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）：蛋白质结构的确定是我们对自然理解的重要组成部分。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。当前大约一半的药物靶标是整体膜蛋白，但是随着已知结构的数量接近50,000个，只有几百个是积分的膜蛋白，在数据流中留下了显着的空隙。显然，整体膜蛋白为当前的结构确定方法带来了独特的挑战，但尚无共识的方法来使用这种特别困难的蛋白质类别。对于NMR光谱法，膜蛋白观察到的动态性质比其他技术（例如X射线晶体学）所带来的样品制备挑战较小。然而，对于NMR而言，局限性是大型结构（例如整体膜蛋白）的缓慢翻滚问题。我们的方法是利用NMR光谱来利用动态蛋白的样品制备的优势，并使用独特的方法来克服缓慢的翻滚问题。它基于我们使用蛋白质反向胶束封装的早期工作。在这种方法中，感兴趣的蛋白质被封装在反向胶束颗粒的保护性水芯中，并且整个组件溶解在低粘度流体中，例如液态乙烷。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更快。这提供了有关现代“三重共振”实验效率的NMR松弛特性的显着改善。该方法允许在可溶性蛋白上获得高性能的NMR光谱，而无需剥离或trosy效应。在这里，我们建议通过采用最近对样品制备至关重要的两次突破来适应整体膜蛋白的研究。我们已经开发了一种封装方法，可以很容易地将其纳入有效，可靠和成本效益的设备中，以制备用于NMR光谱的样品。原型仪器将作为I阶段项目的一部分构建。这项研究途径将表明，不仅可以封装整体膜蛋白，而且可以通过足够的贯穿能力来完成，以使其作为结构确定工具以及生化测定平台变得有意义。为了提高方法的鲁棒性，我们建议使用KCSA钾通道（同型螺旋束）作为模型系统探索和扩展可用的表面活性剂矩阵空间。通过快速筛选各种表面活性剂组合的影响，我们希望能够开发可应用于新系统的预测封装策略。最后，我们将面临封装2-肾上腺素能GPCR并通过NMR光谱显示蛋白质的构象特异性的挑战。这些研究应建立反向胶束溶解方法作为整体膜蛋白结构研究的一般方法。公共卫生相关性：市场目标整体膜蛋白上大约一半的现有药物。在这些蛋白质中，很少有人在原子水平进行结构研究。对高分辨率结构的需求，以详细了解生命分子基础和对疾病的分子基础，需要能够传递分子水平结构信息的工具。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。