自然界存在著一種結構特殊的蛋白，他們在蛋白質結構中存在著”扭結” 的構造而被稱為扭結蛋白。扭結所存在的功能以及為什麼摺疊成為這種特別形狀的原因，一直被科學家所探討，在先前研究扭結蛋白YbeA，透過環形重組技術（CP74）解除了扭結蛋白YbeA的扭結。出乎意料的是，YbeA CP74形成了區域交換的二聚體。為了進一步研究如何形成區域交換的二聚體，我們將五個色胺酸分別突變為苯丙胺酸 (W7F、W19F、W48F、W72F和W100F)以通過圓二色光譜內生性螢光以及核磁共振（NMR）探測局部和全局折疊過程。從色胺酸突變到苯丙胺酸（WtoF）的突變體在小角度X射線散射和X射線晶體學分析下，得知YbeA CP74的整體的外觀結構並未顯著改變。而在熱穩定及化學穩定分析中，一些突變蛋白折疊穩定性明顯降低，並且在分析展開過程中發現，除了W100F外其他WtoF突變體都在熱穩定實驗出現了中間態。藉由15N-1H NMR化學穩定性實驗結果，我們推測CP74的蛋白展開始於W100所在的二聚體交界面。總體來說，透過研究WtoF突變體的實驗貢獻，我們得知了每個色胺酸在結構跟摺疊穩定性的不同重要程度、揭開可能的蛋白質展開過程以及發現W100是維持區域交換重要的胺基酸。

The knotted protein is a protein with a special structure. There are topological knots in all the knotted protein structures. The function and folding pathway of the knotted protein have been explored by scientists. In the previous study of knotted function, the trefoil-knotted protein YbeA was recombinant by the protein-engineering techniques circular permutation to create the new recombinant YbeA CP74. Unexpectedly, YbeA CP74 forms a domain-swapped dimer. To further investigate how the domain-swapped dimerization is achieved, we individually replaced the five endogenous tryptophan residues into phenylalanine – W7F, W19F, W48F, W72F, and W100F – to probe local and global folding events by Circular dichroism (CD) spectroscopy, intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopy. While the tryptophan-to-phenylalanine (WtoF) mutations did not significantly alter the native structures of YbeA CP74, according to small angle X-ray scattering and X-ray crystallography, equilibrium thermal and chemical unfolding analyses showed markedly reduced folding stability for some of the variants. All WtoF variants, except W100F, which is highly destabilized, exhibited two distinct thermal unfolding events evidenced by differential scanning calorimetry. Loss of folding cooperativity was also observed. As well as, the results of Circular dichroism spectroscopy and intrinsic fluorescence of chemical stability showed that the stability of α-helix where W100 is located is flexible. By monitoring tryptophan indole 15N-1H NMR correlations as a function of urea concentration, we speculated that the unfolding of CP74 begins at the hinge region of the dimer interface where W100 resides. In summary, by teasing out the spectral contributions of these WtoF mutants, we knew the importance of the structure and folding stability of each tryptophan, uncovered the possible protein unfolding pathway of YbeA CP74 and confirm W100 is a key point on locking YbeA CP74 domain swapping structure.

