Application of novel labeling method for membrane proteins to aggregational analysis and its sophistication

• Project/Area Number: 24390009
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Kyoto University
• Principal Investigator: MATSUZAKI Katsumi 京都大学, 薬学研究科(研究院), 教授 (00201773)
• Co-Investigator(Kenkyū-buntansha): YANO Yoshiaki 京都大学, 大学院薬学研究科, 助教 (60402799)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥19,110,000 (Direct Cost: ¥14,700,000、Indirect Cost: ¥4,410,000); Fiscal Year 2014: ¥3,380,000 (Direct Cost: ¥2,600,000、Indirect Cost: ¥780,000); Fiscal Year 2013: ¥3,120,000 (Direct Cost: ¥2,400,000、Indirect Cost: ¥720,000); Fiscal Year 2012: ¥12,610,000 (Direct Cost: ¥9,700,000、Indirect Cost: ¥2,910,000)
• Keywords: 膜タンパク質 / オリゴマー化 / 蛍光イメージング / クロスリンク / イメージング

Outline of Final Research Achievements

Membrane proteins include many important proteins for drug targets. Development of better experimental methods for membrane proteins is particularly important because of limitations in the current methods. Here we developed two in-cell analysis methods based on the coiled-coil labeling method. 1) An image analysis method for measuring protein oligomeric state was established and applied for various membrane proteins. 2) A crosslinking method was developed, which was useful for direct comparison of protein oligomeric states in live cells and solubilized micelles.

Research Products

• [Journal Article] Oligomerization-function relationship of EGFR on living cells detected by the coiled-coil labeling and FRET microscopy (2015)
  • Author(s): Yamashita H., Yano Y., Kawano K., Matsuzaki K.
  • Journal Title: Biochim Biophys Acta Volume: 1848 Issue: 6 Pages: 1359-1366
  • DOI: 10.1016/j.bbamem.2015.03.004
  • NAID: 120005621952
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] A dimer is the minimal proton-conducting unit of the influenza a virus M2 channel (2014)
  • Author(s): Kawano K., Yano Y., Matsuzaki K.
  • Journal Title: J. Mol. Biol. Volume: 426 Issue: 14 Pages: 2679-2691
  • DOI: 10.1016/j.jmb.2014.05.002
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Stoichiometric analysis of oligomerization of membrane proteins on living cells using coiled-coil labeling and spectral imaging (2013)
  • Author(s): Kawano K, Omae K, Matsuzaki S, Matsuzaki K
  • Journal Title: Anal. Chem. Volume: 85 Issue: 6 Pages: 3454-3461
  • DOI: 10.1021/ac400177a
  • Peer Reviewed
• [Presentation] 共有結合型コイルドコイルラベル法の確立とグリコフォリンAの会合状態の解明 (2015)
  • Author(s): 古川奈美、矢野義明、小野智史、松崎勝巳
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸サンボーホール（兵庫県）
  • Year and Date: 2015-03-26 – 2015-03-28
• [Presentation] 生細胞におけるアミロイド前駆体タンパク質の会合状態解 (2015)
  • Author(s): 上山和輝、矢野義明、松崎勝巳
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸サンボーホール（兵庫県）
  • Year and Date: 2015-03-26 – 2015-03-28
• [Presentation] コイルドコイル標識法とin-cell蛍光測定法による生細胞膜上での膜タンパク質の会合計測 (2015)
  • Author(s): 松崎勝巳
  • Organizer: 日本薬学会第135年会
  • Place of Presentation: 神戸学院大学（兵庫県）
  • Year and Date: 2015-03-26 – 2015-03-28
  • Invited
• [Presentation] 新規小分子ラベル法とin-cell FRETを用いた膜タンパク質の会合状態の定量的解析 (2014)
  • Author(s): 河野健一、矢野義明、大前薫、松崎紗矢香、松崎勝巳
  • Organizer: 第64回日本薬学会近畿支部大会
  • Place of Presentation: 京都薬科大学（京都府）
  • Year and Date: 2014-10-11
• [Presentation] Dimer is a Minimal Functional Unit for Influenza A Virus M2 Channel on Biomembrane (2014)
  • Author(s): Kenich Kawano, Yoshiaki Yano and Katsumi Matsuzaki
  • Organizer: Biophysical society 58th Annual meeting
  • Place of Presentation: Moscone Center, San Francisco, CA（アメリカ合衆国）
• [Presentation] 抗インフルエンザウイルス薬アマンタジン耐性M2 S31N変異株の生体膜中における会合状態 (2014)
  • Author(s): 河野健一、矢野義明、松崎勝巳
  • Organizer: 日本薬学会第134年会
  • Place of Presentation: 熊本市総合体育館（熊本県）
• [Presentation] 生細胞膜タンパク質の会合・内在化の可視化解析ツール : コイルドコイルラベル法 (2013)
  • Author(s): 矢野義明、河野健一、大前薫、竹田有希、松崎紗矢香、松崎勝巳
  • Organizer: 日本薬学会第133年会
  • Place of Presentation: パシフィコ横浜(神奈川県)(招待講演)
  • Year and Date: 2013-03-28
• [Presentation] コイルドコイル蛍光ラベル法を用いた生細胞での上皮成長因子受容体多量体形成の検出 (2013)
  • Author(s): 山下博隆、河野健一、矢野義明、松崎勝巳
  • Organizer: 第11回次世代を担う若手のためのフィジカル・ファーマフォーラム(PPF)2013
  • Place of Presentation: 清水テルサ（静岡県）
• [Presentation] Dimer is a minimal functional unit for influenza A virus M2 channel on biomembrane (2013)
  • Author(s): Kenich Kawano, Yoshiaki Yano and Katsumi Matsuzaki
  • Organizer: 4th Asia-Pacific International Peptide Symposium
  • Place of Presentation: ホテル阪急エキスポパーク（大阪府）
• [Presentation] コイルドコイル蛍光ラベル法を用いた生細胞での上皮成長因子受容体多量体形成の検出 (2013)
  • Author(s): 山下博隆、河野健一、矢野義明、松崎勝巳
  • Organizer: 第35回生体膜と薬物の相互作用シンポジウム
  • Place of Presentation: 東京大学大学院薬学研究科（東京都）
• [Presentation] Stoichiometric analysis of oligomerization of membrane proteins on living cell surface (2012)
  • Author(s): Kawano K, Omae K, Matsuzaki S, Yano Y, Matsuzaki K
  • Organizer: The 7th Seoul-Kyoto-Osaka joint symposium on pharmaceutical sciences for young scientists
  • Place of Presentation: 京都大学薬学部(京都府)
  • Year and Date: 2012-05-23
• [Remarks] 研究室ホームページ
  • URL: http://www.pharm.kyoto-u.ac.jp/yakkai/