Enrico Luchinat1, Erica Secci2, Francesca Cencetti3, Paola Bruni3. 1. Magnetic Resonance Center-CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy; Department of Biomedical, Experimental and Clinical Sciences "Mario Serio", University of Florence, Viale Morgagni 50, 50134, Florence, Italy. Electronic address: eluchinat@cerm.unifi.it. 2. Magnetic Resonance Center-CERM, University of Florence, Via Luigi Sacconi 6, 50019 Sesto Fiorentino, Florence, Italy. 3. Department of Biomedical, Experimental and Clinical Sciences "Mario Serio", University of Florence, Viale Morgagni 50, 50134, Florence, Italy.
Abstract
BACKGROUND: In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. METHODS: We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (human superoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. RESULTS: SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by (1)H-(15)N heteronuclear NMR. CONCLUSIONS AND GENERAL SIGNIFICANCE: We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein-protein interactions by NMR in human cells.
BACKGROUND: In-cell NMR is a powerful technique to investigate proteins in living human cells at atomic resolution. Ideally, when studying functional processes involving protein-protein interactions by NMR, only one partner should be isotopically labeled. Here we show that constitutive and transient protein expression can be combined with protein silencing to obtain selective protein labeling in human cells. METHODS: We established a human cell line stably overexpressing the copper binding protein HAH1. A second protein (humansuperoxide dismutase 1, SOD1) was overexpressed by transient transfection and isotopically labeled. A silencing vector containing shRNA sequences against the HAH1 gene was used to decrease the rate of HAH1 synthesis during the expression of SOD1. The levels of HAH1 mRNA and protein were measured as a function of time following transfection by RT-PCR and Western Blot, and the final cell samples were analyzed by in-cell NMR. RESULTS:SOD1 was ectopically expressed and labeled in a time window during which HAH1 biosynthesis was strongly decreased by shRNA, thus preventing its labeling. In-cell NMR spectra confirmed that, while both proteins were present, only SOD1 was selectively labeled and could be detected by (1)H-(15)N heteronuclear NMR. CONCLUSIONS AND GENERAL SIGNIFICANCE: We showed that controlling protein expression by specifically silencing a stably expressed protein is a useful strategy to obtain selective isotope labeling of only one protein. This approach relies on established techniques thus permitting the investigation of protein-protein interactions by NMR in human cells.