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

• 批准号: 10301370
• 负责人: Paul W.K. Rothemund
• 金额: $ 20.61万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 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: https://reporter.nih.gov/project-details/10301370
• 关键词: Biological; Biophysics; Boxing; Cell membrane; Cell physiology; Cells; Chemicals; Cholesterol; Collection; Complex; Coupled; Crowding; DNA; DNA receptor; Detergents; Diffuse; Dimerization; Drug Design; Dyes; Environment; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Hand; Health; Heterodimerization; Human; Immune response; In Vitro; Individual; Integral Membrane Protein; Kinetics; Label; Learning; Ligands; Linker DNA; Lipid Bilayers; Lipids; Liposomes; Measurement; Measures; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Metabolism; Methodology; Methods; Modeling; Molecular; Monitor; Nature; Nerve Degeneration; Participant; Process; Protein Subunits; Proteins; Proxy; Reaction; S-nitro-N-acetylpenicillamine; SNAP receptor; Signal Transduction; Site; Structural Models; Surface; Synapses; System; Tacrolimus Binding Proteins; Testing; Time; Transmembrane Domain; Ursidae Family; VAMP-2; Validation; Vesicle; Work; base; design; dimer; ds-DNA; experimental study; fluorophore; insight; instrumentation; interest; monomer; nano; nanometer; neurotransmission; neurotransmitter release; novel strategies; programs; protein complex; protein protein interaction; prototype; receptor; scaffold; single molecule; stoichiometry; syntaxin 1; tool

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 细胞膜填充有蛋白质，其相互作用对于无数细胞很重要 功能，从新陈代谢到信号，包括在此类过程中实现的许多功能 作为神经变性。众所周知，膜蛋白很难处理和研究。对于许多人 膜蛋白尚不清楚它们是否单独存在（单体），成对（作为二聚体）， 或较大的收集（较高的低聚物）。确定数量和布置的方法 其天然脂质膜环境中蛋白质复合物中的亚基将解决 数量现有争议，并最终将产生巨大的影响人类健康。一 解决这个问题的方法是在单分子水平上研究蛋白质相互作用，该蛋白质相互作用 可能需要昂贵且复杂的仪器。另一种方法是采用 相关廉价，化学自我组装的“分子手”以编程相互作用 在精确控制的数字和蛋白质比率之间（其“化学计量”）。接受 第二种方法，我们建议开发一个研究蛋白质蛋白质的通用平台 脂质膜中的相互作用，DNA折纸环 - 模拟脂质体。这个平台将 允许单个脂质中的蛋白质 - 蛋白质相互作用的独家控制和测量 双层，并克服了将单体区分开的现有方法的局限性 二聚体。将构造一个充满椎间盘形脂质体膜的DNA折纸环将被构造 带有感兴趣的单个蛋白质的附着点。沿纳米表格间隔 环的边缘，这些附件将用于定义蛋白质的数量和类型 可以进入膜的亚基。将蛋白质释放到膜上 将通过引入DNA信号来实现，从而破坏DNA接头 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测试分子的相互作用 控制实验。在第二个目标中，我们将用具有的蛋白质代替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 互动。在最终目标中，我们将专注于解决有关的长期问题 SNARE复合蛋白突触2的二聚化，这是重要参与者 释放神经递质所需的膜融合过程。