posters 5th Asia-Pacific NMR Symposium 2013

19F-NMR spectroscopy of proteins in mammalian cultured cells (#179)

Shuhei Murayama 1 , Kohsuke Inomata 2 , Ayako Ohno 3 , Ken-ichi Akagi 4 , Hidehito Tochio 1 , Masahiro Shirakawa 1
  1. Kyoto university, Kyoto, Japan
  2. RIKEN, Osaka, Japan
  3. University of Tokushima, Tokushima, Japan
  4. National Institute of Biomedical Innovation, Osaka, Japan

The inside of cells, where most proteins function, is a crowded and complex environment caused by the high concentration of endogenous biological molecules and organelles. Thus, protein property in cells can differ substantially from in vitro, and direct study of protein function in the cell interior is desired for understanding intrinsic protein functions. In-cell NMR enables observation of proteins in living cells at the atomic level, allowing us to assess their conformations, dynamics and interactions at atomic resolution. Previously, our group has developed a mammalian in-cell NMR technique, where 2D 1H-15N correlation spectra were recorded for uniformly 15N-labeled proteins transduced in living HeLa cells by using the CPP (Cell Penetrating Peptide) tag. In this study, application of amino acid specific 19F-labeling to in-cell NMR was examined. With such labeling strategy, in-cell NMR spectra can be greatly simplified, facilitating spectral analysis. Moreover, shorter measurement time is expected as 1D experiment is sufficient to resolve all NMR resonances in such sparsely labeled systems. FKBP12, 12-kDa FK506-binding protein, was employed as a model system. p-fluoro-L-phenylalanine was incorporated into FKBP12, and the protein was delivered into HeLa cells using our CPP –mediated protein transduction method. Simple one-dimensional 19F-NMR spectra were recorded on the cells with or without administered FK506. The obtained spectra demonstrated formation of the specific complex between FKBP12 and FK506 in the cells. In a same way, in-cell 19F NMR spectra of 19F-FKBP12 were also recorded with rapamycin. Overall, our results demonstrated that in-cell 19F NMR is feasible to monitor proteins functioning in HeLa cells. Because of the simplicity of 1D experiments, higher efficiency is expected in assessing drug candidates targeted to specific cytosolic proteins over the in-cell 2D 1H-15N correlation experiments.