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General light triggered switches and sensors for studying proteins inside the cell

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.

Field of science

  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins
  • /medical and health sciences/clinical medicine/oncology/cancer

Call for proposal

ERC-2010-AdG_20100317
See other projects for this call

Funding Scheme

ERC-AG - ERC Advanced Grant

Host institution

UNIVERSITAT ZURICH
Address
Ramistrasse 71
8006 Zürich
Switzerland
Activity type
Other
EU contribution
€ 2 158 800
Principal investigator
Andreas Georg Plückthun (Prof.)
Administrative Contact
Andreas Plückthun (Prof.)

Beneficiaries (1)

UNIVERSITAT ZURICH
Switzerland
EU contribution
€ 2 158 800
Address
Ramistrasse 71
8006 Zürich
Activity type
Other
Principal investigator
Andreas Georg Plückthun (Prof.)
Administrative Contact
Andreas Plückthun (Prof.)