Bax是一種參與細胞凋亡過程的蛋白質,其中作用機制已經有完整的研究,但是Bax本身的穩定性和變性性質卻很少人探討,因此這篇論文會著重在探討Bax蛋白的穩定性和變性性質。本篇論文會以電子自旋共振光譜儀、旋光光譜儀和Thermofluor來探討Bax在熱和化學作用下的性質,期盼能從根本上了解Bax蛋白質 。

經由電子自旋共振光譜儀的分析,Bax可以分成兩區域：N-terminal和C-terminal。N-terminal是第1到88號氨基酸,此區域在化學作用下會完全失去結構,而剩下的C-terminal卻仍然保有部份結構。從序列的特性來看,百分之九十的含苯環氨基酸都分布在C-terminal,而這些含苯環氨基酸在距離夠近的情況下會產生交互作用,進而穩定蛋白質結構,這就是Bax在化學變性作用下還能保有部份結構的原因。

旋光光譜儀在熱變性作用下顯示Bax仍然存在二級結構和三級結構,這呼應了前面電子自旋共振光譜儀的論點。再者,Thermofluor利用螢光也量化不同突變點的熱穩定性,發現把含苯環氨基酸取代掉的位點（或是空間上靠近含苯環氨基酸的位點）會使蛋白質的熱穩定性下降特別顯著,因此可以證明含苯環氨基酸之間的交互作用是穩定蛋白質結構很重要的因素。

綜合以上, Bax因為有含苯環氨基酸之間的作用力,使得不管是在高溫或是高濃度化學變性劑的環境下都能保有部份結構。

Bcl2 associated X (Bax) protein is an apoptotic member in the Bcl-2 protein family and playing a key role in regulating the apoptotic signaling. However, it remains unclear about the stability and unfolding of Bax protein. This study has reported a comprehensive investigation on the stability of Bax and many Bax variants in 0 and 6 M GdnHCl using ESR, CD, and Thermofluor spectroscopy methods. Nitroxide-based spin label (designated as R1 side chain) was used to probe the changes in local environment of protein with the applied chemical and thermal denaturation. ESR spectra were collected from various sites spanning over the nine α-helices of Bax at temperatures −23, 2, and 25 oC, providing information about how local environment of the respective nine helices is changed with the presence of GdnHCl at varying temperatures. Based on the observed site-specific ESR spectral changes, we found that Bax can be divided into two structural regions, of which respond differently to the presence of GdnHCl. In a solution containing 6 M GdnHCl, the N-terminal region (i.e., the first 88 residues from the N terminus, namely the helices from α1 to α3) was found to unfold largely because the corresponding spectra became similar and exhibited a highly mobile state, whereas the C-terminal region (covering from α4 to α9) of Bax was found to retain to some extent its local structures and remain unfolded. Some of the spectra from the C-terminal region even showed an enhanced immobilization of the R1 side chain either in 6 M GdnHCl or at high temperatures, supporting a view that the C-terminal region retains a well-defined tertiary structure against chemical and thermal denaturation. This finding was evidently supported by the results of CD spectroscopy. The far-UV CD spectra confirmed an appreciable amount of α-helical content of Bax in 0 M GdnHCl at high temperatures (90 oC). Most importantly, CD signal in the near-UV region was observed to be significant in magnitude at 2 oC and continuously increase with increasing temperature, suggesting that aromatic interactions are present within Bax structure and playing an important role in stabilizing Bax against the denaturing effect of increasing temperature. The importance of aromatic interactions within Bax structure was further confirmed by structural calculations to show that a total of 12 aromatic pairs were involved in aromatic interactions in the C-terminal region. Moreover, our Thermofluor assay showed that a point mutation in the interior surrounded by α4, α5, and α6 (particularly in the sequence from 99 to 117) in the C-terminal region would largely disrupt the stability of the whole Bax protein because the interior was spatially crowded with residues involved in aromatic-aromatic and cation-pi interactions in the C-terminal region. As such, we conclude that a molten globule state of Bax protein, which is composed of a coil-like denatured segment in the N-terminal region and a dry core in the C-terminal region, can exist as a stable monomer in 6 M GdnHCl at room temperatures or in 0 M GdnHCl at temperatures up to 90 oC (provided that the core sequence from 99 to 117 is not mutated). Because the aromatic interactions contribute significantly to the stabilization of the C-terminal region, Bax was found to largely unfold only at high temperatures in 6 M GdnHCl. Moreover, our results have evidently ruled out the “indirect interaction mechanism” that protein denaturation occurs because denaturants preferentially solvate hydrophobic residues, which in turn destabilize the native structure of a protein. The C-terminal region of Bax is a highly stable molten globule intermediate and is resistant to the denaturation by either heat or 6 M GdnHCl. Only by combining the effects of thermal and chemical denaturation could we unfold the molten globule intermediate of Bax and distinguish its stabilities.

