Study on cold and heat denaturation of amyloid fibrils of intrinsically disordered alpha-synuclein

• Project/Area Number: 15K18518
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Osaka University
• Principal Investigator: YOUNG-HO LEE 大阪大学, たんぱく質研究所, 講師 (70589431)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2016: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2015: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: Alpha-synuclein / Amyloid fibril / Cold denaturation / Heat denaturation / Kinetics / Protein aggregation / Protein misfolding / Thermodynamics

Outline of Final Research Achievements

I examined cold and heat denaturation of amyloid fibrils of intrinsically disordered alpha-synuclein (aSN) prepared at various temperatures (298, 310, 323, 333, and 343 K). Despite differences in temperature for aSN amyloidogenesis, all fibrils showed similar core regions and secondary structures with the distinct width. As temperature increased from 298 to 383 K, all types of aSN fibrils heat-denatured to monomers from approximately 343 K. Decreasing temperature from 298 to 273 K caused cold denaturation of fibrils to monomers. Intriguingly, the stability of aSN fibrils depended on temperature for preparing fibrils. aSN fibrils generated at higher temperature showed higher stability at high and low temperature as well as slower denaturation kinetics than fibrils formed at lower temperature. Taken together, the stability of aSN amyloid fibrils depended on growing temperature of fibrils which produced differences in higher order structures of amyloid fibrils.

