藉由氫氘交換反應探討植物液泡質子傳送焦磷酸水解酶在質子通道出口區域的結構動態

簡易檢索 / 詳目顯示

回結果列表

研究生：	陳品全 Chan, Pin Chuan
論文名稱：	藉由氫氘交換反應探討植物液泡質子傳送焦磷酸水解酶在質子通道出口區域的結構動態 Structure dynamics of exit regions in proton channel of VrH+-PPase as explored by hydrogen-deuterium exchange
指導教授：	潘榮隆 Pan, Rong Long
口試委員:	許員豪 Hsu, Yuan Hao 劉姿吟 Liu, Tzu Yin
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：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	62
中文關鍵詞：	膜蛋白、焦磷酸水解酶、氫氘交換
外文關鍵詞：	VPPase, HDX, hydrogen-deuterium exchange
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

質子傳送焦磷酸水解酶（簡稱VrH+-PPase; EC 3.6.1.1)存在於植物細胞，細菌、古生菌、以及一些原生生物中。其可藉由水解生物次級代謝產物（焦磷酸)來提供能量驅動質子轉運至胞腔內。近年來已經解出了此酵素的3D構型，但是對於它的動態活動仍然所知甚少。因此，我們運用了氫/氘交換反應結合質譜偵測技術觀察出H+-PPase分別與基質類似物及產物結合後的動態結構改變。當蛋白質置於重水溶液中，氘原子會與蛋白主鏈上NH的氫（Backbone amide hydrogens)做交換，進而被質譜儀所偵測。在此，我們藉由氘原子的攝取量來測定出整體結構動態的改變。而在氫氘交換結果中，與基質類似物和產物結合後，具有高度保守性的酵素活性中心及質子傳送通道會形成較為緊縮的結構，許多區段都不易被氘置換。另外，在結合產物磷酸後，質子通道的出口區域會在短時間內有急劇的變化。這些研究能幫助我們更加了解在基質水解期間酵素的詳細動態機制，以助於將來能夠應用在農業或生物上。

The Vigna radiata H+-translocating pyrophosphatases (VrH+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, archaea, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing PPi, the product of anabolic reactions. Although the crystal structure of H+-PPase has been solved recently, the H+ translocation mechanism of H+-PPase is still unclear. Therefore, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to investigate the dynamic of H+-PPase between the resting (apo form), initiated (bound with substrate analogue) and transient states (bound with Pi). When proteins replaced hydrogen in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we determined the structural dynamic and conformational changes via the deuterium uptake. In the highly conserved substrate binding and exit regions, HDX on H+-PPase showed a compact conformation against deuterium exchange upon binding with substrate analogue and product. In addition, the exit region of proton channel exhibited a rapid-changed deuteration in the short time in the presence of phosphate. These results revealed more details about the mechanism of proton translocating by H+-PPase during PPi hydrolyzing, which are useful for biological and agricultural applications.

Introduction    1
Materials and Methods    7
Heterologous expression of H+-PPase in Saccharomyces cerevisiae    7
Isolation of microsome with H+-PPase    8
Purification of H+-PPase    9
H+-PPase concentration measurement    10
Activity assay of H+-PPase    11
SDS-polyacrylamide gel electrophoresis and Western blotting analysis    11
Silver staining of SDS-PAGE gel    12
HDX experiments of H+-PPase in I and T states    13
Pepsin Digestion of H+-PPase    14
Ultra-Performance Liquid Chromatography (UPLC) separation and Q-TOF 
analysis    14
Results and Discussions    16
Expression and purification of H+-PPase    16
Conformational changes of H+-PPase between R and I states    16
Digestion efficiency of H+-PPase    17
Identification of H+-PPase with pepsin digested.    17
Peptide mapping of H+-PPase    18
Using HDX to compare the three states of H+-PPase    18
Comparisons between the two states of H+-PPase    19
Variation of H+-PPase dynamic between R and I states on HDX    19
Variation of H+-PPase dynamic between I and T states on HDX    21
Variation of H+-PPase dynamic between R and T states on HDX    22
Conclusion    24
References    26
Figures    31
Figure 1. Three types of hydrogens found in peptides or proteins    32
Figure 2. The typical experimental workflow of HDX MS    33
Figure 3. The analysis of purified H+-PPase    34
Figure 4. PPi hydrolysis activity of wild-type H+-PPase    35
Figure 5. SDS-PAGE of the H+-PPase with pepsin digestion    36
Figure 6. Western blotting of the H+-PPase with pepsin digestion    37
Figure 7. Identification of H+-PPase with pepsin digested    38
Figure 8. Peptic peptides coverage map of H+-PPase    39
Figure 9. Deuterium exchange of H+-PPase in the absence and presence of IDP 
or Pi     40
Figure 10. Deuteration behavior of H+-PPase in the R, I and T states    41
Figure 11. Differential deuterium uptake of H+-PPase    43
Figure 12. Deuterium exchange of H+-PPase upon binding with IDP    45
Figure 13. Comparison of deuteration behavior of H+-PPase between R and I 
states (binding with IDP) from the cytosolic view    46
Figure 14. Comparison of deuteration behavior of H+-PPase between T and 
I states    48
Figure 15. Comparison of deuteration behavior in the dimer interface of
 H+-PPase between T and I states    49
Figure 16. Comparison of deuteration behavior of H+-PPase between R and 
T states (binding with Pi) from the cytosolic view    50
Figure 17. Comparison of deuteration behavior in the dimer interface of 
H+-PPase between R and T states from the cytosolic view    51
Figure 18. Comparison of deuteration behavior in exit regions of 
H+-PPase between R and T states from the bottom view    52
Figure 19. Transmembrane domain distribution of H+-PPase    53
Figure 20. A presumable working model for H+-PPase    54
Tables    55
Table 1. YPD medium    55
Table 2. TEL solution    55
Table 3. TEL-PEG solution    56
Table 4. CM medium    56
Table 5. The 2-ME washing solution    56
Table 6. YP lysis medium     57
Table 7. Homogenization medium    57
Table 8. A Buffer    58
Table 9. B Buffer    58
Table 10. Storage Buffer (A* Buffer)    59
Table 11. B* Buffer    59
Supplementary    60
Supplementary Figure 1. HDX kinetics of all H+-PPase peptic peptides    60

