Development of helix-anchored planar membrane

Research Project

Project/Area Number	16K08194
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Multi-year Fund
Section	一般
Research Field	Physical pharmacy
Research Institution	Kyoto University
Principal Investigator	Yano Yoshiaki 京都大学, 薬学研究科, 講師 (60402799)
Research Collaborator	MATSUZAKI Katsumi
Project Period (FY)	2016-04-01 – 2019-03-31
Project Status	Completed (Fiscal Year 2018)
Budget Amount *help	¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000) Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2017: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000) Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Keywords	モデル膜貫通ヘリックス / 膜貫通へリックス / 膜貫通へリックス間相互作用 / 膜貫通ヘリックス固定型tethered膜 / 膜貫通ヘリックス / 繋ぎ止め膜
Outline of Final Research Achievements	In This study, we developed peptide-anchored planar lipid bilayers. Model transmembrane helix was attached to a PEG-coated glass using click chemistry. Liposomes were incubated on the glass surface to form planar lipid bilayers. These bilayers had fluidity but the model peptides were immobile because of tethering. Biotin-avidin interaction revealed that the model peptides were successfully inserted into the bilayers. Thus, we established a preparation method of planar lipid bilayers including immobile peptides with defined insertion topology, which can laterally trap other molecules in the membranes.
Academic Significance and Societal Importance of the Research Achievements	膜受容体など創薬に関連するものも多い膜貫通タンパク質の機能を解明するには、細胞膜疎水部でのタンパク質間相互作用の解析が重要であるが、会合自由エネルギーや会合・解離速度定数を調べる簡便な手法が確立できれば大きな利点がある。本研究では膜内で相互作用する膜貫通ヘリックスの片方をガラス基板上に固定した繋ぎ止め膜（tethered膜）を作製した。この技術は、蛍光標識したもう片方の分子と可逆に相互作用する際に、側方拡散が抑制される事を利用して蛍光相関分光法(FCS)や光褪色後蛍光回復法(FRAP)で相互作用力を迅速に評価する系に応用できる。