Development of localization estimating single particle analysis with GFP label

2015 Fiscal Year Final Research Report

• Project/Area Number: 26650021
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Osaka University
• Principal Investigator: KATO Takayuki 大阪大学, 生命機能研究科, 助教 (20423155)
• Co-Investigator(Renkei-kenkyūsha): MIYATA Tomoko 大阪大学, 生命機能研究科, 特任助教 (30423156) ; TERAHARA Naoya 大阪大学, 生命機能研究科, 特任助教 (40554738)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 低温電子顕微鏡 / 単粒子解析 / 構造解析 / GFPラベル

Outline of Final Research Achievements

To know the orientation and the localization of specific molecule from low resolution map solved by cryoEM, circular permuted GFP was used for the molecular labeling.
The twelve positions for cpGFP fusion with FliM which is rotor component protein in flagella motor were designed. The expression level, function and localization of labeled-FliM in the cell were evaluated by western blotting, phase contrast microscopy and fluorescence microscopy. From their results, we found that the two cpGFP-labeld FliM candidates of them has the FliM function. We demonstrated that the cpGFP can be used for the molecular labeling. Then rotor with cpGFP-labeled FliM in the candidate was purified and observed by electron microscopy. These cpGFP-labeld FliM was dissociated from rotor structure. We suggest that the position is most important factor for cpGFP labeling.

Free Research Field

構造生物学