內含子 (Intein) 具有蛋白質剪接 (Protein splicing) 和切割 (Cleavage reaction) 的功能，常被應用於蛋白質工程，包含蛋白質的放射性標定、蛋白質純化、蛋白質修飾作用和蛋白質的半合成。近年來，內含子被廣泛的應用。Nostoc punctiforme DnaE intein (Npu DnaE intein)，一種來自cyanobacterium Synechocystis的內含子，它被包含在DnaE蛋白內。Npu DnaE 內含子具有高效的反式剪切 (Trans-splicing)及C端切割 (C-terminal cleavage)活性。在我們先前的研究中，我們發現隨著鹽濃度的提升，內含子的切割反應會被抑制。於是，我們想要深入了解鹽是如何調控內含子的切割活性。我們準備了兩種Npu DnaE內含子，完整式內含子 (One-fragment intein) 及分離式內含子 (Split intein)，並利用核磁共振儀 (NMR) 來進行研究。我們進行了化學位移擾動 (Chemical shift perturbation)、縱向弛豫 (Longitudinal relaxation) 和橫向弛豫 (Transverse relaxation)等實驗。藉由實驗結果得知，在高鹽環境下，內含子的翻滾時間 (Tumbling time)增加了，代表可能由於內含子結構內的鹽橋 (Salt bridge) 被破壞而使蛋白質結構擴張。並且，我們發現參與鹽橋和鹽橋附近的殘基其中的氮-氫鍵結 (N-H bond) 振動變快，這可能由於高鹽環境破壞了鹽橋而導致。也發現在高鹽環境下，內含子結構中有些位置可能有結構交換 (Conformational exchange) 。鹽橋可能幫助維持內含子的結構，而蛋白質的摺疊完整性常與活性的完整相關。綜上所述，我們認為這些參與鹽橋的殘基，很有可能成為調控內含子的C端切割活性的熱點。

Intein, Intron-liked protein, a protein segment which can undergo splicing reaction, N- and C-terminal cleavage reactions. Intein plays a popular tool in protein engineering, and has been applied in several ways such as production of recombinant protein, protein labeling, protein purification and protein semi-synthesis. Nostoc punctiforme DnaE intein (Npu DnaE intein), a native split intein, was discovered from cyanobacterium Synechocystis, and exhibited remarkable activity of trans-splicing. In our previous study, we discovered that C-terminal cleavage reaction was inhibited by high salt concentration. Thus, we expected a salt-mediating method to regulate the C-terminal cleavage reaction of Npu DnaE intein. We prepare two types of Npu DnaE intein, one-fragment and split-type, and employed NMR to monitor conformation and dynamics difference. Based on chemical shift difference, we identify residues with significant perturbations which participating in salt bridge. Furthermore, the longitudinal relaxation (T1) show the increased tumbling time in the presence of high concentration of salt, implying the overall intein structure extended. In transverse relaxation (T2) experiment, many residues of one-fragment intein report backbone fluctuation on N-H bonds. We also found some residues containing conformational exchange. We conclude high-salt environment disrupts salt bridge and further decreases structural stability of Npu DnaE intein. The information report potent hot-spot to modulate intein C-terminal cleavage reaction by salt.

中文摘要 1
Abstract 2
Abbreviations 3
1 Introduction 4
1.1 Background of intein 4
1.2 Nostoc punctiforme DnaE intein (Npu DnaE intein) 6
1.3 The salt bridges in Npu DnaE intein 7
1.4 The C1G mutant intein prohibits splicing and N-terminal cleavage reaction 8
1.5 Nuclear magnetic resonance and protein dynamics experiments 8
1.6 The aim of this study 9
2. Materials and methods 22
2.1 Recombinant protein expression and isotope labelling 22
2.2 Protein Purification 23
2.3 NMR sample preparation 23
2.4 Backbone assignment and chemical shift perturbation 24
2.5 T1 and T2 relaxation measurements 24
3 Results 26
3.1 Purification of Npu DnaE intein 26
3.2 Backbone assignment 26
3.3 Chemical shift perturbation 27
3.3-1 Chemical shift perturbation of one-fragment Npu DnaE intein 27
3.3-2 Chemical shift perturbation of split Npu DnaE intein 28
3.4 Longitudinal relaxation 29
3.4-1 Longitudinal relaxation of one-fragment Npu DnaE intein 30
3.4-2 Longitudinal relaxation of split Npu DnaE intein 31
3.5 Transverse relaxation 32
3.5-1 Transverse relaxation of one-fragment Npu DnaE intein 32
3.5-2 Transverse relaxation of split Npu DnaE intein 33
4 Discussion 44
5 Conclusion 46
References 47

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