Ⅰ. Outline

1. Introduction 1
1.1. Study Background and Motivation 1
1.2. Protein Unfolding by Urea and Guanidine Hydrochloride 3
1.2.1. The Difference between Urea and Guanidine Hydrochloride 4
1.2.2. Protein is Partially Unfolded by High Concentration of Denaturant 6
2. Theory 7
2.1. Electron Spin Resonance 7
2.1.1. Historical Perspective 7
2.1.2. Paramagnetic Substances 8
2.1.3. Zeeman Effect and Resonance 9
2.1.4. Hyperfine Interaction 12
2.1.5. ESR Spectra 14
2.1.5.1. Site-Directed Spin Labeling 15
2.1.5.2. The Orientation of Nitroxide Spin Label 16
2.1.5.3. The Motion of Nitroxide Spin Label 17
2.1.5.4. Fast Motion 18
2.1.5.5. Slow Motion 18
2.1.5.6. Very Slow Motion 19
2.2. Circular Dichroism 21
2.2.1. Introduction 21
2.2.2. Singular Value Decomposition (SVD) Analysis. 22
2.3. Thermofluor Essay 23
2.3.1. Introduction 23
3. Material and Methods 27
3.1. Preparation of Bax Cysteine Mutants 27
3.2. Mutant Bax Expression and Purification 27
3.3. EPR Sample Preparation 28
3.4. Cw-ESR Spectroscopy 29
3.4.1. Denaturant Experiment 29
3.4.2. Heating Experiment 29
3.5. Thermofluor Sample Preparation 30
3.5.1. Heating Experiment 30
3.5.2. Denaturant Experiment 30
3.6. Monitoring Protein Thermal Stability by QPCR Machine 31
3.7. Circular Dichroism Sample Preparation and Experiments 31
3.7.1. Heating Experiment 32
3.7.2. Titration Experiment 32
4. Results 33
4.1. Bax Unfolding Study from Macroscopic Views 33
4.1.1. Circular Dichroism Analysis 33
4.1.1.1. Thermal Unfolding by CD spectroscopy 33
4.1.1.2. Chemical Unfolding by CD spectroscopy 39
4.1.2. Thermofluor Analysis 40
4.1.2.1. Thermal Unfolding Experiments with Thermofluor Assay 40
4.1.2.2. Thermofluor Assay in the presence of GdnHCl 44
4.2. Bax Unfolding Study in Local Perspective 47
4.2.1. Electron Spin Resonance 47
4.2.1.1. Bax α1 helix 47
4.2.1.2. Bax α2 helix 49
4.2.1.3. Bax α3 and α4 helices 51
4.2.1.4. Bax α5 helix 54
4.2.1.5. Bax α6 helix 57
4.2.1.6. Bax α7 and α8 helices 59
4.2.1.7. Bax α9 helix 61
4.2.1.8. Low Temperature Line Shape in ESR Spectra 63
4.2.2. The Story from ESR CW Experiments 64
4.2.2.1. Hubbell Plots 64
5. Discussion 72
5.1. Equilibrium Issue 72
5.2. Bax remains in a monomeric state after unfolding 74
5.3. Bax have second hydrophobic core 75
5.4 Bax Unfolded Pathway 77
6. Conclusion 79
7. References 81


