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Content archived on 2024-05-30

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

Final Report Summary - NEXTBINDERS (General light triggered switches and sensors for studying proteins inside the cell)

In this project, novel strategies and technologies have been developed that allow to monitor specific cellular reaction in real time and real space, and to control them by light. These methodologies are completely general and applicable to almost any cellular reaction.

The basis of all approaches have been specific binding proteins which can be expressed in any compartment in any cell. These Designed Ankyrin Repeat Proteins (DARPins) have been developed over the last few years and can be generated to target any component of a cell specifically. A new library of so-called LoopDARPins has been developed, which have allowed to generate picomolar binders in only a single round of ribosome display, something that has never been reported for any library with any selection technology. This has set the stage for generating many DARPins for this project in high-throughput mode.

Several versions of real-time, real-space sensors have been generated. In a first design, an environmentally sensitive fluorophore was attached to the DARPin such that when the DARPins bind to their target, the dye is in a more hydrophobic environment and its fluorescence greatly increases, thereby indicating in real time and real space a binding event. This system was tested with DARPins binding to the phosphorylated and non-phosphorylated form of the important kinase ERK. Indeed, with this system, the activation (phosphorylation) of ERK could be followed directly in the living cell.

In a second design, we developed fluorogen activators, termed FADAs, based on the DARPin scaffold, which also readily folds in the reducing environment of the cytoplasm. We created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. A prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Again, this technology is totally generic.

We have made great progress towards the design of DARPins which can be switched by light, which will offer a generic strategy to control any protein in the cell since DARPins can be made to essentially any target. The basis has been to fuse a LOV domain which can induce conformational changes due to its bound flavin. This domain was heavily engineered, based on crystal structures and directed evolution, to allow light induced switching.

As we have also made very important progress in cell-specific viral delivery and thus to deliver the genetically encodable sensors and switches, the overall project is on an excellent track to achieve the goals. Using an adapter system for adenovirus, efficiently produced in Escherichia coli, Adenovirus can now be converted rapidly to new receptor specificities using any ligand as the receptor-binding moiety. Prefabricated Ads with different payloads thus can be retargeted readily to many cell types of choice.

In summary, the project has achieved the overall objective of this proposal: to develop general strategies to monitor specific cellular reactions in real time and to be able to control them from the outside by light. The methodologies are completely general and will be applicable to almost any cellular reaction.