支鏈鹽橋對於crammer調節半胱胺酸蛋白酶之影響及蛋白特性分析

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研究生：	何沛霖 He, Pei-Lin He
論文名稱：	支鏈鹽橋對於crammer調節半胱胺酸蛋白酶之影響及蛋白特性分析 Salt bridge Impact on Drosophila melanogaster Crammer for Cathepsin Regulation
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	呂平江蘇士哲楊裕雄
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	62
中文關鍵詞：	支鏈鹽橋、長期記憶
相關次數：	點閱：4 下載：0
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果蠅的crammer，是一個具有79個胺基酸的小蛋白，其功能已經被證實是藉由調節半胱胺酸蛋白酶 (cathepsin) 的活性，進而促使果蠅形成長期記憶.。雖然crammer結構在日前已被發表，但crammer詳細的調控機制目前尚未被明確地指出。因此，本實驗利用點突變的方式結合上生物化學與生物物理等方法，探討salt bridge對於crammer抑制半胱胺酸蛋白酶所造成的功能性影響與結構之間的關聯。藉由丙氨酸取代salt bridge之胺基酸，其中E8A、R28A與R29A三個突變蛋白，會造成蛋白穩定性顯著的降低以及使得crammer喪失正確的折疊能力，進而導致功能性的大幅缺失。根據結構分析，Glu8的改變會造成helix-1的結構不穩定，此外，Arg29的替換也會讓原本會與Arg29作用的Ser5、Glu6與Glu62喪失交互作用的引力，因而造成在helix-1和helix-2間所產生出來的hydrophobic core不穩定，而導致crammer整體結構折疊不完美，進一步影響抑制功能。Arg28處於salt bridge network中心位置，而其有固定helix-2與helix-4方位的功能，並且可讓crammer的C端結構擁有正確的位相，使其能夠有效抑制半胱胺酸蛋白酶。根據此研究，我們對於crammer上關鍵性胺基酸的探討，將增加同類型的抑制劑開發潛力，並且能夠有助於對抗阿茲海默症的治療。

Drosophila melanogaster crammer is a small peptide with 79 amino acids, which involves in long-term memory formation through cathepsin regulation. Although the 3D structure of crammer has been reported, the detailed regulatory mechanism in fruit fly is still unclear. In this study, a site directed mutagenesis approach coupled with biochemical and biophysical methods were used to explore potential roles of the salt bridges in crammer. Alanine substitutions at E8A, Arg28 and Arg29 apparently reduce the thermal stability and alter the protein folding, thus losing their cathepsin inhibitory activities. According to structural analysis, the substitution at Glu8 causes the structural instability of helix-1. Moreover, Arg29 makes close contacts with Asp6 and Asp25 to stabilize helices 1 and 2. These two helices act as an essential scaffold for maintaining the hydrophobic core. Moreover, Arg28 lies on the center of a tri-salt bridge network (Glu24-Arg28-Glu67). This network connects helices 2 and 4 to stabilize the C-terminal orientation of crammer, and to maintain the inhibitory potency of crammer. Accordingly, we have already identified the hot spot residues in crammer, which allows us to expand the potential application for pharmaceutical therapy in the Alzheimer’s disease.

Contents
Abstract    1
中文摘要    2
Abbreviations    3
Chapter 1. Introduction    4
1.1 Long-term memory    4
1.2 Propeptide-like cysteine protease inhibitor-crammer    4
1.3 Cathepsin of cysteine proteases    5
1.4 Functionally and structurally critical charged residues of crammer    6
1.5 The theme of the thesis    7
Chapter 2. Materials and Methods    9
2.1 Construction of recombinant crammer mutant    9
2.2 Protein expression and purification    9
2.3 MALDI-TOF MASS analysis    10
2.4 Tricine SDS PAGE    11
2.5 Quantification of protein concentration    12
2.6 Expression of fly procathepsin B    12
2.7 Isolation and solubilization of procathepsin B inclusion bodies    13
2.8 In vitro folding and autoprocessing of proCTSB    13
2.9 Enzymatic assay of cathepsin B    14
2.10 Quantification of cathepsin B concentration by E-64    14
2.11 Circular Dichroism Spectroscopy    15
2.12 Fluorescence measurements    16
2.13 NMR spectroscopy    16
2.14 Molecular modeling and docking    17
Chapter 3. Results and Discussions    18
3.1 Expression and purification of mutant crammer    18
3.2 The effect of salt bridges forming in crammer on inhibitory potency for Drosophila melanogaster cathepsin B    18
3.3 Study the secondary structure of crammer and mutants by using circular dichroism    19
3.4 Intrinsic fluorescence properties of crammer and double mutant proteins    21
3.5 Discussion on the 1H-15N HSQC spectra of double mutant proteins separately    23
Chapter 4. Conclusions    26
Figures and Tables    27
Figure 1. Multiple sequence alignment of crammer and related proteins    27
Figure 2. The residues involve in salt bridges of crammer    28
Figure 3. Experimental strategies    29
Figure 4. The schematic diagram of the plasmid Crammer-pAED4    30
Figure 5. Standard curve for quantifying protein concentration    31
Figure 6. Expression of crammer and mutants    32
Figure 7. HPLC profile for purification of mutant proteins    33
Figure 8. Mass spectra of mutant proteins    34
Figure 9. Progress curves for inhibitory activity of crammer against cathepsin B    35
Figure 10. Resistant analysis of crammer and mutant proteins against cathepsin B in different concentrations.    36
Figure 11. C72S based double mutants at pH 7    37
Figure12. Far-UV CD spectroscopy examines the secondary structure of crammer and mutant proteins at pH 4 and pH 7    38
Figure 13. The impact of salt bridges on protein structural stability in crammer by thermal denaturation experiments    39
Figure 14. Protein packing analysis by intrinsic fluorescence experiment    40
Figure 15. Superposition of 1H-15N HSQC spectra were recorded at 20℃ and pH 6    41
Figure 16. The overlay of the 1H-15N-HSQC spectra for C72S and double mutant proteins    42
Figure 17. The effect of salt bridge mutants through the 1H-15N-HSQC spectra    43
Figure 18. The importance of Arg28 and Glu67    44
Figure 19. Hydrophobic core in crammer    45
Table 1. Compare the identity of cathepsin B of D. melanogaster with cathepsin of other species    46
Table 2. Sequence of oligonucleotide used for site directed mutagenesis    47
Table 3. Observed molecular weight of mutant proteins correlated with the theoretical molecular weights    48
Table 4. The conclusion of functional assay, protein stability and conformational transition in wild-type crammer and mutants    49
Appendixes    50
Appendix 1. The structural change of crammer in the1H-15N-HSQC spectra38    50
Appendix 2. 1H-15N HSQC of C72S at pH 4 and pH 6    52
Appendix 3. The salt bridge network of procathepsin S55.    53
Reference    54

                                

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