Research Products

• [Int'l Joint Research] Eotvos Lorand University(Hungary)
• [Int'l Joint Research] University of Michigan(米国)
• [Int'l Joint Research] Univerisity of Leeds(英国)
• [Int'l Joint Research] Malvern(英国)
• [Int'l Joint Research] Eotvos Lorand University(Hungary)
• [Int'l Joint Research] University of Michigan(米国)
• [Int'l Joint Research] University of Leeds(英国)
• [Int'l Joint Research] Malvern(英国)
• [Journal Article] Kinetics and polymorphs of yeast prion Sup35NM amyloidogenesis International Journal of Biological Macromolecules (2017)
  • Author(s): Misaki Kinoshita, Yuxi Lin, Masatoshi Nakatsuji, Takashi Inui, Young-Ho Lee
  • Journal Title: Int. J. Biol. Macromolec. Volume: -
  • Peer Reviewed
• [Journal Article] Mechanistic and structual basis of bioengineered bovine Cathelicidin-5 with optimized therapeutic activity (2017)
  • Author(s): Bikash R. Sahoo, Kenta Maruyama, Jyotheeswara R. Edula, Takahiro Tougan, Yuxi Lin, Young-Ho Lee, Toshihiro Horii, and Toshimichi Fujiwara
  • Journal Title: Scientific Reports Volume: 7:44781 Issue: 1 Pages: 44781-44796
  • DOI: 10.1038/srep44781
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Practical Tips of Calorimetric Experiments using PEAQ-ITC (2017)
  • Author(s): Misaki Kinoshita, Ju Yaen Kim, Yuxi Lin, Young-Ho Lee
  • Journal Title: Malvern Technical note Volume: -
  • Peer Reviewed
• [Journal Article] Biophysical Applications of Calorimetric Methods to Examination of Protein Misfolding and Aggregation (2017)
  • Author(s): Tatsuya Ikenoue, Misaki Kinoshita, Yuxi Lin, and Young-Ho Lee
  • Journal Title: Malver white paper Volume: -
  • Peer Reviewed
• [Journal Article] A. Reduced Lipid Bilayer Thickness Regulates the Aggregation and Cytotoxicity of Amyloid-β (2017)
  • Author(s): Kyle J. Korshavn, Cristina Satriano, Yuxi Lin, Rongchun Zhang, Mark Dulchavsky, Anirban Bhunia, Magdalena I. Ivanova, Young-Ho Lee, Carmelo La Rosa, Mi Hee Lim, Ayyalusamy Ramamoorthy
  • Journal Title: J. Biol. Chem. Volume: 292 Issue: 11 Pages: 4638-4650
  • DOI: 10.1074/jbc.m116.764092
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Energetic basis on interactions between ferredoxin and ferredoxin NADP+ reductase at varying physiological conditions. (2016)
  • Author(s): Kinoshita, M., Kim, J.Y., S., Lin, Y., Hun Mok K., Kataoka, Y., Ishimori, K., Markova, N., Kurisu, G., Hase, T., Lee, Y.H.
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 482 Issue: 4 Pages: 909-915
  • DOI: 10.1016/j.bbrc.2016.11.132
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Noncovalent Forces Tune the Electron Transfer Complex between Ferredoxin and Sulfite Reductase to Optimize Enzymatic Activity (2016)
  • Author(s): Ju Yaen Kim, Misaki Kinoshita, Satoshi Kume, Guy T. Hanke, Toshihiko Sugiki, John E. Ladbury, Chojiro Kojima, Takahisa Ikegami, Genji Kurisu, Yuji Goto, Toshiharu Hase, Young-Ho Lee
  • Journal Title: Biochem J. Volume: 473 Pages: 3837-3854
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Solution NMR structure and inhibitory effect against amyloid-β fibrillation of Humanin containing a D-isomerized serine residue (2016)
  • Author(s): Nesreen Alsanousi, Toshihiko Sugiki, Kyoko Furuita, Masatomo So, Young-Ho Lee, Toshimichi Fujiwara, and Chojiro Kojima
  • Journal Title: Biochem. Biophys. Res. Commun. Volume: 477 Issue: 4 Pages: 647-653
  • DOI: 10.1016/j.bbrc.2016.06.114
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Mutations affecting the internal equilibrium of the reaction catalyzed by 6-aminohexanoate-dimer hydrolase (2016)
  • Author(s): Negoro S, Kawashima Y, Shibata N, Kobayashi T, Baba T, Lee YH, Kamiya K, Shigeta Y, Nagai K, Takehara I, Kato D, Takeo M, Higuchi Y
  • Journal Title: FEBS Lett Volume: 590 Issue: 18 Pages: 3133-3143
  • DOI: 10.1002/1873-3468.12354
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A linker for the isopeptide mimetics by the peptide ligation (2016)
  • Author(s): Kawakami Toru, Mishima Yuichi, Kinoshita Misaki, Lee Young-Ho, Suetake Isao
  • Journal Title: Tetrahedron Letters Volume: -
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Amorphous Aggregation of Cytochrome c with Inherently Low Amyloidogenicity Is Characterized by the Metastability of Supersaturation and the Phase Diagram (2016)
  • Author(s): Lin Yuxi, Kardos József, Imai Mizue, Ikenoue Tatsuya, Kinoshita Misaki, Sugiki Toshihiko, Ishimori Koichiro, Goto Yuji, and Lee Young-Ho
  • Journal Title: Langmuir Volume: 32 Issue: 8 Pages: 2010-2022
  • DOI: 10.1021/acs.langmuir.5b03810
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] X-ray Structure and Nuclear Magnetic Resonance Analysis of the Interaction Sites of the Ga-Substituted Cyanobacterial Ferredoxin (2015)
