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高速AFMと電子顕微鏡を用いた古細菌オリゴ糖転移酵素の動的構造解析

川﨑, 由貴 KAWASAKI, Yuki カワサキ, ユキ 九州大学

2021.03.24

概要

N 型糖鎖転移反応は、最も重要なタンパク質の翻訳後修飾の 1 つである。オリゴ糖転移酵素は、新生ポリペプチド鎖の糖鎖付加配列を認識し、脂質膜上に組み立てられた糖鎖をアスパラギン残基へ転移する反応を触媒する膜タンパク質酵素である。本研究では、結晶構造解析で得られた構造情報をもとに、高速 AFM や電子顕微鏡を用いて古細菌オリゴ糖転移酵素の構造解析を行った。高速 AFM を用いると、生体分子の構造変化をリアルタイムに観察することができる。高速 AFM観察を行うためには、観察対象とする生体分子の特性に応じて適切な系を用いる必要がある。オリゴ糖転移酵素は膜タンパク質であるため、 AFM の観察ステージへの一定の向きでの固定と脂質二重膜の形成が必要であった。オリゴ糖転移酵素の球状ドメインの構造変化を観察するため、膜貫通領域の細胞質側にビオチン修飾を加えた。AFM の観察ステージ上のマイカ基板にタマビジン二次元結晶を形成し、界面活性剤で可溶化したオリゴ糖転移酵素を球状ドメインが上になるように固定した。オリゴ糖転移酵素固定後に界面活性剤を加えたリポソームをのせて脂質二重膜を形成した。この系を使ってオリゴ糖転移酵素の高さの変化を測定した結果、1.8 nm の振れ幅で二状態を行き来する大きな構造変化が起こることが明らかになった。高さが高い状態を”State 1”, 低い状態を”State 2”としてサバイバルプロットを行うと、単一指数関数の曲線へフィッティングできたことから、オリゴ糖転移酵素の本質的な構造変化を捉えることができたと考える。NMR の解析でもオリゴ糖転移酵素に二状態あることが示唆された。State1 は結晶構造に近い構造で継続時間が長かったのに対し、State2 は結晶構造よりコンパクトな構造で継続時間は短かった。脂質二重膜中にドナー基質である LLO を加えると、State1 が安定化された。AFM で新たに観察された State2 の構造はオリゴ糖転移酵素が酵素反応でできた糖ペプチドをすばやく離して、酵素サイクルを効率的にまわすのに役立つ可能性があると考える。

古細菌オリゴ糖転移酵素の結晶構造解析では、可溶化に界面活性剤が用いられてきた。本研究では、アンフィポールで可溶化した状態とナノディスクの脂質二重膜中に再構成した状態とで電子顕微鏡単粒子解析を行った。その結果、アンフィポールで可溶化した状態のオリゴ糖転移酵素の構造は結晶構造と同等であったが、ナノディスクに埋め込んだオリゴ糖転移酵素の膜貫通領域と球状ドメインの間が開いた構造をとることが明らかとなった。このナノディスク中オリゴ糖転移酵素の構造は結晶構造とも高速 AFM で観察された State2 とも異なる構造であった。

高速 AFM と NMR を用いた動的解析と電子顕微鏡を用いた構造解析により、オリゴ糖転移酵素の膜貫通領域と球状ドメインの配向が非常に可塑的であることが明らかになった。2 つのドメイン間の大きな構造変化がオリゴ糖転移酵素の活性に重要であると考える。

参考文献

[1] M. Aebi, N-linked protein glycosylation in the ER, Biochim Biophys Acta. 1833 (2013) 2430–2437.

[2] Y. Harada, Y. Ohkawa, Y. Kizuka, N. Taniguchi, Oligosaccharyltransferase: A gatekeeper of health and tumor progression, Int. J. Mol. Sci. 20 (2019).

[3] N. Cherepanova, S. Shrimal, R. Gilmore, N-linked glycosylation and homeostasis of the endoplasmic reticulum, Curr. Opin. Cell Biol. 41 (2016) 57–65.

