稻米花藥非特異性脂質傳輸蛋白質之純化、特性與功能研究

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研究生：	陳詩尹 Chen, Shih-Yin
論文名稱：	稻米花藥非特異性脂質傳輸蛋白質之純化、特性與功能研究 Purification, Characterization and Functional Studies of Nonspecific Lipid Transfer Proteins in Rice Anther
指導教授：	呂平江 Lyu, Ping-Chiang
口試委員:	林彩雲黃煥中
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：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	61
中文關鍵詞：	脂質運輸蛋白
外文關鍵詞：	lipid transfer protein
相關次數：	點閱：4 下載：0
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植物非特異性脂質傳輸蛋白質是一群分子量小且多為鹼性的蛋白質。依照他們分子量的差異主要被分為兩種類型，第一型(nsLTP1)的分子量約九千道耳吞(9KDa)，第二型(nsLTP2)的分子量約七千道耳吞(7KDa)。而在近期的研究中，一些花藥特異表現蛋白質因為和非特異性脂質傳輸蛋白質具有序列上的同源性，因此被定義成第三型非特異性脂質傳輸蛋白質。然而，第三型非特異性脂質傳輸蛋白質的脂質傳輸及與脂質結合特性都尚未被研究。在本篇論文中，我們探討兩個被認為和雄性花藥不孕相關的非特異性脂質傳輸蛋白質，分別是LTP49 (第二型, Os01g49650)以及LTP43 (第三型，Os08g43290)。從我們的實驗結果發現，LTP43和LTP49均展現出高度的熱穩定性。而在化學變性實驗中，LTP43的穩定性(Cm>6M GdnHCl) 則高於LTP49 (Cm ~3.2M GdnHCl)。此外，我們也研究這兩個蛋白質的脂質結合與傳輸功能上的生物活性。從結果可以發現LTP49具有與脂質結合及傳輸脂質的活性；相反的，LTP43則不具此活性。從螢光實驗分析，我們推測LTP43的疏水性空腔(hydrophobic cavity)可能較小或此空腔被包埋於蛋白質結構中。此外，我們利用電腦計算模擬出LTP49和脂質之複合體結構且發現這些脂質確實都會結合到LTP49的疏水性空腔中。近期內，我們也將利用核磁共振解出這兩個蛋白質的結構，進一步去探討非特異性脂質傳輸蛋白質之結構與功能的相關性。

Plant non-specific lipid transfer proteins (nsLTPs) are small and basic proteins. They are mainly divided into two subfamilies according to their molecular masses: nsLTP1 (9-kDa) and nsLTP2 (7-kDa). Recently, some anther-specific proteins have considerable homologies with plant nsLTPs, and have been classified as nsLTP3. However, the lipid transfer ability and biophysical properties of nsLTP3 is still unclear. In this study we focused on a nsLTP2 (Os01g49650, abbreviation LTP49) and a nsLTP3 (Os08g43290, abbreviation LTP43), which were reported to be involved in anther male sterility. They both exhibited highly structural stabilities against temperature (up to96℃) from the results of circular dichroism (CD) spectroscopy. In chemical denaturation experiments, a higher concentration of GdnHCl was required to denature LTP3 (Cm > 6M) than the concentration of LTP49 (Cm ~3.2M). Furthermore, the functional assays of lipid binding and lipid transfer activities were performed. The results showed that LTP49 displayed both lipid binding and lipid transfer activities while LTP43 could not interact with lipid molecules. We proposed that the hydrophobic cavity of LTP43 might be small or buried in the structure from the fluorescence data. Besides, the docking model was constructed to mimic the LTP49-lipid complex structure and all the results suggested that some lipids could bind into the hydrophobic cavity of LTP49. In the near future, several 2D and 3D NMR spectra has been carried out and the solution structures of these two nsLTP would be determined to further figure out the relationships between structure and binding/transfer activity of nsLTPs.

