Development of mammalian in cell NMR method employing virus based carrier peptide conjugated to isotope labelled K-Ras protein
To develop mammalian in cell NMR method, we plan to conjugate isotope labelled K-Ras protein with peptides capable to internalise into cells. The K-Ras protein will be expressed in bacteria in isotope-labelled form, as fusion constructs with peptides, and also in itself, making it suitable for native chemical ligation or other conjugation methods with synthetic carrier oligopeptides.
The ability of the peptide–protein constructs to internalise into cells is going to be evaluated with flow citometry, for this we are going to use synthetic conjugates labelled with fluorescent moiety. The use of a spacer moiety between the carrier and protein may be necessary; this can be oligoethyleneglycol derivative and oligoglycine in case of ligation and fusion protein, respectively. The intracellular localisation of the labelled constructs is going to be determined by confocal microscopy.
After the preparation of the constructs, the structure of the 15N-labelled K-Ras protein is going to be studied by NMR under conditions developed earlier to ascertain that peptide conjugation causes no changes in its secondary structure. Then the protein structure is going to be studied in the highly complex mammalian cell lysate resembling in cell environment. After proving the ability of the conjugated peptide to target the protein into the cells without secondary structure changes, large amount of isotope labelled protein is going to be internalised into the mammalian cells to be characterised by in cell NMR*.
Our long-term plans consist of studying the intracellular binding of small molecules to the oncogenic mutant version of K-Ras protein as well.
* Barbieri L, Luchinat E, Banci L (2016): Characterization of proteins by in-cell NMR spectroscopy in cultured mammalian cells, Nat Protoc. 11(6), 1101-1111.
Katalin Uray, Gyula Pálfy, István Vida