High-resolution multi-dimensional NMR spectroscopy of proteins in human cells.
In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein-ligand interactions and conformations. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.
Inomata
Kohsuke
K
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto 615-8510, Japan.
Ohno
Ayako
A
Tochio
Hidehito
H
Isogai
Shin
S
Tenno
Takeshi
T
Nakase
Ikuhiko
I
Takeuchi
Toshihide
T
Futaki
Shiroh
S
Ito
Yutaka
Y
Hiroaki
Hidekazu
H
Shirakawa
Masahiro
M
D000818:Animals; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D041961:Deuterium Exchange Measurement; D004353:Drug Evaluation, Preclinical / Q000379:methods; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007166:Immunosuppressive Agents / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D042541:Intracellular Space / Q000378:metabolism*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D011721:Pyrenes / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
D015696:Gene Products, tat; D007166:Immunosuppressive Agents; D011721:Pyrenes; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; C002218:1-pyrenebutyrate; D022061:Tacrolimus Binding Protein 1A