Contents
Contents    1
Abstract    3
中文摘要    4
Abbreviations    5
Chapter 1. Introduction    6
1.1 Plant nonspecific lipid transfer proteins (nsLTP)    6
1.2 Multiple biological functions of nsLTPs    6
1.3 Structural characteristics of nsLTPs    7
1.4 Previous study in our laboratory    7
1.5 The theme of this thesis    8
Chapter 2. Materials and Method    10
Material    10
2.1 Expression and Purification of nsLTPs    11
2.2 Mass analysis    12
2.3 Quantification of protein concentration    12
2.4 Circular Dichroism (CD) Experiments    13
2.5 Lipid Transfer Assay    14
2.6 Lipid Binding Assay    14
2.7 Nuclear Magnetic Resonance (NMR) Experiments    15
2.8 Molecular Modeling and Docking    15
Chapter 3. Result and Discussion    17
3.1 Expression and Purification    17
3.2 Secondary Structure of nsLTPs    18
3.3 Stability Comparison of LTP49 and LTP43    18
3.4 NMR Spectroscopy    19
3.5 Lipid Transfer Assay    20
3.6 Ligand Binding Assay    20
3.6.1 ANS Competition Assay    20
3.6.2 Isothermal Titration Calorimeter (ITC) Experiments    21
3.6.3 1H-15N HSQC Titration    22
3.7 Molecular Modeling and Docking    22
3.8 LTP43 is a lipid transfer protein or not?    23
Chapter 4. Conclusion    25
Chapter 5. The Attached Tables and Figures    27
Table 1. The property of nonspecific lipid transfer proteins which we discussed in this study.    27
Table 2. The list of lipid molecules we used in this study.    28
Figure 1. Construction of the recombinant plasmid, pET32a-LTP49, and SDS-PAGE of expression and purification.    29
Figure 2. Construction of the recombinant plasmid, pET32a-LTP43 and SDS-PAGE of expression and purification.    30
Figure 3. HPLC profile of LTP49 and LTP43.    31
Figure 4. Mass spectra of LTP49 and LTP43.    32
Figure 5. Sequence alignments of structured nsLTP2s with LTP49 and LTP43.    33
Figure 6. Far-UV CD spectra of LTP49 and LTP43.    34
Figure 7. The thermal denaturation curves of nsLTP49 and nsLTP43.    35
Figure 8. Chemical denaturation curves of the LTP49 and LTP43.    36
Figure 9. Chemical denaturation curves of the nsLTP49 and nsLTP43.    37
Figure 10. pH titration of LTP49 and LTP43.    38
Figure 11. 2D-TOCSY and 2D-NOESY spectra of LTP49 and LTP43    39
Figure 12. pyrPtdCho and Myr2PtdGro    40
Figure 13. Lipid transfer assay of LTP49, LTP43 and nsLTP2.    41
Figure 14. Lipid binding assay by ANS competitive experiment.    42
Figure 15. The diagram of exine synthesis.    43
Figure 16. Lipid binding assay by ANS competitive experiment.    44
Figure 17. Ligands titration with LTP49    45
Figure 18. Ligands titration with LTP43    46
Figure 19. Calorimetric titrations of LTP49 and LTP43 with LysoPC14.    47
Figure 20. The 1H-15N HSQC spectra of LTP49 and LTP43.    48
Figure 21. The 1H-15N HSQC spectra of LTP49 titration with different concentrations of LysoPC14    49
Figure 22. Molecular model and Ramachandran Plot analysis of nsLTP2 and LTP49    50
Figure 23. Molecular docking models    51
Figure 24. The residues on LTP49 and nsLTP2 which interact with ligands    52
Table 3. The residues on LTP49 and nsLTP2 which interact with LysoPC14    53
Table 4. The residues on LTP49 and nsLTP2 which interact with Lauric acid    53
Figure 25. Hydrophobic regions comparison of LTP49 and LTP43.    54
Appendix 1    55
Appendix 2    56
Reference    57

                                

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