| Budget Amount *help |
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2016: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2015: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
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| Outline of Final Research Achievements |
Real time in-cell NMR spectroscopy was applied to quantify kinetics of non-endocytic membrane permeation of octaarginine (R8) into living cells. The non-endocytic membrane translocation of 19F-labeled R8 into a human myeloid leukemia cell line was observed at 4 °C with a time resolution of minute-order. The 19F NMR detected the real time translocation of cationic R8: the binding to anionic glycosaminoglycan at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. Our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of membrane-permeable peptides like R8 in cell membranes.
The methods were also applied to analyze spontaneous peptide bond cleavage at natural L-α-Asp and abnormal D-β-Asp isomers in eye lens α-crystallin fragments in real time. Kinetic analysis showed how tough the uncommon D-β-Asp residue was to allow abnormal accumulation.
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