Ayako Ohno, Kohsuke Inomata, Hidehito Tochio and Masahiro Shirakawa Pages 68 - 73 ( 6 )
“In-cell nuclear magnetic resonance (NMR)” is a unique method for characterization of conformation, interaction and dynamics of proteins inside living cells at atomic level. Since the method was proposed by Dotch and co-workers in 2001(1), its application had been limited to bacterial cells and oocytes of Xenopus laevis(2). Recently, we reported the method for efficient delivery of 15N-labeled proteins into human HeLa cells by using cell-penetrating peptides, and measured high-resolution two-dimensional 1H-15N correlation spectra of proteins in the cells. The in-cell NMR spectroscopy in human cells is capable of analyzing structures, interactions, dynamics and stability of proteins inside cells. Of its possible applications, we propose that in-cell NMR spectroscopy can be utilized as an effective step in protein-targeted drug development process, by demonstrating that interaction of FKBP12 with immunosuppressants administered extracellularly was successfully observed in living cells. This observation suggests that drug delivery and capability of target proteins inside cells for interaction with drugs can be investigated by in-cell NMR spectroscopy. More recently, an alternative way for intracellular delivery of labeled proteins for in-cell NMR was reported on 293F cells by Shimada and co-workers. Here, we review recent technical development of in-cell NMR spectroscopy, and discuss potential usefulness for protein chemistry and drug screening process.
In-cell NMR, drug delivery, protein interaction, drug discovery, nuclear magnetic resonance, NMR, cell-penetrating peptide, CPP, deubiquitination proteases, DUBs, pyrenbutyrate, CPP-ubiquitin-cargo protein, streptolysin O, SLO, tymosin 4, Thermus thermophilus, Escherichia coli, Synuclein, Ubiquitin, Chymotrypsin Inhibitor 2, Calmodulin, Green Fluorescence Protein, Histidinol Dehydrogenase, ri-19F-m-phenylalanine, tmf-Phe
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto 615-8510, Japan.