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研究生： 劉振凰
Liu, Tseng-Huang
論文名稱： The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and gold nanoparticle technique
利用原子力顯微鏡和金奈米粒子技術解析雙元體液泡質子傳送焦磷酸水解酶之羧酸端間距
指導教授： 潘榮隆
Pan, Rong-Long
曾繁根
Tseng, Fan-Gang
口試委員:
學位類別： 博士
Doctor
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： 2010
畢業學年度： 98
語文別： 英文
論文頁數： 58
中文關鍵詞： 焦磷酸水解酶 、原子力顯微鏡 、奈米金粒子 、電子顯微鏡
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    • Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis for the storage of ions, sugars, and other metabolites. Dimerization of V-PPase is necessary for full proton translocation function although the structural details of V-PPase within the vacuolar membrane remain uncertain. The C-terminus presumably plays a crucial role in sustaining enzymatic and proton translocating reactions. We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological conditions. V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V-PPase protrusions were 2.8 ± 0.3 and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 and 21.7 ± 3.6 nm, respectively. Moreover, both C-termini of dimeric subunits of V-PPase are on the same side of membrane and close to each other as visualized with antibody and gold nanoparticles against 6□His tag on C-ends of the enzyme. The distance between the V-PPase C-terminal ends was determined to be approximately 4.2 ± 1.4 nm. Thus our study is the first to provide the structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini.

    Table of Contents Abbreviations…………………………………………………………………………….4 Introduction………………………………………………………………………………5 Materials and Methods…………………………………………………………………..9 Cloning, expression, and purification………………………………………………….9 Enzyme assay and protein determination……………………………………………..10 Measurement of proton translocation…………………………………………………11 SDS–PAGE, western analysis and size exclusion gel filtration……………………12 Reconstitution of V-PPase into lipid bilayer………………………………………….13 Immunofluorescence microscopy……………………………………………………14 Immunolabeling of reconstituted V-PPase……………………………………………15 Ni-NTA GNP-labeling of reconstituted V-PPase…………………………………….15 Atomic force microscopy……………………………………………………………15 Molecular volume measurements of membrane proteins…………………………….17 TEM image analysis…………………………………………………………………..18 Results and Discussions………………………………………………………………19 AFM analysis of purified V-PPase adsorbed onto mica……………………………. .19 AFM analysis of V-PPase reconstituted into liposomes……………………………...22 Proximity of V-PPase C-termini in reconstituted membranes……………………… .25 References……………………………………………………………………………….32 Tables and Figures……………………………………………………………………...40 Table 1. Dimensions of free and membrane-bound recombinant V-PPase determined by AFM. ..................................................................................40 Fig. 1. Purification and proton transport activity of V-PPase………………………...41 Fig. 2. AFM analysis of purified V-PPase…………………………………………....42 Fig. 3. Reconstitution of V-PPase into proteoliposomes……………………………..43 Fig.4. High-resolution AFM image of V-PPase dimers in a reconstituted membrane...………………………………………………………………….45 Fig. 5. AFM analysis of V-PPase in a reconstituted lipid bilayer immunolabeled with an anti-His antibody to detect the C-terminal 6×His tag of V-PPase…...46 Fig. 6. AFM analysis of Ni-NTA GNPs bound to the C-terminal 6×His tag of V-PPase…………………………………………………………………….…47 Fig. 7. TEM analysis of Ni-NTA GNP–labeled V-PPase………………………….…49 Fig. 8. A working model of V-PPase…………………………………………………50 Appendix…………………………………………………………………………….…..51 Fig. S1. Size exclusion gel filtration chromatography of V-PPase……………….…..52 Fig. S2. Force-distance curves recorded traced from the reconstituted V-PPase.…....53 Fig. S3. AFM images of liposomes and planar lipid bilayer…………….……………54 Fig. S4. The ratios of volume, amino acid numbers and measured height for cytosolic/lumenal sides of V-PPase..…………………………………………………55 Fig. S5. AFM images of GNPs supported on mica surface…………………………..56 Fig. S6. AFM image simulation of GNPs bound to V-PPase C-termini……………..57

    1 Maeshima M (2000) Vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1465, 37-51.
    2 Tzeng CM, Yang CY, Yang SJ, Jiang SS, Kuo SY, Hung SS, Ma JT & Pan RL (1996) Subunit structure of vacuolar proton pyrophosphatase as determined by radiation inactivation. Biochem J 316, 143-147.
