方式生产此类样品，而无需任何高压应用经验。在这项提案中，我们试图开发一种仪器，克服目前的关键限制，定期使用，这是一个看似艰巨的任务，找到新的蛋白质的封装条件。目前，包封条件（表面活性剂混合物;样品缓冲液;等）通常以材料密集的方式手动优化。这对于大多数非学术应用程序来说是不可接受的，并且通常肯定是不理想的。需要采取一种更加精简、人力和物力密集程度较低的办法。我们建议开发一种仪器，这将允许相对自动化的封装条件的阵列的检查，并将确定使用各种光谱探针的最佳组合，并以最小的消耗或试剂。该仪器将建立在Daedalus Innovations的成熟技术基础上。我们的目标是为那些对蛋白质封装技术没有深入了解的研究人员提供利用这种强大技术的机会。拟议的仪器将完成Daedalus Innovations提供的一套仪器，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：生物医学研究继续扩大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。在分子一级确定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，这项技术可以作为一个强大的平台，用于合理设计治疗一系列人类疾病的药物。

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.