翻译后修饰(PTM),如磷酸化和去磷酸化可以迅速改变蛋白质表面化学和结构构象,它可以在信号网络内切换蛋白质-蛋白质相互作用(PPI)。最近,已经创建了从头设计的磷酸化响应蛋白开关,该开关利用激酶和磷酸酶介导的磷酸化来调节PPI。PTM驱动的蛋白质开关是研究活细胞中PTM动力学的有前途的工具,开发生物相容性纳米设备,和工程信号通路来编程细胞行为。然而,对PTM驱动的蛋白质开关的物理和动力学约束知之甚少,这限制了它们的实际应用。在这项研究中,我们提出了一个框架来评估基于四个性能指标的两组分PTM驱动的蛋白质开关:有效浓度,动态范围,响应时间,和可逆性。我们的计算模型揭示了结合动力学之间的复杂关系,磷酸化动力学,和开关浓度,控制PTM驱动的蛋白质开关的灵敏度和可逆性。基于交互建模的洞察力,我们建立并评估了新型的磷酸化驱动的蛋白质开关,该开关由磷酸化敏感的卷曲线圈组成,作为传感器域融合到荧光蛋白作为执行器域。通过用特定的蛋白激酶和磷酸酶调节开关的磷酸化状态,我们展示得很快,“开”和“关”状态之间的可逆转换。开关的响应与激酶浓度线性相关,证明了其作为实时测量激酶的生物传感器的潜力。按照预期,开关响应特定的激酶活性,荧光信号增加,我们的模型可以用来区分开关激活的两种机制:二聚化或结构重排。此处开发的蛋白质开关动力学模型应使PTM驱动的开关能够被设计为具有针对特定应用的理想性能。
Post-translational modifications (PTMs) such as phosphorylation and dephosphorylation can rapidly alter protein surface chemistry and structural conformation, which can switch protein-protein interactions (PPIs) within signaling networks. Recently, de novo-designed phosphorylation-responsive protein switches have been created that harness kinase- and phosphatase-mediated phosphorylation to modulate PPIs. PTM-driven protein switches are promising tools for investigating PTM dynamics in living cells, developing biocompatible nanodevices, and engineering signaling pathways to program cell behavior. However, little is known about the physical and kinetic constraints of PTM-driven protein switches, which limits their practical application. In this study, we present a framework to evaluate two-component PTM-driven protein switches based on four performance metrics: effective concentration, dynamic range, response time, and reversibility. Our computational models reveal an intricate relationship between the binding kinetics, phosphorylation kinetics, and switch concentration that governs the sensitivity and reversibility of PTM-driven protein switches. Building upon the insights of the interaction modeling, we built and evaluated novel phosphorylation-driven protein switches consisting of phosphorylation-sensitive coiled coils as sensor domains fused to fluorescent proteins as actuator domains. By modulating the phosphorylation state of the switches with a specific protein kinase and phosphatase, we demonstrate fast, reversible transitions between \"on\" and \"off\" states. The response of the switches linearly correlated to the kinase concentration, demonstrating its potential as a biosensor for kinase measurements in real time. As intended, the switches responded to specific kinase activity with an increase in the fluorescence signal and our model could be used to distinguish between two mechanisms of switch activation: dimerization or a structural rearrangement. The protein switch kinetics model developed here should enable PTM-driven switches to be designed with ideal performance for specific applications.