                                

Asaoka M, Segami S, Maeshima M. (2014). Identification of the critical residues for the function of vacuolar H+-pyrophosphatase by mutational analysis based on the 3D structure. J Biochem 46, 1-12.
Bao AK, Wang SM, Wu GQ, Xi JJ, Zhang JL, Wang CM. (2009). Overexpression of the Arabidopsis H+-PPase enhanced resistance to salt and drought stress in transgenic alfalfa (Medicago sativa L.). Plant Science 176, 232-240.
Baykov AA, Bakuleva NP, Rea PA. (1993). Steady-state kinetics of substrate hydrolysis by vacuolar H+-pyrophosphatase. A simple three-state model. Eur J Biochem 217, 755-762.
Belogurov GA, Lahti R. (2002). A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans. J Biol Chem 277, 49651-4.
Beyenbach KW, Wieczorek H. (2006). The V-type H+ ATPase: molecular structure and function, physiological roles and regulation. J Exp Biol, 209 (Pt 4), 577-589.
Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254.
Coleman J, Blake-Kalff M, Davies E. (1997). Detoxification of xenobiotics by plants: chemical modification and vacuolar compartmentation. Trends Plant Sci 2, 144-151.
Cherian S, Oliveira MM. (2005). Transgenic plants in phytoremediation: recent advances and new possibilities. Environ Sci Technol, 39(24), 9377-9390.
Drozdowicz YM, Kissinger JC, Rea PA. (2000). AVP2, a sequence-divergent, K+-insensitive H+-translocating inorganic pyrophosphatase from Arabidopsis. Plant Physiol, 123 (1), 353-362.
Drozdowicz YM, Rea PA. (2001). Vacuolar H+ pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6, 206–211.
Englander SW, Mayne L, Bai Y, Sosnick TR. (1997). Hydrogen exchange: the modern legacy of Linderstrøm-Lang. Protein Sci 6, 1101-09.
Fiske CH, Subbarow Y. (1925). The colorimetric determination of phosphorous, J Biol Chem 66, 378–400.
Fruton JS. (1970). The specificity and mechanism of pepsin action, Adv. Enzymol. Relat. Areas Mol. Biol 33, 401–443.
Gaxiola RA, Palmgren MG, Schumacher K. (2007). Plant proton pumps. FEBS Lett, 581 (12), 2204-2214.
Gaxiola RA, Regmi K, Hirschi KD. (2016). Moving on up: H+-PPase mediated crop improvement. Trends, 34 (5), 347-349.
Gerstein M, Lesk AM, Chothia C. (1994). Structural mechanisms for domain movements in proteins. Biochemistry 33, 6739-49.
Hirono M, Mimura H, Nakanishi Y, Maeshima M. (2005). Expression of functional Streptomyces coelicolor H+-pyrophosphatase and characterization of its molecular properties. J Biochem, 138 (2), 183-191.
Hsiao YY, Van RC, Hung SH, Lin HH, Pan RL. (2004). Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1608, 190-9.
Hsu SH, Lo YY, Liu TH, Pan YJ, Huang YT, Sun YJ, Hung CC, Tseng FG, Yang CW, Pan RL. (2015). Substrate-induced changes in domain interaction of vacuolar H+-pyrophosphatase. J Biol Chem, 290 (2), 1197-1209.
Huang YT, Liu TH, Lin SM, Chen YW, Pan YJ, Lee CH, Sun YJ, Tseng FG, Pan RL. (2013). Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding. J Biol Chem 288, 19312-20.
Kellosalo J. (2012). The structure and catalytic cycle of a sodium-pumping pyrophosphatase. Science 337, 473-6.
Laemmli UK. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-5.
Li KM, Wilkinson C, Kellosalo J, Tsai JY, Kajander T, Jeuken LJ, Sun YJ, Goldman A. (2016). Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism. Nature Communications, 7.
Lin SM, Tsai JY, Hsiao CD, Huang YT, Chiu CL, Liu MH, Tung JY, Liu TH, Pan RL, Sun YJ. (2012). Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature 484, 399-403.
