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

摘要 蛋白质-蛋白质相互作用(PPI)是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工作。与生物相关的PPI的强度差异很大 与亚NM离解常数的近永久相互作用到与MM离解的弱瞬时相互作用。 电离常数。尽管瞬时PPI对于控制单元功能的许多关键过程很重要， 目前尚缺乏对其进行原位表征的方法。为了解决这个问题，我们将利用我们的模数- 用于无背景信号恢复和量化以开发调制的BLE荧光蛋白(FP) 转移-Förster共振能量转移(MT-FRET)。调制施主(一个可光开关的FP，激发子- 同时在488 nm和405 nm)激发)编码了两个激光器的和和差 调制频率对施主发射的影响。这种调制传递给接受者，接受者本身也被调制 在900 nm激发下，全部在一个宽视场中，荧光几何图形。结合的络合物、给体和受体 所有信号都以独特的调制频率出现，其幅度与系数成正比。 各自为政。然后，这种MT-FRET方法会产生真正的无背景平衡常数，即使是 微弱的瞬时PPI，因为未结合的荧光团信号出现在不同于 这个建筑群。既适用于自由扩散的物种，也适用于结合的物种，这是相对于现有AP的关键进步。 确定PPI相互作用的方法，因为它抑制了压倒性的背景特征 瞬时PPI和活细胞中的PPI。具体地说，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将不局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，用于测量和测量瞬变 PPI在复杂的活细胞环境中。

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.