扭結蛋白是一種特殊的蛋白家族，其可以折疊成不同複雜程度的拓樸結結構。YibK是一種屬於甲基轉移酶家族的三葉型扭結蛋白。為了要研究蛋白扭結對於結構、折疊及功能的重要性，我們基於YibK建構出環形序列重組突變體CP82。YibK的序列被重新排列，其原始的 N- 和 C- 端被連接，並在結上形成新的開口作為新的N- 和C- 端。利用X光晶體繞射及小角度X光散射 (SAXS) 可以證實環形序列重組不會顯著破壞YibK的三維空間結構。儘管總體結構具相似性，藉由圓二色光譜儀及螢光光譜儀偵測到CP82顯現出熱穩定性及化學穩定性的下降。此外，藉由氫氘交換質譜儀可以在高的結構解析度下觀察到CP82的不穩定效應。重要的是，YibK的紐結區域相關於甲基轉移酶活性之輔因子的鍵結，CP82中扭結的解開完全破壞與輔因子鍵結的能力。我們的實驗結果表明，環形序列重組不會破壞YibK的總體結構，但在摺疊穩定性和功能上具有顯著的影響。

Knotted proteins are a special family of proteins that can fold into topological knots with different complexities. YibK is a trefoil-knotted protein belonging to a superfamily of methyltransferases. To investigate the importance of a protein knot to the structure, folding and function, we generated a circular permutant, CP82, based on YibK. The sequence of YibK was rearranged such that the original N- and C- termini were linked and a new opening was introduced at knotting loop to form the new N- and C- termini. The circular permutation (CP) did not significantly perturb the three-dimensional structure of YibK, which was confirmed by X-ray crystallography and small angle X-ray scattering (SAXS). Despite the overall structural similarity, CP82 exhibited a clear reduction in the thermal and chemical stabilities monitored by far-UV CD and intrinsic fluorescence spectroscopy. Furthermore, the destabilization effect of CP82 was observed at a high structural resolution by hydrogen-deuterium exchange mass spectrometry (HDX-MS). Importantly, the knotted region of YibK is responsible for cofactor binding required for its methyltransferase activity. The knotting loop opening in CP82 completely abolished the cofactor binding capacity. Our experimental results indicated that, while CP did not perturb the overall structure of YibK, it generated profound impacts on the folding stability and function.

中文摘要 i
Abstract ii
誌謝 iii
List of Figures and Tables vi
Abbreviations ix
Chapter 1. Introduction 1
1.1 Knotted proteins 1
1.2 SPOUT superfamily 2
1.3 Trefoil knotted protein YibK 5
1.4 Evolutionary conservation 6
1.5 Circular permutation 8
1.6 Aim of the study 9
1.7 Experimental flowchart 10
Chapter 2. Materials and Methods 11
2.1 Circular permutation 11
2.1.1 CP sites selection 11
2.1.2 Construction of circular permutation protein plasmids 13
2.2 Protein expression and purification 15
2.2.1 Small-scale expression test 15
2.2.2 Large-scale expression for production 15
2.2.3 Protein purification 16
2.2.4 Protein refolding 19
2.2.5 Mass spectrometry 20
2.3 Structural analysis 20
2.3.1 Secondary structure analysis by far-UV CD spectroscopy 20
2.3.2 Size-exclusion chromatography coupled with multiangle light scattering (SEC-MALS) 21
2.3.3 Small angle X-ray scattering (SAXS) 21
2.3.4 X-ray crystallography 21
2.4 Stability analysis 23
2.4.1 Thermal stability 23
2.4.2 Chemical stability 23
2.4.3 Native state folding dynamics 24
2.5 Isothermal titration calorimetry (ITC) 26
Chapter 3. Results 27
3.1 Protein Expression and Purification of CPs 27
3.2 Structural analysis 29
3.2.1 Secondary structure 29
3.2.2 Dimerization 30
3.2.3 Global conformation 31
3.2.5 Structural comparison 33
3.3 Folding stability 40
3.3.1 Thermal stability 40
3.3.2 Chemical stability 42
3.3.3 Native state folding dynamics 44
3.4 Co-factor binding capacity assessed by ITC 47
Chapter 4. Discussions 48
4.1 Comparison of CPs 48
4.2 Structural comparison of CP82 with WT 48
4.3 Functional role of protein knot 51
4.4 Protein folding stability 55
Chapter 5. Conclusion 56
Bibliographies 57
Appendices 60

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