Cellular synthesis of protein pretzelanes
Cellular synthesis of protein pretzelanes
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DOI：
10.1016/j.giant.2022.100092
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发表时间：
2022-02-22
期刊：
影响因子：
7
通讯作者：
Zhang, Wen-Bin
中科院分区：
文献类型：
--
作者：
Bai, Xilin;Liu, Yajie;Zhang, Wen-Bin
拓扑结构已被公认为是分子工程中的一个独特维度，但拓扑结构的多样性在很大程度上仍未得到开发，特别是在大分子中。在这里，我们报告的分子设计，细胞合成，和详细的表征蛋白pretzelanes与化学拓扑结构的一个桥接的霍普夫链接。由p53 dim（X）结构域引导的分子内链缠绕和由SpyTag（A）-SpyCatcher（B）反应遗传编码的侧链偶联之间的协同作用促进了模型蛋白pretzelane BXA-BXA在大肠杆菌中的直接合成。该方法容许在两个环之间的桥区域插入各种感兴趣的蛋白质，例如弹性蛋白样蛋白（ELP）、超折叠绿色荧光蛋白（GFP）和二氢叶酸还原酶（DHFR），产生三种蛋白质pretzelane BXA-ELP-BXA、BXA-GFP-BXA和BXA-DHFR-BXA。它们的拓扑结构已经通过MALDI-TOF质谱、离子迁移率质谱、位点特异性突变和正交蛋白水解消化实验的组合技术进行了验证。不仅GFP的荧光性质和DHFR的催化性质完全保留，pretzelane拓扑结构还使BXA-DHFR-BXA比野生型DHFR更具热弹性。这些结果扩大了蛋白质的拓扑多样性，并证明蛋白质稳定性作为一个潜在的功能优势的pretzelane拓扑结构。
Topology has been recognized as a unique dimension in molecular engineering, yet the topological diversity remains largely untapped, especially in macromolecules. Herein, we report the molecular design, cellular synthesis, and detailed characterization of protein pretzelanes with a chemical topology of a bridged Hopf link. The synergy between the intramolecular chain entwining guided by the p53dim (X) domains and the genetically encoded side-chain coupling by SpyTag(A)-SpyCatcher(B) reaction facilitates the direct synthesis of the model protein pretzelane BXA-BXA in Escherichia coli. The approach tolerates the insertion of various proteins-of interest, such as elastin-like protein (ELP), superfolder green fluorescent protein (GFP) and dihydrofolate reductase (DHFR), at the bridge region between two rings, giving rise to three protein pretzelanes BXA-ELP-BXA, BXA-GFP-BXA, and BXA-DHFR-BXA. Their topology has been verified by combined techniques of MALDI-TOF mass spectrometry, ion mobility-mass spectrometry, site-specific mutation, and orthogonal proteolytic digestion experiments. Not only are the fluorescent properties of GFP and the catalytic properties of DHFR fully retained, the pretzelane topology also renders BXA-DHFR-BXA more thermally resilient than the wild-type DHFR. These results expand the topological diversity of proteins and demonstrate protein stabilization as a potential functional benefit for the pretzelane topology.