Visualization of molecular interactions at the nanometer scale

Research Project

Project/Area Number	25650062
Research Category	Grant-in-Aid for Challenging Exploratory Research
Allocation Type	Multi-year Fund
Research Field	Cell biology
Research Institution	Nagoya University
Principal Investigator	FUJIMOTO Toyoshi 名古屋大学, 医学(系)研究科(研究院), 教授 (50115929)
Project Period (FY)	2013-04-01 – 2015-03-31
Project Status	Completed (Fiscal Year 2014)
Budget Amount *help	¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000) Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000) Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Keywords	生体膜 / 電子顕微鏡 / 膜タンパク質 / 凍結割断レプリカ / 膜蛋白質 / 凍結割断レプリカ法 / ビオチン化
Outline of Final Research Achievements	Methods to visualize the location where two different proteins interact include fluorescence resonance energy transfer, bimolecular fluorescence complementation, and proximity ligation assay, but their spatial resolution is not high. In this study, we conceived an idea of a method based on quick-freezing and freeze-fracture replica labeling method. The principle of the method was: 1) tagging of biotinylation enzyme BirA and its acceptor peptide AcP to the cytoplasmic domain of two transmembrane proteins A and B, respectively; 2) biotinylation of AcP in freeze-fracture replicas; 3) immunolabeling and electron microscopic observation of biotins. Although various conditions were examined, satisfactory results were not obtained so far. Further trials using different conditions are needed.