[4] A. Yan, W.J. Lennarz, Unraveling the mechanism of protein N-glycosylation, J. Biol. Chem. 280 (2005) 3121–3124.

[5] A. Helenius, M. Aebi, Roles of N-linked glycans in the endoplasmic reticulum, Annu Rev Biochem. 73 (2004) 1019–1049.

[6] F. Schwarz, M. Aebi, Mechanisms and principles of N-linked protein glycosylation, Curr Opin Struct Biol. 21 (2011) 576–582.

[7] K.F. Jarrell, Y. Ding, B.H. Meyer, S. V Albers, L. Kaminski, J. Eichler, N-linked glycosylation in Archaea: a structural, functional, and genetic analysis, Microbiol Mol Biol Rev. 78 (2014) 304–341.

[8] H. Nothaft, C.M. Szymanski, Bacterial protein N-glycosylation: new perspectives and applications, J Biol Chem. 288 (2013) 6912–6920.

[9] A. Larkin, B. Imperiali, The expanding horizons of asparagine-linked glycosylation, Biochemistry. 50 (2011) 4411–4426.

[10] D.N. Hebert, M. Molinari, In and out of the ER: protein folding, quality control, degradation, and related human diseases, Physiol Rev. 87 (2007) 1377–1408.

[11] N.G. Jayaprakash, A. Surolia, Role of glycosylation in nucleating protein folding and stability, Biochem. J. 474 (2017) 2333–2347.

[12] Y. Gavel, G. von Heijne, Sequence differences between glycosylated and non-glycosylated Asn-X-Thr/Ser acceptor sites: implications for protein engineering, Protein Eng. 3 (1990) 433–442.

[13] D.J. Kelleher, R. Gilmore, An evolving view of the eukaryotic oligosaccharyltransferase, Glycobiology. 16 (2006) 47R-62R.

[14] S. Shrimal, R. Gilmore, Oligosaccharyltransferase structures provide novel insight into the mechanism of asparagine-linked glycosylation in prokaryotic and eukaryotic cells, Glycobiology. (2018).

[15] J. Breitling, M. Aebi, N-linked protein glycosylation in the endoplasmic reticulum, Cold Spring Harb Perspect Biol. 5 (2013) a013359.

[16] A. Larkin, M.M. Chang, G.E. Whitworth, B. Imperiali, Biochemical evidence for an alternate pathway in N-linked glycoprotein biosynthesis, Nat Chem Biol. 9 (2013) 367– 373.

[17] Y. Taguchi, D. Fujinami, D. Kohda, Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation, J Biol Chem. 291 (2016) 11042–11054.

[18] J. Eichler, Z. Guan, Lipid sugar carriers at the extremes: The phosphodolichols Archaea use in N-glycosylation, Biochim. Biophys. Acta - Mol. Cell Biol. Lipids. 1862 (2017) 589–599.

[19] D. Kohda, Structural Basis of Protein Asn-Glycosylation by Oligosaccharyltransferases, Adv Exp Med Biol. 1104 (2018) 171–199.

[20] C. Lizak, S. Gerber, S. Numao, M. Aebi, K.P. Locher, X-ray structure of a bacterial oligosaccharyltransferase, Nature. 474 (2011) 350–355.

[21] M. Napiorkowska, J. Boilevin, T. Sovdat, T. Darbre, J.L. Reymond, M. Aebi, K.P. Locher, Molecular basis of lipid-linked oligosaccharide recognition and processing by bacterial oligosaccharyltransferase, Nat Struct Mol Biol. 24 (2017) 1100–1106.

[22] M. Napiorkowska, J. Boilevin, T. Darbre, J.L. Reymond, K.P. Locher, Structure of bacterial oligosaccharyltransferase PglB bound to a reactive LLO and an inhibitory peptide, Sci Rep. 8 (2018) 16297.

[23] S. Matsumoto, A. Shimada, J. Nyirenda, M. Igura, Y. Kawano, D. Kohda, Crystal structures of an archaeal oligosaccharyltransferase provide insights into the catalytic cycle of N-linked protein glycosylation, Proc Natl Acad Sci U S A. 110 (2013) 17868– 17873.

