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
基本信息
- 批准号:7745172
- 负责人:
- 金额:$ 24.96万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AchievementAdrenergic AgentsAdrenergic ReceptorAlkanesAreaAwardBindingBiochemicalBiochemistryBiological AssayBiological ModelsBrainConsensusCrystallographyDataDevelopmentDiseaseDrug Delivery SystemsDrug DesignEligibility DeterminationEncapsulatedEnvironmentEscherichia coliEthaneG Protein-Coupled Receptor GenesGoalsHandHeteronuclear NMRIntegral Membrane ProteinLeftLibrariesLibrary SurveysLifeLigand BindingLiquid substanceMarketingMedicineMembraneMembrane ProteinsMethodologyMethodsMicellesMolecularNIH Program AnnouncementsNMR SpectroscopyNatureNeurotensin ReceptorsNuclear Magnetic ResonancePennsylvaniaPerformancePharmacologic SubstancePhasePositioning AttributePotassium ChannelPreparationProceduresPropertyProtein DynamicsProtein NMR SpectroscopyProteinsRelaxationResearchResolutionRestRouteSamplingScreening procedureSmall Business Innovation Research GrantSolutionsSolventsSpecificityStagingStreamStructureSystemTechniquesTechnologyTestingUniversitiesValidationViscosityWaterWorkX-Ray Crystallographyadrenergicaqueousbasebeta-adrenergic receptorcostdesignhuman diseaseimprovedinnovationinstrumentinterestmimeticsnovelnovel strategiesparticleprofessorprotein aggregationprotein structureprototypepublic health relevancerapid techniquereceptorresearch studysmall moleculestructural biologysuccesssurfactanttool
项目摘要
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点是整合膜蛋白,但随着已知结构的数量接近50,000,其中只有几百个是整合膜蛋白,在数据流中留下了显著的空白。很明显,整合膜蛋白提供了独特的挑战,目前的结构确定方法,并没有共识的方法与这类特别困难的蛋白质。对于NMR光谱学,观察到的膜蛋白的动态性质比其他技术如X射线晶体学对样品制备的挑战更小。然而,核磁共振的局限性是大结构(如整合膜蛋白)的缓慢翻滚问题。我们的方法是利用NMR光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。它是基于我们早期的工作,使用反胶束包封蛋白质。在该方法中,感兴趣的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。该方法允许高性能的NMR光谱上获得的可溶性蛋白质大至100 kDa,而没有氘代或TROSY效应的好处。在这里,我们建议采用最近的两个突破,关键的样品制备,以适应这种方法的研究,膜蛋白的整合。我们已经开发了一种封装方法,可以很容易地纳入一个有效的,可靠的和具有成本效益的装置,用于制备样品的NMR光谱。原型仪器将作为第一阶段项目的一部分建造。这条研究途径将证明,不仅是封装膜蛋白是可行的,但可以做足够的吞吐量,它成为有意义的结构确定工具,以及生化分析平台。为了提高该方法的鲁棒性,我们建议探索和扩大可用的表面活性剂基质空间使用KcsA钾通道,一个同源四聚体螺旋束,作为一个模型系统。通过快速筛选各种表面活性剂组合的影响,我们希望能够开发出可应用于新系统的预测性包封策略。最后,我们将采取的封装的挑战?2-肾上腺素能GPCR,并通过NMR光谱显示蛋白质的构象特异性。这些研究应建立反胶束增溶方法作为一般的方法,完整的膜蛋白的结构研究。公共卫生相关性:市场上现有的药物中约有一半针对膜蛋白。在这些蛋白质中,很少有人在原子水平上进行结构研究。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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
数据更新时间:{{ journalArticles.updateTime }}
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