1. Introduction 1
1.1 Knotted protein 1
1.2 SPOUT superfamily 2
1.3 Circular permutation 2
1.4 YbeA and YbeA CP74 3
1.5 Tryptophan biochemistry of protein 4
1.6 Aim of the study 5
Figure 1.1 Examples of knotted proteins 6
Figure 1.2 Examples of circular permutation. 6
Figure 1.3 Structural alignment of WT and CP74. 7
Figure 1.4 YbeA forms domain swapping dimer after circular permutation. 7
2. Materials and methods 8
2.1 Materials 8
2.2 Construction of YbeA CP74 mutants 9
2.3 Protein expression and purification 9
2.3.1 Expression test 9
2.3.2 Purification 10
2.4 Far-UV CD spectroscopy 12
2.5 Small angle X-ray scattering (SAXS) 13
2.6 X-ray crystallography 14
2.7 Thermal stability: far-UV CD spectroscopy 15
2.8 Thermal stability: differential scanning calorimetry (DSC) 17
2.9 Chemical stability: intrinsic fluorescence spectroscopy 18
2.10 Chemical stability: far-UV CD spectroscopy 19
2.11 Solubility and stability screen of DSF 20
2.12 Nuclear magnetic resonance (NMR) spectroscopy 20
2.13 Protein-related information of YbeA CP74 variants 22
Table 2.1 Primers 24
Table 2.2 Data collection and refinement statistics of crystal. 25
3. Results 26
3.1 Construction, expression and purification of YbeA CP74 and variants 26
3.2 Secondary Structural analysis of far-UV CD spectroscopy 27
3.3 X-ray analysis of CP74 and variants 27
3.3.1 Global conformations of CP74 radiant assessed by SAXS 27
3.3.2 CP74 variants crystal structure comparison 29
3.4 Tryptophan assignment by 2D NMR spectrometer of SOFAST-HMQC 30
3.5 Thermal stability 32
3.5.1 Thermal stability of DSC 32
3.5.2 Thermal stability of CD 33
3.5.3 Solubility and stability screen of Differential scanning fluorimetry (DSF) 34
3.6 Chemical stability 35
3.6.1 Chemical stability of intrinsic fluorescence spectroscopy 35
3.6.2 Chemical stability of CD 35
3.6.3 Chemical stability of CP74 variants monitored by NMR 36
Figure 3.1 Plasmid map of pET21b-YbeA-CP74 39
Figure 3.2 Expression test of YbeA CP74, W7F, W19F, W48F, W72F, and W100F 40
Figure 3.3.1 Purification of CP74 with SDS-PAGE analysis 41
Figure 3.3.2 Purification of W7F with SDS-PAGE analysis 41
Figure 3.3.3 Purification of W19F with SDS-PAGE analysis 42
Figure 3.3.4 Purification of W48F with SDS-PAGE analysis 42
Figure 3.3.5 Purification of W72F with SDS-PAGE analysis 43
Figure 3.3.6 Purification of W100F with SDS-PAGE analysis 43
Figure 3.4.1 Expression test and purification of W100A, W100Y , W100H and W100P 44
Figure 3.4.2 Expression test and purification of W100V, W100G 45
Figure 3.5 Secondary structure comparing of CD 46
Table 3.1 The values of Dmax and Rg collected by SAXS 47
Figure 3.6 The small angle X-ray scattering (SAXS) revealed YbeA CP74 and variants of the structure at the solution state 48
Figure 3.7.1 Crystal and diffraction images pattern of W7F, W48F, and W72F 49
Figure 3.7.2 Crystal structure and structure alignment 50
Table 3.2 The RMSD value of comparing the overall structure of YbeA CP74, W7F, W19F and W72F 51
Figure 3.7.3 Comparison of the crystal structures of YbeA CP74 (yellow), W7F (pink), and W48F (red) 52
Figure 3.7.4 Comparison of the crystal structures of YbeA CP74 (blue) and W7F (cyan) 53
Figure 3.7.5 Comparison of the B-factor of YbeA CP74, W7F, W48F, and W72F 54
Figure 3.8.1 NMR 1D spectroscopy of YbeA CP74 variants 55
Figure 3.8.2 NMR 1D spectroscopy of YbeA CP74 variants 55
Figure 3.8.3 NMR 1D spectroscopy of YbeA CP74 by labeling 19F on tryptophan 56
Figure 3.9 15N-1H SOFAST-HMQC of YbeA CP74 variants tryptophan assignment 57
Figure 3.10.1 Thermal stability compared between YbeA CP74 variants of DSC and CD 58
Figure 3.10.2 Thermal stability compared between YbeA CP74 variants of CD by Gaussians fitting 59
Table 3.3 Thermodynamic parameters of thermal unfolding compared between DSC and CD 60
Figure 3.11 Differential scanning fluorimetry of CP74, W7F, W19F, W48F, W72F, and W100F analyzed by the heat map 61
Table 3.4 Differential scanning fluorimetry of CP74, W7F, W19F, W48F, W72F, and W100F 62
Figure 3.12.1 Equilibrium chemical unfolding of urea-induce wavelength by intrinsic fluorescence spectroscopy and CD 63
Figure 3.12.2 Equilibrium chemical unfolding of intrinsic fluorescence and CD 64
Table 3.5 Chemical melting parameter compared between intrinsic fluorescence and CD 65
Figure 3.13 15N-1H SOFAST-HMQC of YbeA CP74 and its tryptophan variants assignment in 7.2M urea 66
Figure 3.14 Chemical stabilities of each tryptophan in YbeA CP74 by NMR 67
Figure 3.15 YbeA CP74 variants equilibrium chemical unfolding of urea-induce by 15N-1H SOFAST-HMQC 68
4. Discussion 69
4.1 Structural comparison of YbeA and its variants 69
4.2 Stability comparison of YbeA and its variants 69
4.3 Important residue W100, W72, W19 of protein folding stability 71
4.4 The possibly unfolding process of YbeA CP74 73
Table 4.1 The distance of CH–π interactions between V62 and W100, and the hydrogen bonds between E66 and Trp indole-N of W100 74
Figure 4.1 Comparing the YbeA CP74 different chain of the tryptophan side chain 74
Figure 4.2 Interaction of W100 with E66 and V62 from different chains 75
Table 4.2 The distance of methionine-aromatic interaction 75
Figure 4.4 W19 locates in the hydrophobic core 77
Figure 4.5 The possible intermediate structure of CP74 in unfolding process 77
5. Conclusion 78
6. Reference 79

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