Development of Non-Supported Membrane over Nanowells for Visualization of Functional Dynamics of Membrane Proteins with High-Speed AFM

2015 Fiscal Year Final Research Report

• Project/Area Number: 26600052
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Nanobioscience
• Research Institution: Kanazawa University
• Principal Investigator: UCHIHASHI TAKAYUKI 金沢大学, 数物科学系, 教授 (30326300)
• Co-Investigator(Kenkyū-buntansha): IINO Ryota 自然科学研究機構, 岡崎統合バイオサイエンスセンター, 教授 (70403003)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 一分子計測 / 膜タンパク質 / 高速原子間力顕微鏡

Outline of Final Research Achievements

We prepared nano-pore arrays with a diameter of 100nm on glass and silicon substrate using electron-beam lithography and focused ion beam. Also we succeeded in sealing the nano pores with lipid bilayers. Purple membranes in which two dimensional crystals of bacteriorhodopsin are embedded were used as a test sample and the individual bacteriorhodopsin molecules were resolved on the nano pore with high-speed AFM. We also developed a method to conveniently prepare the nano pores on a silicone elastomer using small beads with a diameter of 100nm as a template. We observed ion-channel membrane protein KcsA on a supported lipid bilayer and visualized individual KcsA tetramers and theirs diffusion dynamics in the membrane. As the next step, we will reconstitute KcsA molecules into the membrane on the nano pore and reveal structural dynamics of KcsA..

Free Research Field

生物物理学