High-resolution multi-dimensional NMR spectroscopy of proteins in human cells

In-cell NMR: protein structure in human cells Until now, in-cell NMR spectroscopy of living cells has been limited to bacteria and Xenopus laevis oocytes. Wider application to living eukaryote cells has been limited by the relatively inefficient delivery of isotope-labelled proteins into these cells. Now Inomata et al . show that it is possible to deliver suitably labelled proteins into the cytosol of human cells via the pyrenebutyrate-mediated action of cell-penetrating peptides that have been linked covalently to the proteins of interest. When the proteins are released by endogenous enzymes or autonomous reductive cleavage, high-resolution two-dimensional heteronuclear NMR spectra of proteins inside living human cells can be obtained. This technique could be a powerful tool for the design and screening of drugs targeted to intracellular proteins. In-cell NMR is a relatively new technique that can be used to examine the conformations of proteins in living cells at the atomic level. This method has been successfully used in bacteria and Xenopus laevis oocytes, but wider application of the technique to living eukaryote cells has been limited by the inefficient delivery of isotope-labeled proteins into eukaryote somatic cells. This study shows that it is possible to deliver suitably labelled proteins into the cytosol of human cells through the pyrenebutyrate-mediated action of cell-penetrating peptides that have been linked covalently to the proteins of interest. 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 1 . This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein–ligand interactions 2 and conformations 3 , 4 . However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes 5 , 6 , 7 . 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 8 linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronu