Tools to generate and study molecular switches in living cells
Many cellular switches, including conformational changes, posttranslational modifications, translocations, ion channel openings/closures etc., are happening on the second to minute scale. Investigation of single switches, but also complex signaling events, are often experimentally accessible by inducing the switch on the molecular level inside intact cells. During the last funding period, the expiring project Z1 has provided an extended arsenal of switching tools that is constantly being developed further. The most prominent examples are photoactivat- able, membrane-permeant small signaling molecules including many lipids and chemical dimerizer systems for manipulating events by light, or the addition of a small molecule to cells, respectively, within less than 30 sec. In addition, a series of fluorescent sensors and reporters was developed, useful to study fast changes in cells in real-time. Within this TRR, these tools have been made available to various research groups over the past four years in the context of project Z1. It is the long-term objective of the new project Z04 to 1) provide existing tools originally developed by the Schultz lab to the consortium and 2) to develop new tools adapted to the needs of the consortium members.
To broaden the scope of the Z-project, we furthermore will develop a new technology platform for switching protein function with small molecules in cells. The approach is based on inserting a circularly permutated bacterial dihydrofolate reductase (cpDHFR) into proteins; switching of the activity of the protein is achieved through addition or removal of the cell-permeable drug trimethoprim, which induces a major conformational change in the cpDHFR structure. We have already shown how the affinity of a nanobody towards GFP can be switched on and off in cells. By controlling the cellular localization of GFP fusion proteins, the engineered nanobody allows to study their role in basic biological processes, an approach that should be applicable to numerous GFP fusions. We now want to expand this approach to other nanobodies and enzymes, such as lipases, that are of relevance for this consortium. Furthermore, we will pursue other mechanisms for switching protein function with small molecules.