[24] S. Matsumoto, Y. Taguchi, A. Shimada, M. Igura, D. Kohda, Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex, Biochemistry. 56 (2017) 602–611.

[25] L. Bai, T. Wang, G. Zhao, A. Kovach, H. Li, The atomic structure of a eukaryotic oligosaccharyltransferase complex, Nature. 555 (2018) 328–333.

[26] R. Wild, J. Kowal, J. Eyring, E.M. Ngwa, M. Aebi, K.P. Locher, Structure of the yeast oligosaccharyltransferase complex gives insight into eukaryotic N-glycosylation, Science (80-. ). 359 (2018) 545–550.

[27] A.S. Ramirez, J. Kowal, K.P. Locher, Cryo-electron microscopy structures of human oligosaccharyltransferase complexes OST-A and OST-B, 1375 (2019) 1372–1375.

[28] C. Lizak, S. Gerber, D. Zinne, G. Michaud, M. Schubert, F. Chen, M. Bucher, T. Darbre,R. Zenobi, J.L. Reymond, K.P. Locher, A catalytically essential motif in external loop 5 of the bacterial oligosaccharyltransferase PglB, J Biol Chem. 289 (2014) 735–746.

[29] J. Nyirenda, S. Matsumoto, T. Saitoh, N. Maita, N.N. Noda, F. Inagaki, D. Kohda, Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance, Structure. 21 (2013) 32–41.

[30] T. Ando, N. Kodera, E. Takai, D. Maruyama, K. Saito, A. Toda, A high-speed atomic force microscope for studying biological macromolecules, Proc Natl Acad Sci U S A. 98 (2001)

[31] T. Ando, T. Uchihashi, T. Fukuma, High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes, Prog. Surf. Sci. 83 (2008) 337– 437.

[32] T. Ando, T. Uchihashi, S. Scheuring, Filming biomolecular processes by high-speed atomic force microscopy, Chem Rev. 114 (2014) 3120–3188.

[33] T. Ando, T. Uchihashi, N. Kodera, D. Yamamoto, A. Miyagi, M. Taniguchi, H. Yamashita, High-speed AFM and nano-visualization of biomolecular processes, Pflugers Arch. 456 (2008) 211–225.

[34] B.O. Alunda, Y.J. Lee, Review: Cantilever-based sensors for high speed atomic force microscopy, Sensors (Switzerland). 20 (2020) 1–39.

[35] M. Li, N. Xi, Y. Wang, L. Liu, Atomic Force Microscopy as a Powerful Multifunctional Tool for Probing the Behaviors of Single Proteins, IEEE Trans. Nanobioscience. 19 (2020) 78–99.

[36] N. Kodera, D. Yamamoto, R. Ishikawa, T. Ando, Video imaging of walking myosin V by high-speed atomic force microscopy, Nature 468 (2010) 72–76

[37] N. Kodera, T. Ando, The path to visualization of walking myosin V by high-speed atomic force microscopy, Biophys Rev 6 (2014) 237–260

[38] T. Uchihashi, R. Iino, T. Ando, H. Noji, High-speed atomic force microscopy reveals rotary catalysis of rotorless F1-ATPase, Science 333 (2011) 755−758

[39] D. Yamamoto, T. Ando, Chaperonin GroEL-GroES functions as both alternating and non-alternating engines, J Mol Biol 428 (2016) 3090–3101

[40] T. Uchihashi, N. Kodera, T. Ando, Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy, Nat Protoc. 7 (2012) 1193–1206.

[41] Q. Zhang, V. Cherezov, Chemical tools for membrane protein structural biology, Curr Opin Struct Biol 58 (2019) 278-285

[42] T.H. Bayburt, S.G. Sligar, Membrane protein assembly into Nanodiscs, FEBS Lett 584 (2010) 1721-1727

[43] I.G. Denisov, S.G. Sligar S.G, Nanodiscs for structural and functional studies of membrane proteins, Nat Struct Mol Biol 23 (2016) 481-486

[44] J.E. Rouck, J.E. Krapf, J. Roy, H.C. Huff, A. Das, Recent advances in nanodisc technology for membrane protein studies (2012-2017), FEBS Lett 591 (2017) 2057-2088

[45] Y. Gohon, F. Giusti, C. Prata, D. Charvolin, P. Timmin, C. Ebel, C. Tribet, J.L. Popot, Well-defined nanoparticles formed by hydrophobic assembly of a short and polydisperse random terpolymer, amphipol A8-35, Langmuir 22 (2006) 1281-1290

[46] N. Planchard, E. Point, T. Dahmane, F. Giusti, M. Renault, C.L. Bon, G. Durand, A. Milon, E. Guittet, M. Zoonens, J.L. Popot, L.J. Catoire, The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other olubilizing media, J Membr Biol 247 (2014) 827-842

[47] T. Haruyama, Y. Sugano, N. Kodera, T. Uchihashi, T. Ando, Y. Tanaka, H. Konno, T. Tsukazaki, Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy, Structure. 27 (2019) 152-160 e3.

[48] A. Sumino, T. Uchihashi, S. Oiki, Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging, J Phys Chem Lett. 8 (2017) 785–793.

[49] T. Uchihashi, S. Scheuring, Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes, Biochim Biophys Acta Gen Subj. 1862 (2018) 229–240.

[50] G.R. Heath, S. Scheuring, Advances in high-speed atomic force microscopy (HS-AFM) reveal dynamics of transmembrane channels and transporters, Curr. Opin. Struct. Biol. 57 (2019) 93–102.

[51] K.X. Ngo, N. Kodera, E. Katayama, T. Ando, T.Q. Uyeda, Cofilin-induced unidirectional cooperative conformational changes in actin filaments revealed by high-speed atomic force microscopy, Elife. 4 (2015).

[52] J. Schindelin, I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.Y. Tinevez, D.J. White, V. Hartenstein,K. Eliceiri, P. Tomancak, A. Cardona, Fiji: an open-source platform for biological-image analysis, Nat Methods. 9 (2012) 676–682.

[53] J.Y. Tinevez, N. Perry, J. Schindelin, G.M. Hoopes, G.D. Reynolds, E. Laplantine, S.Y. Bednarek, S.L. Shorte, K.W. Eliceiri, TrackMate: An open and extensible platform for single-particle tracking, Methods. 115 (2017) 80–90.

[54] T. Benaglia, D. Chauveau, D.R. Hunter, D.S. Young, mixtools: An R Package for Analyzing Finite Mixture Models, J. Stat. Softw. 32 (2009).

[55] R. Killick, I.A. Eckley, changepoint: An R Package for Changepoint Analysis, J. Stat. Softw. 58 (2014).

[56] D. Kohda, M. Yamada, M. Igura, J. Kamishikiryo, K. Maenaka, New oligosaccharyltransferase assay method, Glycobiology. 17 (2007) 1175–1182.

[57] T. Yamasaki, D. Kohda, A Radioisotope-free Oligosaccharyltransferase Assay Method, Bio-Protocol. 9 (2019) e3186.

[58] F. Delaglio, S. Grzesiek, G.W. Vuister, G. Zhu, J. Pfeifer, A. Bax, NMRPipe: a multidimensional spectral processing system based on UNIX pipes, J Biomol NMR. 6 (1995) 277–293.

[59] C. Amero, P. Schanda, M.A. Dura, I. Ayala, D. Marion, B. Franzetti, B. Brutscher, J. Boisbouvier, Fast two-dimensional NMR spectroscopy of high molecular weight protein assemblies, J Am Chem Soc. 131 (2009) 3448–3449.

[60] W. Zheng, B.R. Brooks, Modeling Protein Conformational Changes by Iterative Fitting of Distance Constraints Using Reoriented Normal Modes, Biophys. J. 90 (2006) 4327– 4336.

[61] A. Allouche, Software News and Updates Gabedit — A Graphical User Interface for Computational Chemistry Softwares, J. Comput. Chem. 32 (2012) 174–182.

[62] M.A. Lomize, I.D. Pogozheva, H. Joo, H.I. Mosberg, A.L. Lomize, OPM database and PPM web server: Resources for positioning of proteins in membranes, Nucleic Acids Res. 40 (2012) 370–376.

[63] A.E. Knight, C. Veigel, C. Chambers, J.E. Molloy, Analysis of single-molecule mechanical recordings: Application to acto-myosin interactions, Prog. Biophys. Mol. Biol. 77 (2001) 45–72.

[64] M. Capitanio, M. Canepari, M. Maffei, D. Beneventi, C. Monico, F. Vanzi, R. Bottinelli,F.S. Pavone, Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke, Nat. Methods. 9 (2012) 1013–1019.

[65] G.I. Mashanov, M. Nobles, S.C. Harmer, J.E. Molloy, A. Tinker, Direct observation of individual KCNQ1 potassium channels reveals their distinctive diffusive behavior, J. Biol. Chem. 285 (2010) 3664–3675.

[66] V. Biju, M. Micic, D. Hu, H.P. Lu, Intermittent single-molecule interfacial electron transfer dynamics, J. Am. Chem. Soc. 126 (2004) 9374–9381.

[67] D. Fujinami, J. Nyirenda, S. Matsumoto, D. Kohda, Structural elucidation of an asparagine-linked oligosaccharide from the hyperthermophilic archaeon, Archaeoglobus fulgidus, Carbohydr Res. 413 (2015) 55–62.

[68] S. Shrimal, N.A. Cherepanova, R. Gilmore, One flexible loop in OST lassos both substrates, Nat. Struct. Mol. Biol. 24 (2017) 1009–1010.

[69] W. Zheng, B.R. Brooks, Modeling Protein Conformational Changes by Iterative Fitting of Distance Constraints Using Reoriented Normal Modes, Biophys. J. 90 (2006) 4327– 4336.

[70] H.S. Lee, W. Im, Transmembrane motions of PglB induced by LLO are coupled with EL5 loop conformational changes necessary for OST activity, Glycobiology. 27 (2017) 734–742.

[71] Y. Kobashigawa, K. Harada, N. Yoshida, K. Ogura, F.I nagaki,Phosphoinositide-incorporated lipid-protein nanodiscs: A tool for studying protein-lipid interactions Anal Biochem 410 (2011) 77-83

[72] S.J. Ludtke, P.R. Baldwin, W. Chiu W, EMAN: semiautomated software for high-resolution single-particle reconstructions J Struct Biol 128 (1999) 82-97

[73] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng ,T.E. Ferrin, UCSF Chimera--a visualization system for exploratory research and analysis J Comput Chem 25 (2004) 1605-1612

[74] G.D. Pintilie, J. Zhang, T.D. Goddard, W. Chiu, D.C. Gossard, Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions J Struct Biol 170 (2010) 427-438

[75] W. Wriggers W, Conventions and workflows for using Situs Acta Crystallogr D Biol Crystallogr 68 (2012) 344-351

[76] L.G. Trabuco, E. Villa, K. Mitra, J. Frank, K. Schulten, Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics Structure 16 (2008) 673-683

[77] A. Rohou, N. Grigorieff, CTFFIND4: Fast and accurate defocus estimation from electron micrographs. Journal of Structural Biology 192 (2015) 216–221

[78] S.H.W. Scheres, RELION: implementation of a Bayesian approach to cryo-EM structure determination. Journal of Structural Biology 180 (2012) 519-530

[79] D. Kimanius, B.O. Forsberg, S.H. Scheres, E. Lindahl, Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5 (2016) e18722

[80] I.G. Denisov, Y.V. Grinkova, A.A. Lazarides, S.G. Sligar SG, Directed self-assembly of monodisperse phospholipid bilayer Nanodiscs with controlled size J Am Chem Soc 126 (2004) 3477-3487

[81] B. Chen, B. Shen, J. Frank, Particle migration analysis in iterative classification of cryo-EM single-particle data J Struct Biol 188 (2014) 267-273

[82] S.H. Scheres, H. Gao, M. Valle, G.T. Herman, P.P. Eggermont, J. Frank, J.M. Carazo, Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization Nat Methods 4 (2007) 27-29

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