Efficient scouting instrumentation for the determination of reverse micelle encap

描述（由申请人提供）：详细的结构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继续推动核磁共振可达到的尺寸限制，而巧妙的样品制备方法为研究诸如完整膜蛋白等其他顽固性蛋白质打开了大门。然而，进展在很大程度上仍然是渐进式的，很明显，可能需要彻底改变方法，以充分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。反胶束技术最初是为了解决大可溶性蛋白对溶液核磁共振方法的缓慢翻滚问题而设计的。从最初的概念开始，它已被证明可用于研究广泛的传统难治性蛋白质，如积分和锚定膜蛋白，聚集倾向蛋白和边缘稳定蛋白。基本思路是将感兴趣的蛋白质封装在反胶束颗粒的保护性水性核心中，并将整个组装溶解在低粘度流体中，如液态乙烷。在低粘度流体中，反向胶束颗粒比溶解在散装水中的蛋白质翻滚得快。这为控制现代“三重共振”实验效率的核磁共振弛豫特性提供了显著的改进。通过这种方法，可以研究大至150 kDa的蛋白质结构，而无需氘化或TROSY效应，从而可以获得更全面的结构和动力学信息。为了最大限度地发挥这种效果，必须在液态乙烷中制备反胶束样品，这需要在显著压力下制备样品，并将加压样品保持在核磁共振样管中。代达罗斯创新公司通过开发硬件解决方案，克服了实施这种方法的最初障碍，使研究人员能够以安全和可重复的方式生产这种样品，而不需要任何以前的高压应用经验。在本提案中，我们寻求开发一种仪器，克服当前常规使用的关键限制，这是寻找新蛋白质封装条件的看似艰巨的任务。目前，包封条件（表面活性剂混合物、样品缓冲液等）的优化通常是人工进行的，而且往往需要大量的材料。这对于大多数非学术应用来说是不可接受的，并且通常是不理想的。需要采取一种更精简和较少耗费人力和物力的办法。我们建议开发一种仪器，该仪器将允许对一系列封装条件进行相对自动化的检查，并将使用各种光谱探针确定最佳组合，并且以最小的消耗或试剂完成。该仪器将建立在代达罗斯创新公司成熟的技术基础上。目标是提供研究人员没有亲密的知识的蛋白质封装艺术，使利用这一强大的技术。该仪器将完成代达罗斯创新公司提供的整套仪器，该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。

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.