Liu TH, Hsu SH, Huang YT, Lin SM, Huang TW, Chuang TH, Fan SK, Fu CC, Tseng FG, Pan RL. (2009). The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique. FEBS J 276, 4381-94.
Liu Q, Zhang Q, Burton RA, Shirley NJ, Atwell BJ. (2010). Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice. Plant Mol Biol 72:47–60.
Li Z, Baldwin CM, Hu Q, Liu H, Luo H. (2010). Heterologous expression of Arabidopsis H+‐pyrophosphatase enhances salt tolerance in transgenic creeping bentgrass (Agrostis stolonifera L.). Plant, Cell Environ, 33 (2), 272-289.
Maeshima M. (2000). Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465. 37-51.
Maeshima M. (2001). Tonoplast transporters. Organization and function. Annu Rev Plant Physiol Plant Mol. Biol 52, 469–497.
Marcsisin SR, Engen JR. (2010). Hydrogen exchange mass spectrometry: what is it and what can it tell us? Anal Bioanal Chem 397, 967-72.
Martinoia E, Massonneau A, Frangne N. (2000). Transport processes of solutes across the vacuolar membrane of higher plants. Plant Cell Physiol, 41 (11), 1175-1186.
Martinoia E, Maeshima M, Neuhaus HE. (2007). Vacuolar transporters and their essential role in plant metabolism. J Exp Bot, 58(1), 83-102.
Marty F. (1999). Plant vacuoles. Plant Cell 11, 587-600.
Nakanishi Y, Matsuda N, Aizawa K, Kashiyama T, Yamamoto K, Mimura T, Ikeda M, Maeshima M. (1999). Molecular cloning and sequencing of the cDNA for vacuolar H+-pyrophosphatase from Chara corallina. Biochim Biophys Acta-Biomembranes, 1418 (1), 245-250.
Nakanishi Y, Saijo T, Wada Y, Maeshima M. (2001). Mutagenic analysis of functional residues in putative substrate-binding site and acidic domains of vacuolar H+-pyrophosphatase. J Biol Chem 276, 7654-60.
Pasapula V, Shen G, Kuppu S, Paez‐Valencia J, Mendoza M, Hou P, Chen J, Qiu X, Zhu L, Zhang X, Auld D, Blumwald E, Zhang H, Gaxiola R, Payton P. (2011). Expression of an Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) in cotton improves drought‐and salt tolerance and increases fibre yield in the field conditions. Plant Biotechn J, 9 (1), 88-99.
Rea PA, Kim Y, Sarafian V, Poole RJ, Davies JM, Sanders D. (1992). Vacuolar H+-translocating pyrophosphatases: a new category of ion translocase. Trends Biochem Sci 17, 348-53.
Segami, S., Nakanishi, Y., Sato, M. H., & Maeshima, M. (2010). Quantification, organ-specific accumulation and intracellular localization of type II H+-pyrophosphatase in Arabidopsis thaliana. Plant and cell physiology, 51(8), 1350-1360.
Silva P, Gerós H. (2009). Regulation by salt of vacuolar H+-ATPase and H+-pyrophosphatase activities and Na+/H+ exchange. Plant Signal Behav 4, 718-26.
Tsai JY, Kellosalo J, Sun YJ, Goldman A. (2014). Proton/sodium pumping pyrophosphatases: the last of the primary ion pumps. Curr Opin Struct Biol 27, 38-47.
Yang SJ, Jiang SS, Hsiao YY, Van RC, Pan YJ, Pan RL. (2004). Thermoinactivaion analysis of vacuolar H+-pyrophosphatase. Biochim Biophysica Acta-Bioenergetics, 1656(2), 88-95.
Zhang J, Li J, Wang X, Chen J. (2011). OVP1, a vacuolar H+-translocating inorganic pyrophosphatase (V-PPase), overexpression improved rice cold tolerance. Plant Physiol Biochem 49, 33-38.
Zhang Z, Smith DL. (1993). Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation. Protein Sci 2, 522-531.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文