    3 Drozdowicz YM & Rea PA (2001) Vacuolar proton-pyrophosphatases: from the evolutionary backwaters into the mainstream. Trends Plant Sci 6, 206-211.
    4 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, 348-353.
    5 Gordon-Weeks R, Steele SH & Leigh RA (1996) The role of magnesium, pyrophosphate, and their complexes as substrates and activators of the vacuolar H+-pumping inorganic pyrophosphatase (studies using ligand protection from covalent inhibitors). Plant Physiol 111, 195-202.
    6 Walker RR & Leigh RA (1981) Mg2+-dependent, cation-stimulated inorganic pyrophosphatase associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.). Planta 153, 150-155.
    7 Maeshima M (1991) H+-translocating inorganic pyrophosphatase of plant vacuoles: inhibition by Ca2+, stabilization by Mg2+ and immunological comparison with other inorganic pyrophosphatases. Eur J Biochem 196, 11-17.
    8 Baykov AA, Dubnova EB, Bakuleva NP, Evtushenko OA, Zhen RG & Rea PA (1993) Steady-state kinetics of substrate hydrolysis by vacuolar H+-pyrophosphatase. A simple three-state model. FEBS Lett 327, 199-202.
    9 Lin HH, Pan YJ, Hsu SH, Van RC, Hsiao YY, Chen JH & Pan RL (2005) Deletion mutation analysis on C-terminal domain of plant vacuolar H+- pyrophosphatase. Arch Biochem Biophys 442(2), 206-13.
    10 Yang SJ, Ko SJ, Tsai YR, Jiang SS, Kuo SY, Hung SH & Pan RL (1998) Subunit interaction of vacuolar H+-pyrophosphatase as determined by high hydrostatic pressure. Biochem J 331, 395-402.
    11 Maeshima M (1990) Oligomeric structure of H+-translocating inorganic pyrophosphatase of plant vacuoles. Biochem Biophys Res Commun 168, 1157-1162.
    12 Sarafian V, Potier M & Poole RJ (1992) Radiation-inactivation analysis of vacuolar H+-ATPase and H+-pyrophosphatase from Beta vulgaris L. Functional sizes for substrate hydrolysis and for H+ transport. Biochem J 283, 493-497.
    13 Sato MH, Kasahara M, Ishii N, Homareda H, Matsui H & Yoshida M (1994) Purified vacuolar inorganic pyrophosphatase consisting of a 75-kDa polypeptide can pump H+ into reconstituted proteoliposomes. J Biol Chem 269, 6725-6758.
    14 López-Marqués RL, Pérez-Castiñeira JR, Buch-Pedersen JB, Marco S, Rigaud JL, Palmaren MG & Serrano A (2005) Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritina: A "Hot-Solve" method for isolation of recombinant thermophilic membrane proteins. Biochim Biophys Acta 1716, 69-76.
    15 Dufrene YF & Lee GU (2000) Advances in the characterization of supported lipid films with the atomic force microscope. Biochim Biophys Acta 1509, 14-41.
    16 Rinia HA & de Kruijff B (2001) Imaging domains in model membranes with atomic force microscopy. FEBS Lett 504, 194-199.
    17 Yuan C & Johnston LJ (2001) Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers. Biophys J 81, 1059-1069.
    18 Slade A, Luh J, Ho S & Yip CM (2002) Single molecule imaging of supported planar lipid bilayer-reconstituted human insulin receptors by in situ scanning probe microscopy. J Struct Biol 137, 283-291.
    19 Saslowsky DE, Lawrence J, Ren X, Brown DA, Henderson RM & Edwardson JM (2002) Placental alkaline phosphatase is efficiently targeted to rafts in supported lipid bilayers. J Biol Chem 277, 26966-26970.
    20 Milhiet PE, Giocondi MC, Baghdadi O, Ronzon F, Roux B & LeGrimellec C (2002) Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts. EMBO Rep 3, 485-490.
    21 Liu LN, Aartsma TJ & Frese RN (2008) Dimers of light-harvesting complex 2 from Rhodobacter sphaeroides characterized in reconstituted 2D crystals with atomic force microscopy. FEBS J 275, 3157-3166.
