Objective The overall objective is to develop general strategies for creating sensors and switches that work inside cells. They will be engineered from Designed Ankyrin Repeat Proteins (DARPins) as general binding proteins that function inside cells and can be generated against any target by selection and directed evolution. Light-triggered switches will be engineered by placing a LOV domain from phototropin in such a way across the DARPin that it is blocked, and only the light-triggered conformational change makes the DARPin accessible. As a second strategy, DARPins specifically recognizing each one of the two isomers of azobenzene, which can be interconverted by light, will be used within light-dependent cross-linkers. Third, DARPins selected to tightly bind fluorogens, by which these small molecules increase their fluorescence by several orders of magnitude, will be converted into general sensors of the conformation of a target protein working within the cell: large DARPins will be created with overlapping binding sites for the protein of interest and the fluorogen. By using DARPins which can selectively distinguish conformations of the target protein, the conformational changes are made visible in a spatiotemporal manner in an individual cell. As proof of principle, we will generate sensors and switches for the kinase domains of the four ErbB receptors, pivotal in signal transduction in human cancers. To increase the impact of this research further, these novel switches and sensors have to be efficiently brought into cells. For this purpose, adenovirus will be engineered for novel cell tropism, also by using DARPins, to homogenously infect tumor cells to study ErbB signaling and the effect of therapeutics in real time in a receptor-differentiating manner. While tested for the ErbB family as a prototype, the strategies to be developed will be totally general and should open up novel ways of studying signaling within cells in real time and with high spatial resolution. Fields of science natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsengineering and technologyelectrical engineering, electronic engineering, information engineeringelectronic engineeringsensorsmedical and health sciencesclinical medicineoncology Programme(s) FP7-IDEAS-ERC - Specific programme: "Ideas" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013) Topic(s) ERC-AG-LS1 - ERC Advanced Grant - Molecular and Structural Biology and Biochemistry Call for proposal ERC-2010-AdG_20100317 See other projects for this call Funding Scheme ERC-AG - ERC Advanced Grant Coordinator University of Zurich Address Ramistrasse 71 8006 Zurich Switzerland See on map Activity type Higher or Secondary Education Establishments Principal investigator Andreas Georg Plückthun (Prof.) Administrative Contact Andreas Plückthun (Prof.) Links Contact the organisation Opens in new window Website Opens in new window EU contribution No data Beneficiaries (1) Sort alphabetically Sort by EU Contribution Expand all Collapse all University of Zurich Switzerland EU contribution € 2 158 800,00 Address Ramistrasse 71 8006 Zurich See on map Activity type Higher or Secondary Education Establishments Principal investigator Andreas Georg Plückthun (Prof.) Administrative Contact Andreas Plückthun (Prof.) Links Contact the organisation Opens in new window Website Opens in new window Other funding No data
