De novo protein design is delivering new peptide and protein structures at a rapid pace. Many of these synthetic polypeptides form well-defined and hyperthermal-stable structures. Generally, however, less is known about the dynamic properties of the de novo designed structures. Here, we explore one aspect of dynamics in a series of de novo coiled-coil peptide assemblies: namely, peptide exchange within and between different oligomers from dimers through to heptamers. First, we develop a fluorescence-based reporter assay for peptide exchange that is straightforward to implement, and, thus, would be useful to others examining similar systems. We apply this assay to explore both homotypic exchange within single species, and heterotypic exchange between coiled coils of different oligomeric states. For the former, we provide detailed study for a dimeric coiled coil, CC-Di, finding a half-life for exchange of 4.2 ± 0.3 minutes at a peptide concentration of 200 µM. Interestingly, more broadly when assessing exchange across all of the oligomeric states, we find that some of the designs are faithful and only undergo homotypic strand exchange, whereas others are promiscuous and exchange to form unexpected hetero-oligomers. Finally, we develop two design strategies to improve the orthogonality of the different oligomers: (i) using alternate positioning of salt bridge interactions; and (ii) incorporating non-canonical repeats into the designed sequences. In so doing, we reconcile the promiscuity and deliver a set of faithful homo-oligomeric de novo coiled-coil peptides. Our findings have implications for the application of these and other coiled coils as modules in chemical and synthetic biology.

中文翻译：

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从头设计的卷曲螺旋肽组装体中的交换、混杂和正交性


De novo protein design 正在快速提供新的肽和蛋白质结构。这些合成多肽中的许多形成定义明确且高温稳定的结构。然而，一般来说，人们对从头设计结构的动力学特性知之甚少。在这里，我们探讨了一系列从头卷曲螺旋肽组装体中动力学的一个方面：即从二聚体到七聚体的不同寡聚体内部和之间的肽交换。首先，我们开发了一种基于荧光的肽交换报告基因测定法，该测定法易于实施，因此对其他检查类似系统的人很有用。我们应用该测定法来探索单个物种内的同型交换以及不同寡聚状态的卷曲螺旋之间的异型交换。对于前者，我们提供了二聚体卷曲线圈 CC-Di 的详细研究，发现在 200 μM 的肽浓度下交换的半衰期为 4.2 ± 0.3 分钟。有趣的是，更广泛地说，在评估所有寡聚体状态之间的交换时，我们发现一些设计是忠实的，并且只进行同型链交换， 而其他的则是混杂的，并交换形成意想不到的异源寡聚体。最后，我们开发了两种设计策略来提高不同低聚物的正交性：（i） 使用盐桥相互作用的交替定位;（ii） 将非经典重复序列纳入设计的序列中。在此过程中，我们调和了混杂性，并提供了一组忠实的同源寡聚从头卷曲螺旋肽。我们的研究结果对这些和其他卷曲线圈作为模块在化学和合成生物学中的应用具有意义。