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

Abstract

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.

Residue level quantification of protein stability in living cells

“…Inomata et al (51) used this technique to measure k obs values in human cells for four residues in ubiquitin, but experiments confirming the exchange mechanism were not reported and opening free energies were not quantified. Our results fill this void and provide quantitative residue-level protein stability measurements in living cells under nonperturbing conditions.…”

Soft interactions and crowding

“…The inability of the cytoplasm to fold ProtL was discussed above (Schlesinger et al 2011). Inomata et al (2009 used in-cell NMR to assess the amide proton exchange rates for ubiquitin in HeLa cells.…”

In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants

“…I n-cell NMR represents a unique tool for characterizing physiological processes and their alteration in the 'natural' environment, that is, within a live cellular context, with an atomic-level approach [1][2][3][4][5][6][7][8][9] . This level of characterization is necessary to understand the molecular cause of cell malfunction in pathological states and to develop treatments and therapeutic protocols.…”