    22 Bahatyrova S, Frese RN, Siebert CA, Olsen JD, van Der Werf KO, van Grondelle R, Niederman RA, Bullough PA, Otto C & Hunter CN (2004) The native architecture of a photosynthetic membrane. Nature 430, 1058-1062.
    23 Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A & Palczewski K (2003) Atomic-force microscopy: Rhodopsin dimers in native disc membranes. Nature 421, 127-128.
    24 Hung SH, Chiu SJ, Lin LY & Pan R L (1995) A cDNA clone encoding the A subunit of the vacuolar H+-ATPase from etiolated mung bean seedlings. Plant Physiol 109, 1125-1127.
    25 Hsiao YY, Van RC, Hung SH, Lin HH & Pan RL (2004) Roles of histidine residues in plant vacuolar H+-pyrophosphatase. Biochim Biophys Acta 1608, 190-199.
    26 Kim EJ, Zhen RG & Rea PA (1995) Site-directed mutagenesis of vacuolar H+-pyrophosphatase: necessity of Cys634 for inhibition by maleimides but not catalysis. J Biol Chem 270, 2630-2635.
    27 Fiske CH & Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66, 375– 400.
    28 Wang MY, Lin YH, Chow WM, Chung TP & Pan RL (1989) Purification and characterization of tonoplast ATPase from etiolated mung bean seedlings. Plant Physiol 90, 475-481.
    29 Bradford M (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.
    30 Rea PA & Turner JC (1990) Tonoplast adenosine triphosphatase and inorganic pyrophosphatase. In Methods Plant Biochem, vol. 3 (Lea PJ, Ed.), pp 385-405. Academic Press, London.
    31 Rottenberg H (1979) The measurement of membrane potential and deltapH in cells, organelles, and vesicles. In Methods Enzymol, vol. 55 (Fleischer S & Packer L, Eds.), pp 547-569. Academic Press, New York.
    32 Clerc S & Barenholz Y (1998) A quantitative model for using acridine orange as a transmembrane pH gradient probe. Anal Biochem 259, 104-111.
    33 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 222, 680-685.
    34 Lin H, Zhu YJ & Lal R (1999) Amyloid beta protein (1-40) forms calcium-permeable Zn2+-sensitive channel in reconstituted lipid vesicles. Biochemistry 38, 11189-11196.
    35 Schneider S, Folprecht G, Krohne G & Oberleithner H (1995) Immunolocalization of lamins and nuclear pore complex proteins by atomic force microscopy, Pflügers Arch. 430, 795-801.
    36 Edstrom RD, Meinke MH, Yang R, Yang X, Elings V & Evans DF (1990) Direct visualization of phosphorylase-phosphorylase kinase complexes by scanning tunneling and atomic force microscopy. Biophys J 58, 1437-1448.
    37 Schneider SW, Lärmer J, Henderson RM & Oberleithner H (1998) Molecular weights of individual proteins correlate with molecular volumes measured by atomic force microscopy. Pflügers Arch 435, 362-367.
    38 Geisse NA, Cover TL, Henderson RM & Edwardson JM (2004) Targeting of Helicobacter pylori vacuolating toxin to lipid raft membrane domains analysed by atomic force microscopy. Biochem J 381, 911-917.
    39 Gallová J, Uhríková D, Islamov A, Kuklin A & Balgavý P (2004) Effect of cholesterol on the bilayer thickness in unilamellar extruded DLPC and DOPC liposomes. Gen Physiol Biophys 23, 113-128.
    40 Hörber JKH & Miles MJ (2003) Scanning probe evolution in biology. Science 302, 1002-1005.
    41 Engel A & Müller DJ (2000) Observing single biomolecules at work with the atomic force microscope. Nature Struct Biol 7, 715-718.
    42 Rapp M, Seppälä S, Granseth E & von Heijne G (2007) Emulating membrane protein evolution by rational design. Science 315, 1282-1284.
    43 Brekke OH, Michaelsen TE & Sandlie I (1995) The structural requirements for complement activation by IgG: does it hinge on the hinge? Immunol Today 16(2), 85-90.
    44 Takasu A, Nakanishi Y, Yamauchi T & Maeshima M (1997) Analysis of the substrate binding site and carboxyl terminal region of vacuolar H+-pyrophosphatase of mung bean with peptide antibodies. J Biochem 122, 883-889.

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