Ⅱ. Figures
Figure 1.1. Bax activation mechanism proposed by Youle et al. Bax N-terminal was engaged in translocation and oligomerization while α5, α6 and α9 were insert into membrane.1 2
Figure 1.2. Model demonstrating Bax dimerization structure and possible activation mechanism. 2
Figure 2.1 Energy level of free electron in magnetic field B. 10
Figure 2.2. Commonly used nitroxide spin label. 12
Figure 2.3 Energy levels of electron spin in magnetic field B considering interaction with nearby nuclei. 14
Figure 2.4. MTSSL spin label interacts with protein cysteine side chain to form covalent bond. 15
Figure 2.5 Powder spectra of ESR spectroscopy in different orientation. 16
Figure 2.6. The spectra are made by EasySpin simulation. 20
Figure 2.7. Circular dichroism spectra of proteins secondary structure. 22
Figure 2.8. The structure of Sypro Orange, which is not disclosed by manufacturer. 24
Figure 2.9. Thermofluor transition in protein unfolding by heat. Black dot represents Sypro Orange in quenched status. Star means Sypro Orange binding to hydrophobic residues with fluorescence emission. 25
Figure 2.10. 26
Figure 3.1. ESR experiment sample preparation. 29
Figure 3.2. Sample preparation for thermofluor experiment. 31
Figure 4.1. Bax denatured by heating from 2 oC to 90 oC in the step of 2 oC. (Only show the results by 10 oC increment) 34
Figure 4.2. Bax unfolding at 222 nm CD signal. 35
Figure 4.3. Bax melting temperature extrat by SVD. V represents the row vector in VT, which contain crucial information from CD spectra. 35
Figure 4.4. Bax heating experiment near-UV CD spectra. 36
Figure 4.5. Aromatic residues (color red) distribute mainly on Bax C-terminal domain (color blue). 37
Figure 4.6. Mechanisms of breaking disulfied bond. 38
Figure 4.7. Bax heating experiment with TCEP. The strong near-UV signals still exsist. 38
Figure 4.8. Bax was titrating from 0 M to 8 M monitoring 222 nm. 39
Figure 4.9. Bax mutants at 76, 99, 113, 114, 117, 137, and 144 are colored red. They form an important region to stabilize Bax structure. 42
Figure 4.10. Bax WT thermal unfolding transitions in triplicate experiments. 43
Figure 4.11. Bax WT in first derivative. 44
Figure 4.12. The Tm differences between mutants and Bax wild type. 44
Figure 4.13. Bax WT thermofluor data in different concentration of GdnHCl. 45
Figure 4.14. Bax first derivative data. Black arrows point out ambiguous slope changing point. 46
Figure 4.15. Bax α1 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 48
Figure 4.16. Bax α1 helix mutants (Red). 49
Figure 4.17. Bax α2 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 50
Figure 4.18. Bax α2 helix mutants (Red). 51
Figure 4.19. Bax α3 and α4 helices in 0 M (Blue) and 6 M (Red) GdnHCl. 53
Figure 4.20. Bax α3 and α4 helix mutants (Red). 54
Figure 4.21. π-π aromatic interaction between α4 (Blue) and α6 (Red). 54
Figure 4.22. Bax α5 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 55
Figure 4.23. Bax α5 helix mutants (Red). 56
Figure 4.24. π-π stacking among α5 (Blue), (from up-right to left) α7, α8 and α9 (Red). 56
Figure 4.25. Bax α6 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 58
Figure 4.26. Bax α6 helix mutants (Red). 58
Figure 4.27. α6 helix (Blue) π-π interaction with α4 (Red, up) and α5 (Red, down) 59
Figure 4.28. Bax α7 and α8 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 60
Figure 4.29. Bax α7 and α8 helix mutants (Red). 60
Figure 4.30. Bax α9 helix in 0 M (Blue) and 6 M (Red) GdnHCl. 62
Figure 4.31. Bax α9 helix mutants (Red). 62
Figure 4.32. π-π interaction between M188 and F116 resulted to M191R1 slow motion comparing to S4R1 motion. 63
Figure 4.33. Bax mutants in 0M GdnHCl Hubbell plot at 25oC. 65
Figure 4.34. Bax mutants in 6 M GdnHCl Hubbell plot. The plot is separated into three regions. 66
Figure 4.35. Central linewidth (ΔH0-1) with Bax mutants. 67
Figure 4.36. Subtraction central line width by using each residue’s unfolded value as standard. 68
Figure 4.37. Second moments (2)-1 with Bax mutants. 69
Figure 4.38. Subtraction second moment by using each residue’s unfolded value as standard. 69
Figure 5.1. G150R1 unfolded spectra within one day (Blue) and after one week in 25 oC. (Red) 73
Figure 5.2. R94R1 unfolded spectra within one day (Blue) and after two weeks in 25 oC. (Red) 73
Figure 5.3. Carbonic Anhydrase (Blue) was bigger than Bax (Red) in unfolded state. 74
Figure 5.4. F114R1 in 0M GdnHCl heating from 30 oC (Red) to 80oC (Black). 76
Figure 5.5. F114R1 in 6M GdnHCl heating from 30 oC (Red) to 80 oC (Black). 76
Figure 5.6. The most left peak of F114R1 intensity changes from ESR heating-denaturant experiment. 77
Figure 6.1 Bax native structure. 80
Figure 6.2 Bax unfolded structure, in molten globular state. 80




Ⅲ. Tables
Table 4.1. Tm points for all Bax mutants by Thermofluor experiment. Wild type is denoted as WT and Cysteine Free is abbreviated by CF. ΔTm were generated by subtracting Bax wild type as standard. 41
Figure 4.39. Hubbell plot of Bax. The region refers to prior research and experiment spectra. (Blue: 0 M, Red: 6 M GdnHCl ) 70
Table 4.2. Hubbell plot parameters. 70
Table 4.3. Hubbell plot parameters. 71

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