发展新型蛋白质定点双标记方法研究β-Arrestin-1动态构象变化

The study of conformational dynamics in proteins through single molecule fluorescence resonance energy transfer (smFRET) may have great importance for proteins. However, site-specific dual-labling of proteins constrict the application of smFRET. We synthesized 2-amino-3-(4-mercaptophenyl)propanoic acid, and incorporated them into β-arrestin-1 at distinct sites through genetic codon expansion. We also inserted a binding motif (CCPGCC) into β-arrestin-1. These two positions can react with BODIPY and FLAsH specifically in biocompatible conditions, permitting the introduction of FRET donor and acceptor fluorophphores into β-arrestin-1. We will probe conformational dynamics in dual-labled β-arrestin-1 under combination with different proteins. Because this method is convenient and biocompatible, we hope it could become a universal method for dual-labling of proteins and have great applications in developing new drugs.

利用单分子荧光共振能量转移（smFRET）方法研究蛋白质的动态构象变化对于蛋白质科学具有重要意义。限制smFRET应用范围的主要瓶颈是蛋白质的定点双标记。针对此问题，我们合成了对巯基苯丙氨酸，通过基因密码子扩展方法插入到β-arrestin-1的特定位点，同时引入四半胱氨酸序列（CCPGCC）。这两个位点可以在生物兼容条件下分别与BODIPY和FLAsH染料特异性的反应，实现在β-arrestin-1上同时标记荧光供体和受体探针。我们将利用smFRET技术检测双标记的β-arrestin-1突变体在结合不同蛋白情况下的动态构象变化，解析β-arrestin-1介导的信号传递机制。由于此双标记方法简便、生物兼容，我们希望此方法成为蛋白质双标记的通用方法，并在新药发展方面获得重要应用。