MT-FRET to decode transient protein-protein interactions in Cu homeostasis

MT-FRET 解码铜稳态中瞬时蛋白质-蛋白质相互作用

• 批准号: 9979477
• 负责人: ROBERT M DICKSON
• 金额: $ 22.59万
• 依托单位: GEORGIA 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: 2020-04-01 至 2022-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9979477
• 关键词: Address; Biochemical; Biological; Biology; Calibration; Cell physiology; Cells; Characteristics; Chemicals; Communities; Complex; Copper; Coupled; Data; Detection; Development; Diffuse; Dimerization; Dissociation; Energy Transfer; Environment; Equilibrium; Fluorescence; Fluorescence Resonance Energy Transfer; Frequencies; Gene Expression; Geometry; Goals; Homeostasis; Homodimerization; Immunoprecipitation; In Situ; In Vitro; Ions; Label; Lasers; Lighting; Measurable; Measures; Membrane; Methods; Microscopy; Modeling; Molecular Chaperones; Names; Optics; Pathway interactions; Process; Proteins; Recovery; Regulation; Research; Residual state; Role; Salvelinus; Scheme; Signal Transduction; Sum; Surveys; Techniques; Thermodynamics; Time; Transition Elements; Work; base; cellular targeting; combat; crosslink; fluorescence imaging; fluorophore; human disease; imprint; improved; insight; live cell imaging; new technology; novel diagnostics; novel therapeutics; protein protein interaction; spatiotemporal; tool; trafficking; uptake

Abstract Protein-protein interactions (PPIs) represent the fundamental components of highly complex and dynamic net- works that govern nearly all cellular processes. The strength of biologically relevant PPIs varies widely from near permanent interactions with sub-nM dissociation constants to weak transient interactions with mM disso- ciation constants. Despite the importance of transient PPIs for many key processes governing cell functions, methods for their in situ characterization are still lacking. To address this problem, we will utilize our modulata- ble fluorescent proteins (FPs) for background-free signal recovery and quantification to develop Modulation Transfer-Förster Resonance Energy Transfer (MT-FRET). Modulating the Donor (a photoswitchable FP, excit- ed at both 488 and 405 nm simultaneously) excitations encodes the sum and difference of the two lasers’ modulation frequencies on donor emission. This modulation transfers to the acceptor, which itself is modulated with 900nm excitation, all in a widefield, epifluorescence geometry. The bound complex, donor, and acceptor signals all appear at unique modulation frequencies with amplitudes that are directly proportional to the con- centration of each. This MT-FRET approach then yields true background-free equilibrium constants, even of weak, transient PPIs, as unbound fluorophore signals appear at different modulation frequencies from that of the complex. Applicable to both freely diffusing and bound species, this is a key advance over existing ap- proaches for determining PPI interactions as it suppresses the overwhelming background characteristic of transient PPIs and those in live cells. Specifically, we will apply these general methods to characterize selected proteins involved in dynamic trafficking of copper in vitro and in live cells. The materials and methods devel- oped will not be limited to the characterization of PPIs relevant to copper homeostasis, but will simultaneously provide a more general set of tools, methods, and overall framework for surveying and measuring transient PPIs within the complex environment of live cells.

摘要 蛋白质-蛋白质相互作用（PPI）代表了高度复杂和动态网络的基本组成部分， 控制着几乎所有的细胞过程。生物学相关PPI的强度差异很大， 与亚nM解离常数的接近永久的相互作用到与mM解离常数的弱瞬时相互作用 引用常数。尽管瞬时PPI对于控制细胞功能的许多关键过程很重要， 但是仍然缺乏用于它们的原位表征的方法。为了解决这个问题，我们将利用我们的模块化- 用于无背景信号恢复和定量的荧光蛋白（FP）， 转移-福斯特共振能量转移（MT-FRET）。调节供体（光开关FP，激发- 艾德同时在488和405 nm处）激发对两个激光器的 对施主发射的调制频率。这种调制传递到受体，受体本身也被调制 用900 nm激发，全部在宽视场、落射荧光几何结构中。结合复合物、供体和受体 信号都以独特的调制频率出现，其幅度直接与信号的频率成比例。 每个人的中心。然后，这种MT-FRET方法产生真正的无背景平衡常数，甚至 弱的、瞬时的PPI，因为未结合的荧光团信号出现在与 复杂的。适用于自由扩散和约束的物种，这是一个关键的进步，现有的ap- 方法确定PPI的相互作用，因为它抑制了压倒性的背景特征， 瞬时PPI和活细胞中的PPI。具体而言，我们将应用这些一般方法来表征选定的 参与铜在体外和活细胞中动态运输的蛋白质。材料和方法的发展- oped将不限于与铜稳态相关的PPI的表征，但同时将 提供了一套更通用的工具、方法和总体框架，用于勘测和测量瞬态 活细胞复杂环境中的PPI。