NMR Spectroscopic Studies Reveal the Critical Role of Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.
NMR Spectroscopic Studies Reveal the Critical Role of Isopeptide Bond in Forming the Otherwise Unstable SpyTag-SpyCatcher Mutant Complexes.
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核磁共振波谱研究揭示了异肽键在形成不稳定的 SpyTag-SpyCatcher 突变体复合物中的关键作用。
DOI：
10.1021/acs.biochem.0c00287
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Wen-Bin Zhang
中科院分区：
文献类型：
--
作者：
Nan Zhang;Jing Liu;Yajie Liu;Wen-Hao Wu;Jing Fang;Xiao-Di Da;Shenlin Wang;Wen-Bin Zhang
蛋白质折叠和化学反应之间的相互作用一直是一个有趣的课题。在这方面的贡献，我们报告的SpyTag和SpyCatcher反应突变体的研究，使用组合技术的SDS-PAGE，LC-MS，圆二色谱，和NMR光谱。发现野生型SpyCatcher在溶液中折叠良好，并与SpyTag对接形成促进异肽形成的中间体。相比之下，双突变体SpyCatcherVA在溶液中是无序的，但仍然对SpyTag具有反应性，形成良好折叠的共价复合物。使用催化失活突变体的对照实验进一步揭示了异肽键在稳定原本松散的SpyTag-SpyCatcherVA复合物中的关键作用，放大了微小序列差异的影响。我们相信蛋白质折叠和异肽键合之间的协同作用是增强蛋白质稳定性和设计蛋白质-蛋白质相互作用的有效途径。
The interplay between protein folding and chemical reaction has been an intriguing subject. In this contribution, we report the study of SpyTag and SpyCatcher reactive mutants using combined techniques of SDS-PAGE, LC-MS, circular dichroism, and NMR spectroscopy. It was found that the wild-type SpyCatcher is well-folded in solution and docks with SpyTag to form an intermediate that promotes isopeptide formation. By contrast, the double mutant SpyCatcherVA is disordered in solution, yet remains reactive toward SpyTag, forming a well-folded covalent complex. Control experiments using the catalytically inactive mutants further reveal the critical role of the isopeptide bond in stabilizing the otherwise loose SpyTag-SpyCatcherVA complex, amplifying the effect of minute sequence disparity. We believe that the synergy between protein folding and isopeptide bonding is an effective way to enhance protein stability and engineer protein-protein interactions.