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

基本信息

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
项目类别：
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

项目摘要

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.

抽象的蛋白质-蛋白质相互作用（PPI）代表了高度复杂和动态的网络的基本组成部分。控制几乎所有细胞过程的生物学相关 PPI 的强度差异很大。与亚纳米解离常数的近永久相互作用到与毫米解离常数的弱瞬时相互作用尽管瞬态 PPI 对于许多控制细胞功能的关键过程很重要，仍然缺乏对其进行原位表征的方法，为了解决这个问题，我们将利用我们的调制器。 ble 荧光蛋白 (FP) 用于无背景信号恢复和定量以开发调制 Transfer-Förster 共振能量转移 (MT-FRET)。同时在 488 和 405 nm 处编辑）激发编码了两个激光的和与差供体发射的调制频率该调制转移到受体，受体本身也被调制。 900 nm 激发，全部处于宽场落射荧光几何结构中。信号都出现在独特的调制频率，其幅度与反面成正比然后，这种 MT-FRET 方法会产生真正的无背景平衡常数，甚至是弱、瞬态 PPI，因为未结合的荧光团信号出现在与荧光团不同的调制频率下该复合物适用于自由扩散和束缚物种，这是现有应用程序的一个关键进步。确定 PPI 相互作用的方法，因为它抑制了压倒性的背景特征具体来说，我们将应用这些通用方法来表征选定的 PPI。参与体外和活细胞内铜动态运输的蛋白质。 OPED 将不仅限于与铜稳态相关的 PPI 的表征，而是同时提供一套更通用的工具、方法和总体框架，用于测量和测量瞬态活细胞复杂环境中的 PPI。