Apparatus for encapsulating integral membrane proteins for structural studies by

用于封装完整膜蛋白以进行结构研究的装置

• 批准号: 7745172
• 负责人: Ronald William Peterson
• 金额: $ 24.96万
• 依托单位: DAEDALUS INNOVATIONS, LLC
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-03 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7745172
• 关键词: Achievement; Adrenergic Agents; Adrenergic Receptor; Alkanes; Area; Award; Binding; Biochemical; Biochemistry; Biological Assay; Biological Models; Brain; Consensus; Crystallography; Data; Development; Disease; Drug Delivery Systems; Drug Design; Eligibility Determination; Encapsulated; Environment; Escherichia coli; Ethane; G Protein-Coupled Receptor Genes; Goals; Hand; Heteronuclear NMR; Integral Membrane Protein; Left; Libraries; Library Surveys; Life; Ligand Binding; Liquid substance; Marketing; Medicine; Membrane; Membrane Proteins; Methodology; Methods; Micelles; Molecular; NIH Program Announcements; NMR Spectroscopy; Nature; Neurotensin Receptors; Nuclear Magnetic Resonance; Pennsylvania; Performance; Pharmacologic Substance; Phase; Positioning Attribute; Potassium Channel; Preparation; Procedures; Property; Protein Dynamics; Protein NMR Spectroscopy; Proteins; Relaxation; Research; Resolution; Rest; Route; Sampling; Screening procedure; Small Business Innovation Research Grant; Solutions; Solvents; Specificity; Staging; Stream; Structure; System; Techniques; Technology; Testing; Universities; Validation; Viscosity; Water; Work; X-Ray Crystallography; adrenergic; aqueous; base; beta-adrenergic receptor; cost; design; human disease; improved; innovation; instrument; interest; mimetics; novel; novel strategies; particle; professor; protein aggregation; protein structure; prototype; public health relevance; rapid technique; receptor; research study; small molecule; structural biology; success; surfactant; tool

DESCRIPTION (provided by applicant): The determination of protein structures is a vital component of our understanding of nature. In the area of medicine and drug design the structure of the protein can greatly facilitate rational design of effective pharmaceuticals. Approximately half of current drug targets are integral membrane proteins, yet as the number of known structures approaches 50,000 only a few hundred of those are of integral membrane proteins, leaving a significant void in the data stream. It is clear that integral membrane proteins offer unique challenges to current methods of structure determination, and as yet there is no consensus approach for working with this especially difficult class of proteins. For NMR spectroscopy the dynamic nature observed for membrane proteins poses less of a sample preparation challenge than for other techniques such as X-ray crystallography. The limitation, however, for NMR has been the slow tumbling problem of large constructs such as integral membrane proteins. Our approach is to utilize NMR spectroscopy to take advantage of the ease of sample preparation of dynamic proteins, and uses a unique approach to overcome the slow tumbling problem. It is based on our earlier work using reverse micelle encapsulation of proteins. In that approach, the protein of interest is encapsulated within the protective aqueous core of a reverse micelle particle and the entire assembly is dissolved 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. The method allows high performance NMR spectra to be obtained on soluble proteins as large as 100 kDa without benefit of deuteration or the TROSY effect. Here we propose to adapt this approach to studies of integral membrane proteins by employing two recent breakthroughs critical to sample preparation. We have developed a method of encapsulation that can be readily incorporated into an efficient, reliable and cost-effective apparatus for the preparation of samples for NMR spectroscopy. The prototype instrument will be built as part of this Phase I project. This avenue of research will demonstrate that not only is encapsulating integral membrane proteins viable, but can be done with sufficient through-put that it becomes meaningful as a structure determination tool as well as biochemical assay platform. To improve the robustness of the method we propose to explore and expand the available surfactant matrix space using the KcsA potassium channel, a homotetrameric helical bundle, as a model system. By rapidly-screening the effects of a variety of surfactant combinations we expect to be able to develop predictive encapsulation strategies that can be applied to new systems. Finally, we will take on the challenge of encapsulating the ?2-adrenergic GPCR and show conformational specificity of the protein by NMR spectroscopy. These studies should establish the reverse micelle solubilization method as general approach to structural studies of integral membrane proteins. PUBLIC HEALTH RELEVANCE: Approximately half of existing pharmaceuticals on the market target integral membrane proteins. Of these proteins very few have been studied structurally at the atomic level. The demand for high resolution structures for developing a detailed understanding of the molecular basis for life and for disease requires tools capable of delivering molecular level structural information. 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）：蛋白质结构的测定是我们理解自然的重要组成部分。在医学和药物设计领域，蛋白质的结构可以极大地促进有效药物的合理设计。目前大约一半的药物靶标是整合膜蛋白，但随着已知结构的数量接近 50,000 个，其中只有几百个是整合膜蛋白，从而在数据流中留下了显着的空白。显然，整合膜蛋白对当前的结构测定方法提出了独特的挑战，并且迄今为止还没有一致的方法来处理这一类特别困难的蛋白质。与 X 射线晶体学等其他技术相比，对于 NMR 波谱而言，观察到的膜蛋白的动态性质对样品制备的挑战较小。然而，NMR 的局限性在于大型结构（例如完整膜蛋白）的缓慢翻滚问题。我们的方法是利用核磁共振波谱法来利用动态蛋白质样品制备的便利性，并使用独特的方法来克服缓慢翻滚的问题。它基于我们早期使用蛋白质反胶束封装的工作。在该方法中，目标蛋白质被封装在反胶束颗粒的保护性水性核心内，并且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。该方法可以在大至 100 kDa 的可溶性蛋白质上获得高性能 NMR 谱，而无需借助氘化或 TROSY 效应。在这里，我们建议通过采用对样品制备至关重要的两项最新突破，将这种方法应用于整合膜蛋白的研究。我们开发了一种封装方法，可以轻松融入高效、可靠且经济高效的设备中，用于制备核磁共振波谱样品。原型仪器将作为第一阶段项目的一部分建造。这一研究途径将证明，封装完整膜蛋白不仅是可行的，而且可以以足够的通量完成，使其作为结构测定工具和生化分析平台变得有意义。为了提高该方法的稳健性，我们建议使用 KcsA 钾通道（同四聚体螺旋束）作为模型系统来探索和扩展可用的表面活性剂基质空间。通过快速筛选各种表面活性剂组合的效果，我们期望能够开发出可应用于新系统的预测封装策略。最后，我们将接受封装 β2-肾上腺素 GPCR 的挑战，并通过 NMR 光谱显示蛋白质的构象特异性。这些研究应将反胶束溶解方法确立为完整膜蛋白结构研究的通用方法。公共健康相关性：市场上大约一半的现有药物以整合膜蛋白为目标。在这些蛋白质中，很少有人在原子水平上进行过结构研究。为了详细了解生命和疾病的分子基础，对高分辨率结构的需求需要能够提供分子水平结构信息的工具。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。