Structural studies of proteins in intracellular environment: design and characterization of suitable cellular systems

The aim of this PhD project was to design a suitable system to study a protein in its physiological conditions. This methodology becomes more intriguing when the selected molecule is an intrinsically disordered domain (IDD), whose folding may be induced by its interaction with the partners. Our target has been the IDD c-prune. Firstly we tried to obtain a fusion protein able to introduce c-prune in the cell to perform in cell-NMR spectra. To achieve this goal we have studied the mechanism of internalization of this domain in eukaryotic HeLa cell line using different CPPs. Successively the behavior of c-prune in cell lysates of eukaryotic cells has been evaluated. In particular we focused on the use of gH625 for delivery of c-prune. The peculiar properties of gH625 render it an optimal candidate to act as a carrier for net acidic charged molecules by comparison with the positively charged TAT. Furthermore this study is focused also on the interaction studies between c-prune and its different partners over-expressed in human cells via NMR spectroscopy. Besides Nm23-H1, the interaction with GSK-3beta, ASAP1 and gelsolin has also been studied. All these proteins are involved in different physiological processes that enable cells to control their shape and motility and that are strictly linked to tumor progression.