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Content archived on 2024-05-30

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.

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Topic(s)

Calls for proposals are divided into topics. A topic defines a specific subject or area for which applicants can submit proposals. The description of a topic comprises its specific scope and the expected impact of the funded project.

Call for proposal

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ERC-2010-AdG_20100317
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Funding Scheme

Funding scheme (or “Type of Action”) inside a programme with common features. It specifies: the scope of what is funded; the reimbursement rate; specific evaluation criteria to qualify for funding; and the use of simplified forms of costs like lump sums.

ERC-AG - ERC Advanced Grant

Host institution

University of Zurich
EU contribution
€ 2 158 800,00
Address
RAMISTRASSE 71
8006 ZURICH
Switzerland

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Activity type
Higher or Secondary Education Establishments
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Total cost

The total costs incurred by this organisation to participate in the project, including direct and indirect costs. This amount is a subset of the overall project budget.

No data

Beneficiaries (1)

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