数据更新时间:{{ journalArticles.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ monograph.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ sciAawards.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ conferencePapers.updateTime }}
{{ item.title }}
- 作者:
{{ item.author }}
数据更新时间:{{ patent.updateTime }}
Ronald William Peterson其他文献
Ronald William Peterson的其他文献
{{
item.title }}
{{ item.translation_title }}
- DOI:
{{ item.doi }} - 发表时间:
{{ item.publish_year }} - 期刊:
- 影响因子:{{ item.factor }}
- 作者:
{{ item.authors }} - 通讯作者:
{{ item.author }}
{{ truncateString('Ronald William Peterson', 18)}}的其他基金
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 - 财政年份:2012
- 资助金额:
$ 24.96万 - 项目类别:
Apparatus for NMR spectroscopy of encapsulated proteins
封装蛋白质的核磁共振波谱仪
- 批准号:
7937172 - 财政年份:2009
- 资助金额:
$ 24.96万 - 项目类别:
Apparatus for NMR spectroscopy of encapsulated proteins
封装蛋白质的核磁共振波谱仪
- 批准号:
7463930 - 财政年份:2005
- 资助金额:
$ 24.96万 - 项目类别:
Apparatus for NMR spectroscopy of encapsulated proteins
封装蛋白质的核磁共振波谱仪
- 批准号:
7325872 - 财政年份:2005
- 资助金额:
$ 24.96万 - 项目类别:
Apparatus for NMR spectroscopy of encapsulated proteins
封装蛋白质的核磁共振波谱仪
- 批准号:
6932630 - 财政年份:2005
- 资助金额:
$ 24.96万 - 项目类别:
相似海外基金
Preclinical test for the efficacy of adrenergic agents in treatment of AD
肾上腺素能药物治疗AD疗效的临床前试验
- 批准号:
8358448 - 财政年份:2012
- 资助金额:
$ 24.96万 - 项目类别:
Preclinical test for the efficacy of adrenergic agents in treatment of AD
肾上腺素能药物治疗AD疗效的临床前试验
- 批准号:
8517552 - 财政年份:2012
- 资助金额:
$ 24.96万 - 项目类别:
MODULATING FLUID THERAPY WITH ADRENERGIC AGENTS AND CYCLIC AMP ENHANCERS IN
使用肾上腺素能药物和环放大器增强剂调节液体治疗
- 批准号:
7952159 - 财政年份:2009
- 资助金额:
$ 24.96万 - 项目类别:
THE EFFECT OF BETA-ADRENERGIC AGENTS AND FLUID THERAPY IN HUMANS
β-肾上腺素能药物和液体疗法对人体的影响
- 批准号:
7952152 - 财政年份:2009
- 资助金额:
$ 24.96万 - 项目类别:
MODULATING FLUID THERAPY WITH ADRENERGIC AGENTS AND CYCLIC AMP ENHANCERS IN
使用肾上腺素能药物和环放大器增强剂调节液体治疗
- 批准号:
7719194 - 财政年份:2008
- 资助金额:
$ 24.96万 - 项目类别:
THE EFFECT OF BETA-ADRENERGIC AGENTS AND FLUID THERAPY IN HUMANS
β-肾上腺素能药物和液体疗法对人体的影响
- 批准号:
7605416 - 财政年份:2007
- 资助金额:
$ 24.96万 - 项目类别:
MODULATING FLUID THERAPY WITH ADRENERGIC AGENTS AND CYCLIC AMP ENHANCERS IN
使用肾上腺素能药物和环放大器增强剂调节液体治疗
- 批准号:
7605425 - 财政年份:2007
- 资助金额:
$ 24.96万 - 项目类别:
THE EFFECT OF BETA-ADRENERGIC AGENTS AND FLUID THERAPY IN HUMANS
β-肾上腺素能药物和液体疗法对人体的影响
- 批准号:
7378753 - 财政年份:2006
- 资助金额:
$ 24.96万 - 项目类别:
Adrenergic Agents for Methamphetamine: Outpatient Trials
甲基苯丙胺肾上腺素药物:门诊试验
- 批准号:
6825160 - 财政年份:2004
- 资助金额:
$ 24.96万 - 项目类别:
ADRENERGIC AGENTS FOR CARDIOPULMONARY RESUSCITATION
用于心肺复苏的肾上腺素能药物
- 批准号:
2702283 - 财政年份:1997
- 资助金额:
$ 24.96万 - 项目类别: