2012 Fiscal Year Final Research Report
Analysis of structural change and ligand binding of membrane receptors by single molecule spectroscopy
| Project/Area Number |
23790044
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Physical pharmacy
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| Research Institution | Kyoto University |
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Principal Investigator |
YOSHIAKI Yano 京都大学, 大学院・薬学研究科, 助教 (60402799)
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| Project Period (FY) |
2011 – 2012
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| Keywords | 一分子蛍光 / GPCR / 無細胞合 |
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| Research Abstract |
In vitro synthesis of G protein coupled receptors (GPCRs) was examined to develop experimental systems useful for single-molecule fluorescence measurements. Firstly, lipid nanodiscs proteins were prepared as a platform for folding of GPCRs. Secondary, β2 adrenergic receptors were synthesized in insect-derived cell-free translational system in the presence of liposomes and ligand-binding ability was assessed by binding of fluorescent ligand. However, a commercially available fluorescent ligand had a significant nonspecific binding to lipid membranes. To find fluorescent ligands with little nonspecific binding, Cy3- and Alexa647- labeled noradrenaline were synthesized. Both Cy3-NA and Alexa647-NA did not have sufficient binding affinity to the receptors that were expressed on CHO cells, suggesting importance of further development of probes.
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