在生物巨分子中,核磁共振技術(NMR, Nuclear Magnetic Resonance)常被用來測量蛋白質的結構。然而,核磁共振技術卻因分子太大縮短了自由感應衰減(FID, free induction decay),造成了傅立葉轉換(FT, Fourier Transform)後的半高寬上升,使得光譜的訊雜比變差。在過去十年間,核磁共振領域發展出利用分離式內含蛋白(split INTEIN)將蛋白質做部分片段的同位素標定,再利用核磁共振原理進行蛋白質結構的特性測量。因此,分離式內含蛋白在這項技術中扮演重要的角色。在這篇論文中,我們選擇有較高蛋白反式剪接(PTS, Protein trans-splicing)反應效率的Nostoc punctiforme (Npu) DnaE內含蛋白做為研究對象。然而,在過去的蛋白質資料庫(PDB, Protein Data bank),沒有任何的分離式內含蛋白的結構,並發現原本的Npu DnaE 內含蛋白因第一個半胱胺酸(Cysteine)被置換成丙胺酸(Alanine)而喪失蛋白反式剪接反應。然而,有功能的分離式內含蛋白的結構能夠使我們了解到分離式內含蛋白在蛋白反式剪接反應過程前後結構的運動方式以及反應介面的反應變化。因此,我們設計出一套可快速且簡潔的方式進行具有功能的分離式內含蛋白的純化,再利用傳統的核磁共振技術計算內含蛋白的結構,並運用ROSETTA結合少量的核磁共振數據,快速且準確的預測完整內含蛋白的結構。
Nuclear magnetic resonance (NMR) is used to determine protein structure in solution. The potential application is usually restricted by the presence of severe line broadening and resonance overlapping in a macromolecule. In the past decade, scientists widely used the strategy of segmental isotope labeling to overcome the problem. By employing split INTEIN to mediate protein trans-splicing (PTS), we can solve the problem by labeling one domain and leaving the other domain unlabeled. There is still no available split INTEIN structure in Protein Data Bank. Therefore, we reported the first solution structure of split INTEIN here. We developed a streamlined method to simply and quickly prepare Npu DnaE split INEIN and used NMR method to determine the solution structure. The demonstrated structure revealed differences to native INTEIN. It helps us to evaluate the interaction within the enzymatic site including the critical residues and the N- and C-terminus. Meanwhile, it might provide structural basis in understanding structural difference before and after PTS reaction. We also setup CS-ROSETTA system to allow us quickly predict the correct fold of native INTEIN based on very limited NOEs constraints. The strategy can dramatically curtail the time of structure determination and meanwhile, compromise the usage between conventional NMR structure determination and computer simulation.