Abstract:
Assembly of de novo peptides designed from scratch is in a semi-rational manner and creates artificial supramolecular structures with unique properties. Considering that the functions of various proteins in living cells are highly regulated by their assemblies, building artificial assemblies within cells holds the potential to simulate the functions of natural protein assemblies and engineer cellular activities for controlled manipulation. How can we evaluate the self-assembly of designed peptides in cells? The most effective approach involves the genetic fusion of fluorescent proteins (FPs). Expressing a self-assembling peptide fused with an FP within cells allows for evaluating assemblies through fluorescence signal. When µm-scale assemblies such as condensates are formed, the peptide assemblies can be directly observed by imaging. For sub-µm-scale assemblies, fluorescence correlation spectroscopy analysis is more practical. Additionally, the fluorescence resonance energy transfer (FRET) signal between FPs is valuable evidence of proximity. The decrease in fluorescence anisotropy associated with homo-FRET reveals the properties of self-assembly. Furthermore, by combining two FPs, one acting as a donor and the other as an acceptor, the heteromeric interaction between two different components can be studied through the FRET signal. In this chapter, we provide detailed protocols, from designing and constructing plasmid DNA expressing the peptide-fused protein to analysis of self-assembly in living cells.
摘要:
从头设计的从头肽的组装是半理性的方式,并产生具有独特性质的人工超分子结构。考虑到活细胞中各种蛋白质的功能受到其组装的高度调节,在细胞内构建人工组件具有模拟天然蛋白质组件的功能和工程细胞活动以进行受控操作的潜力。我们如何评估设计肽在细胞中的自组装?最有效的方法涉及荧光蛋白(FPs)的遗传融合。在细胞内表达与FP融合的自组装肽允许通过荧光信号评估组装体。当形成微米级的组件如冷凝物时,肽组装体可以通过成像直接观察。对于亚微米级组件,荧光相关光谱分析更加实用。此外,FPs之间的荧光共振能量转移(FRET)信号是邻近的有价值的证据。与同源FRET相关的荧光各向异性的降低揭示了自组装的性质。此外,通过结合两个FP,一个作为捐赠者,另一个作为接受者,可以通过FRET信号研究两种不同组分之间的异聚相互作用。在这一章中,我们提供详细的协议,从设计和构建表达肽融合蛋白的质粒DNA到分析活细胞中的自组装。