FRET-FLIM to Determine Protein Interactions and Membrane Topology of Enzyme Complexes.
FRET-FLIM to Determine Protein Interactions and Membrane Topology of Enzyme Complexes.
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FRET-FLIM 用于确定酶复合物的蛋白质相互作用和膜拓扑结构。
DOI：
10.1002/cpz1.598
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Spatola Rossi T
中科院分区：
文献类型：
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作者：
Spatola Rossi T
确定蛋白质之间的相互作用对于了解细胞和代谢过程，包括酶复合体和代谢物至关重要。Förster共振能量转移荧光寿命成像显微镜(FRET-FLIM)是一种先进的成像方法，可以定量检测蛋白质之间的相互作用。在这种方法中，相互作用研究中感兴趣的蛋白质被融合到不同的荧光团，如增强型绿色荧光蛋白(EGFP；供体分子)和单体红色荧光蛋白(MRFP；受体分子)。只有当感兴趣的蛋白质在物理上非常接近时，两个荧光团之间的能量转移才能有效地发生，大约在≤10 nm左右，因此最有可能相互作用。FRET-FLIM测量了在受体(MRFP)存在和不存在的情况下供体荧光团(EGFP)激发态寿命的减少，因此可以提供关于蛋白质-蛋白质相互作用和被测蛋白质膜拓扑的信息。在这里，我们描述了利用农杆菌介导的植物转化和植物细胞中的FRET-FLIM数据采集和分析方法来生产用于烟草叶表皮细胞FRET-FLIM分析的荧光蛋白融合。这些方案适用于不同细胞定位的膜蛋白和可溶性蛋白。©2022作者。目前由Wiley期刊LLC发表的方案：基本方案1：通过瞬时农杆菌介导的植物转化在烟草叶细胞中表达蛋白质基本方案2：FRET-FLIM数据采集和分析
Determining protein‐protein interactions is vital for gaining knowledge on cellular and metabolic processes including enzyme complexes and metabolons. Förster resonance energy transfer with fluorescence lifetime imaging microscopy (FRET‐FLIM) is an advanced imaging methodology that allows for the quantitative detection of protein‐protein interactions. In this method, proteins of interest for interaction studies are fused to different fluorophores such as enhanced green fluorescent protein (eGFP; donor molecule) and monomeric red fluorescent protein (mRFP; acceptor molecule). Energy transfer between the two fluorophore groups can only occur efficiently when the proteins of interest are in close physical proximity, around ≤10 nm, and therefore are most likely interacting. FRET‐FLIM measures the decrease in excited‐state lifetime of the donor fluorophore (eGFP) with and without the presence of the acceptor (mRFP) and can therefore give information on protein‐protein interactions and the membrane topology of the tested protein. Here we describe the production of fluorescent protein fusions for FRET‐FLIM analysis in tobacco leaf epidermal cells usingAgrobacterium‐mediated plant transformation and a FRET‐FLIM data acquisition and analysis protocol in plant cells. These protocols are applicable and can be adapted for both membrane and soluble proteins in different cellular localizations. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Protein expression in tobacco leaf cells via transientAgrobacterium‐mediated plant transformationBasic Protocol 2: FRET‐FLIM data acquisition and analysis