A DNA origami platform for measuring membrane protein interactions
用于测量膜蛋白相互作用的 DNA 折纸平台
基本信息
- 批准号:10301370
- 负责人:
- 金额:$ 20.61万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-11-12 至 2022-10-31
- 项目状态:已结题
- 来源:
- 关键词:BiologicalBiophysicsBoxingCell membraneCell physiologyCellsChemicalsCholesterolCollectionComplexCoupledCrowdingDNADNA receptorDetergentsDiffuseDimerizationDrug DesignDyesEnvironmentFluorescenceFluorescence Resonance Energy TransferGoalsHandHealthHeterodimerizationHumanImmune responseIn VitroIndividualIntegral Membrane ProteinKineticsLabelLearningLigandsLinker DNALipid BilayersLipidsLiposomesMeasurementMeasuresMediatingMembraneMembrane FusionMembrane LipidsMembrane ProteinsMetabolismMethodologyMethodsModelingMolecularMonitorNatureNerve DegenerationParticipantProcessProtein SubunitsProteinsProxyReactionS-nitro-N-acetylpenicillamineSNAP receptorSignal TransductionSiteStructural ModelsSurfaceSynapsesSystemTacrolimus Binding ProteinsTestingTimeTransmembrane DomainUrsidae FamilyVAMP-2ValidationVesicleWorkbasedesigndimerds-DNAexperimental studyfluorophoreinsightinstrumentationinterestmonomernanonanometerneurotransmissionneurotransmitter releasenovel strategiesprogramsprotein complexprotein protein interactionprototypereceptorscaffoldsingle moleculestoichiometrysyntaxin 1tool
项目摘要
Project Summary/Abstract
Cell membranes are populated with proteins whose interactions are important for myriad cellular
functions, from metabolism to signaling, including many functions implicated in processes such
as neurodegeneration. Membrane proteins are notoriously difficult to handle and study. For many
membrane proteins it is unknown whether they exist singly (as monomers), in pairs (as dimers),
or larger collections (higher oligomers). A method to determine the number and arrangement of
subunits in a protein complex within its native lipid membrane environment would resolve a
number existing controversies, and would eventually have a large impact human health. One
approach to this problem would be to study protein interactions at the single molecule level, which
can require expensive and complex instrumentation. Another approach would be to employ a
relatively inexpensive, chemically self-assembled “molecular hand” to program the interactions
between precisely controlled numbers and ratios of proteins (their “stoichiometry”). Taking the
second approach, we propose to develop a general platform for studying protein-protein
interactions in lipid membranes, the DNA origami ring-templated liposome. This platform will
allow exquisite control and measurement of protein-protein interactions within a single lipid
bilayer, and overcome the limitations of existing methods for differentiating monomers from
dimers. A DNA origami ring, filled with a disc-shaped liposomal membrane, will be constructed
with attachment points for individual proteins of interest. Spaced with nanometer-precision along
the edge of the ring, these attachment points will be used to define the number and type of protein
subunits that can enter the membrane. Programmed release of the proteins into the membrane
will be achieved through the introduction of DNA signals, that break DNA linkers between the
proteins and the edge of the DNA ring. In our first aim, we will prototype and troubleshoot the
platform by studying the interactions of fluorescently labelled DNA test molecules in a number of
control experiments. In a second aim, we will replace the DNA test molecules with proteins having
a known interaction, and verify that the platform can be used to measure protein-protein
interactions. In a final aim, we will focus on resolving a long-standing question regarding the
dimerization of a SNARE complex protein Synaptobrevin 2, which is an important participant in
the membrane fusion process required for the release of neurotransmitters.
项目摘要/摘要
细胞膜上充满了蛋白质,这些蛋白质的相互作用对无数细胞很重要。
功能,从新陈代谢到信号,包括许多过程中涉及的功能,如
神经退行性变。膜蛋白是出了名的难以处理和研究。对许多人来说
膜蛋白尚不清楚它们是单独存在(作为单体),还是成对存在(作为二聚体),
或更大的集合(更高的低聚物)。一种确定数字电视节目数量和排列方式的方法
蛋白质复合体中的亚基在其天然的脂膜环境中将分解一个
许多现有的争议,并最终将对人类健康产生很大影响。一
解决这个问题的方法是在单分子水平上研究蛋白质的相互作用,这是
可能需要昂贵而复杂的仪器。另一种方法是使用
相对便宜的、化学上自组装的“分子手”来编程相互作用
精确控制的蛋白质数量和比例(它们的“化学计量比”)之间的差异。参加了
第二种方法,我们建议开发一个研究蛋白质的通用平台
脂膜中的相互作用,DNA折纸环状模板脂质体。这个平台将
允许精确控制和测量单一脂质内的蛋白质-蛋白质相互作用
双分子层,克服了现有方法区分单体和
二聚体。将构建一个DNA折纸环,里面填充着一个圆盘状的脂质体薄膜
带有单个感兴趣蛋白质的附着点。以纳米级的精度沿
环的边缘,这些连接点将被用来定义蛋白质的数量和类型
可以进入细胞膜的亚基。蛋白质的程序性释放进入细胞膜
将通过引入DNA信号来实现,这些信号打破了
蛋白质和DNA环的边缘。在我们的第一个目标中,我们将原型和故障排除
通过研究荧光标记的DNA测试分子在许多
对照实验。在第二个目标中,我们将用具有以下特性的蛋白质取代DNA测试分子
已知的相互作用,并验证该平台可用于测量蛋白质-蛋白质
互动。在最后一个目标中,我们将重点解决一个长期存在的关于
SNARE复合蛋白Synaptobrevin 2的二聚化
释放神经递质所需的膜融合过程。
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)
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Paul W.K. Rothemund其他文献
Paul W.K. Rothemund的其他文献
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- 资助金额:
$ 20.61万 - 项目类别:
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