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研究生:李昆謀
研究生(外文):Li, Kun Mou
論文名稱:探討質子泵焦磷酸水解酶核心穿膜區在質子轉運過程中扮演的角色
論文名稱(外文):Role of Core Transmembrane Domains of H+-PPase in Proton Translocation
指導教授:孫玉珠
指導教授(外文):Sun, Yuh Ju
學位類別:博士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:71
中文關鍵詞:綠豆離子泵焦磷酸水解酶質子泵
外文關鍵詞:VrH+-PPaseMembrane-embedded pyrophosphataseH+-pumping
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離子泵焦磷酸水解酶藉由水解焦磷酸來帶動質子或/和鈉離子的轉運通過細胞質膜或液泡膜。依據已解析的綠豆質子泵焦磷酸水解酶晶體結構,其具有16 個穿膜螺旋(16 TMs)並由核心的TMs 5/6/11/12/15/16建構離子轉運途徑。綠豆質子泵焦磷酸水解酶離子轉運途徑上TMs 5/6/16的關鍵性胺基酸R242/D294/K742/E301已經被明確定義出來,但是詳細的離子轉運機制尚未明朗。在本研究中,我們解析了綠豆質子泵焦磷酸水解酶結合單一磷酸鹽和兩磷酸鹽的複合物晶體分子結構。從內圈TMs 5/6和外圈TMs 13/14中得知,焦磷酸結合區的構形變化是透過在細胞質區穿膜螺旋的移動來調控。自綠豆質子泵焦磷酸水解酶的疏水性閘門胺基酸TM5之T228和TM12之L555突變實驗中,發現有水分子出現在T228D和L555K突變體結構的疏水性閘門區域。這顯示綠豆質子泵焦磷酸水解酶的疏水性閘門有可能被水合化進而允許質子通過閘門然後進入出口通道。另外,在綠豆質子泵焦磷酸水解酶的出口通道胺基酸TM5之E225突變實驗中,顯示E225S 和E225H突變體結構在出口通道終端靠近液泡腔區都保有與TM12之R562的交互作用。綜觀,TM5上必需性質子攜帶者R242接受來自上方TMs 6/16之D287/D731的質子來作為質子轉運的入口,以及E225與R562所形成的作用力落在離子轉運的出口。這顯示R242和R562可能分別作為質子轉運的起始者和終端者。
Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes PPi to drive the transportation of H+ or/and Na+ across plasma/vacuolar membranes. M-PPase is composed of 16 transmembrane helices (TMs) with the ion translocation pathway constructed by core TMs 5/6/11/12/15/16. The key residues R242/D294/K742/E301 in TMs 5/6/16 in proton translocation pathway of Vigna radiata H+-pumping M-PPase (VrH+-PPase) have been identified. However, the detailed ion transport mechanism is still unclear. In this study, the crystal structures of the VrH+-PPase-2Pi and VrH+-PPase-Pi complexes were determined. It suggests that opening and closing of the PPi-binding pocket are mediated by motions in inner TMs 5/6 and outer TMs 13/14. From the mutagenesis study of T228D in TM5 and L555K in TM12 at the hydrophobic gate of VrH+-PPase, water molecules were observed at the hydrophobic gates of T228D and L555K mutants’ structures. It suggests that the hydrophobic gate of VrH+-PPase could potentially be hydrated to allow the proton passing through the exit channel. Furthermore, the mutagenesis study of E225 in TM5 at the exit channel of VrH+-PPase shows that E225S and E225H mutants’ structures preserve the essential interaction of E225 with R562 in TM12 at the end of the exit channel to the lumen. Overall, the essential proton carrier R242 in TM5 accepts the proton from D287/D731 in TMs 6/16 at the entrance of proton transport, and the salt-bridge of E225-R562 is located at the exit of proton release. It proposes that R242 and R562 might act as the initiator and terminator of proton translocation, respectively.
SUMMARY I
中文摘要 II
謝誌 III
ABBREVIATIONS VI
CHAPTER 1. INTRODUCTION 1
CHAPTER 2. EXPERIMENTAL PROCEDURES 8
2.1 Expression of VrH+-PPase 8
2.2 Microsome isolation and protein purification of VrH+-PPase 9
2.3 Site-directed mutagenesis 10
2.4 PPi-hydrolytic activity 11
2.5 Crystallization and structure determination 11
2.6 Reconstitution of VrH+-PPase into liposome 14
CHAPTER 3. RESULTS and DISUCUSSION 16
3.1 Crystal structure of 2Pi-bound VrH+-PPase 16
3.2 Immediate-product-bound state of VrH+-PPase 17
3.3 Crystal structure of Pi-bound VrH+-PPase 19
3.4 Structural changes between Pi- and IDP-binding VrH+-PPase 20
3.5 Inner-outer-dimer interface interaction by TM5, TM13 and neighboring TM10 21
3.6 Releasing salt-bridge network by TM 5 and TM12 23
3.7 The closure of the PPi-binding pocket upon substrate binding 24
3.8 The role of essential tryptophan in TM15 26
3.9 Proteoliposome of VrH+-PPase 28
3.10 Hydration of hydrophobic gate regulated by L555 28
3.11 Proton translocation checked by T228 at the exit channel 31
3.12 Proton release mediated by salt bridge E225-R562 32
3.13 Comparison of the H+/Na+ translocation specific residues 35
3.14 Q301 at the ion transport pathway 37
CHAPTER 4. CONCLUSION 40
TABLES 41
Table 1. Mutagenic standard primers of mutated residues 41
Table 2. Data collection and refinement statistics of 2Pi- and Pi-bound VrH+-PPase complexes 42
Table 3. X-ray diffraction data and refinement statistics of E225S/H, T228D, E301Q and L555M/K mutants 43
Table 4. Mutagenesis of residues in the monomer-monomer interface 44
Table 5. Detailed conformational change of TM5 and TM12 44
FIGURES 45
Figure 1. Overview of M-PPase. 45
Figure 2. Overall structures of 2Pi- and Pi-bound VrH+-PPase complexes. 47
Figure 3. Comparison of three ligands in VrH+-PPase. 48
Figure 4. Comparison of Pi-bound with IDP-bound VrH+-PPase complexes. 49
Figure 5. Conformational change from TM5TM13neighboring TM10. 50
Figure 6. The salt-bridge network of inner six core TMs. 51
Figure 7. The closure of PPi-binding pocket of M-PPase upon substrate binding. 52
Figure 8. The five states of catalytic cycle of VrH+-PPase. 53
Figure 9. The highly conserved Trp on TM5. 54
Figure 10. Reconstitution of VrH+-PPase into liposome. 55
Figure 12. Multiple sequence alignment of M-PPase. 59
Figure 13. Electron density maps of of M555 of L555M mutant and K555 of L555K mutant at the hydrophobic gate. 61
Figure 14. Comparison of the L555M/K mutants with the VrH+-PPase -2Pi complex. 62
Figure 15. Electron density maps of D228 of T228D mutant, S225 of E225S mutant and H225 of E225H mutant at the exit channel. 63
Figure 16. Comparison of the T228D mutant with VrH+-PPase-2Pi complex and L555K mutant. 64
Figure 17. Comparison of the E225S/H mutants with the VrH+-PPase -2Pi complex. 65
Figure 18. Comparison of the E301Q mutant with the VrH+-PPase -2Pi complex. 66
REFERENCES 67
PUBLICATION LIST 71

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