  • Author(s): Mutoh Risa, Muraki Norifumi, Shinmura Kanako, Kubota-Kawai Hisako, Lee Young-Ho, Marc M. Nowaczyk, Matthias Rögner, Hase Toshiharu, Ikegami Takahisa, Kurisu Genji
  • Journal Title: Biochemistry Volume: 54 Issue: 39 Pages: 6052-6061
  • DOI: 10.1021/acs.biochem.5b00601
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Physicochemical Nature of Interfaces Controlling Ferredoxin NADP+ Reductase Activity through Its Interprotein Interactions with Ferredoxin (2015)
  • Author(s): Kinoshita Misaki, Kim Ju Yaen, Kume Satoshi, Sakakibara Yukiko, Sugiki Toshihiko, Kojima Chojiro, Kurisu Genji, Ikegami Takahisa, Hase Toshiharu, Kimata-Ariga Yoko, Lee Young-Ho
  • Journal Title: Biochimica et Biophysica Acta-Bioenergetics Volume: 1847 Issue: 10 Pages: 1200-1211
  • DOI: 10.1016/j.bbabio.2015.05.023
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Principle of protein misfolding and aggregation ‐ new findings of amyloid formation from thermodynamics (2016)
  • Author(s): Lee Young-Ho
  • Organizer: RIKEN CLST invited seminar
  • Place of Presentation: Kobe, Japan
  • Year and Date: 2016-03-18
  • Invited
• [Presentation] The interaction of sulfite reductase with ferredoxin and its relation to enzyme activity (2016)
  • Author(s): Lee Young-Ho
  • Organizer: Invited seminar in Hokkaido University
  • Place of Presentation: Hokkaido, Japan
  • Year and Date: 2016-03-12
  • Invited
• [Presentation] Thermodynamic study on interprotein interaction between oppositely-charged electron transfer proteins (2016)
  • Author(s): Lee Young-Ho
  • Organizer: Institute for Protein Research seminar
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2016-02-02
  • Invited
• [Presentation] New findings on thermodynamic properties of disease-inducing protein aggregation, Japan-Korea Bilateral Symposium on Multi-Scale Structural Biology (2016)
  • Author(s): Young-Ho Lee
  • Organizer: International seminar at Institute for Protein Research
  • Place of Presentation: 大阪大学（大阪府吹田市）
  • Invited
• [Presentation] 1. 等温滴定熱量測定を用いた分子間相互作用の熱力学 (2016)
  • Author(s): 李 映昊
  • Organizer: Malvern ウェブナー
  • Place of Presentation: 神戸国際ビジネスセンター（兵庫県、神戸市）
  • Invited
• [Presentation] The study on thermodynamic property of protein misfolding and aggregation (2015)
  • Author(s): Lee Young-Ho
  • Organizer: 第38回日本分子生物学会・第88回日本生化学会大会
  • Place of Presentation: Kobe, Japan
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] Toward the establishment of thermodynamics on protein misfolding and aggregatio (2015)
  • Author(s): Lee Young-Ho
  • Organizer: Open seminar in Osaka Prefecture University
  • Place of Presentation: Osaka, Japan
  • Year and Date: 2015-04-22
  • Invited
• [Book] Advanced Techniques to Detect Protein-Protein Interactions using Solution Nuclear Magnetic Resonance (Part II: Cross-saturation, Paramagnetic Effects, and In-cell NMR) Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis (2017)
  • Author(s): Toshihiko Sugiki, Young-Ho Lee
  • Total Pages: 21
  • Publisher: Nova Science Publishers, Inc.
• [Book] Advanced Techniques to Detect Protein-Protein Interactions using Solution Nuclear Magnetic Resonance (Part I: Chemical Shift Perturbation and Residual Dipolar Coupling) Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis (2017)
  • Author(s): Young-Ho Lee, Toshihiko Sugiki
  • Total Pages: 21
  • Publisher: Nova Science Publishers, Inc.
• [Book] Molecular Structure of Amyloid Fibrils Revealed by Thermodynamics Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis (2017)
  • Author(s): Tatsuya Ikenoue, Yuxi Lin, Misaki Kinoshita, Kazumasa Sakurai, Ayyalusamy Ramamoorthy, Yuji Goto, Young-Ho Lee
  • Total Pages: 13
  • Publisher: Nova Science Publishers, Inc.
• [Book] Thermodynamics of Protein-Protein Interactions Examined by Isothermal Titration Calorimetry Protein-Protein Interactions (PPIs): Types, Methods for Detection and Analysis (2017)
  • Author(s): Ju Yaen Kim, Misaki Kinoshita, Takashi Inui, Genji Kurisu, Yuji Goto, Toshiharu Hase, Koichiro Ishimori, Young-Ho Lee
  • Total Pages: 16
  • Publisher: Nova Science Publishers, Inc.
• [Book] Methods in Molecular Biology: Biochemical and Biophysical Methods to Examine Effects of Site-directed Mutagenesis on Enzymatic Activities and Interprotein Interactions (2017)
  • Author(s): Misaki Kinoshita, Ju Yaen Kim, Yuxi Lin, Natalia Markova, Toshiharu Hase, Young-Ho Lee
  • Total Pages: 22
  • Publisher: Springer Nature
• [Book] Methods in Molecular Biology (In vitro mutagenesis: Methods and Protocol) (2016)
  • Author(s): Kinoshita Misaki, Kim Ju Yaen, Lin Yuxi, Natalia Markova, Toshiharu Hase, Lee Young-Ho
  • Total Pages: 500
  • Publisher: Springer Science+Business Media