Covalent labeling and chemical induced dimerization of fusion proteins

A new method was developed for the specific and covalent labeling of fusion proteins with small organic probes in vitro and in vivo. The method is based on the construction of fusion proteins of the human O6-alkylguanine-DNA alkyltransferase (hAGT) with a protein of interest and the subsequent tethering of a probe to hAGT. Apart from its natural substrate, O6-alkylguanine-DNA, hAGT specifically reacts with DNA-free O6-benzylguanine (BG) derivatives, leading to the covalent attachment of the benzyl group to a reactive cysteine residue. It was demonstrated in the course of this project that the use of BG derivatives, which are modified with a desired reporter group in the para position of the benzyl moiety, enables the labeling of hAGT fusion proteins with this reporter group. Several BG derivatives carrying different reporter groups were synthesized and two major applications of the hAGT tag were established in vitro and in vivo. In the first application the covalent labeling of hAGT fusion proteins by either fluorescent probes or affinity tags was demonstrated. The fluorescence labeling of hAGT fusion proteins was achieved using BG derivatives of the fluorescent probes fluorescein and BODIPY, whereas affinity labeling was realized using BG-derivatives of biotin and digoxigenin. It was shown that all substrates were capable of labeling hAGT in vitro, but labeling in E. coli, yeast or mammalian cells strongly depended on the molecular properties, such as cell-permeability and solubility, of the BG derivative. In a second application, the feasibility of the hAGT tag to trigger the chemical induced dimerization of fusion proteins was investigated. Such an application might potentially be valuable for controlling cellular processes that depend on the dimerization of two biological subunits. First, different BG-methotrexate (BGMtx) hetero-dimers were synthesized to induce the dimerization of hAGT fusion proteins with fusion proteins of the enzyme dihydrofolate reductase (DHFR). The capability of these molecules to induce dimerization in living cells was demonstrated in a so-called yeast three-hybrid system. In this assay, the transcriptional activation of a reporter gene depends on the efficient dimerization of fusion proteins of hAGT and DHFR by BGMtx. In a further approach, the covalent homo-dimerization (cross-linking) of several hAGT fusion proteins was achieved using different BG-BG homo-dimers. The cross-linking was demonstrated in vitro as well as in living cells. The use of dimeric (interacting) proteins as fusion partners of hAGT demonstrated that the hAGT tag together with BG-BG derivatives could be used as proximity sensor for protein-protein interactions. Furthermore, the cross-linking of hAGT fusion proteins targeted to different cellular compartments revealed that the system could also be used to indicate the spatial proximity of fusion proteins that do not necessarily interact with each other, but are located in the same cellular compart