Photoaktivierbare Proteine
Final Report Abstract
Within the project we intended to use the photo-caged, non-natural amino acid DMNB-L-Ser as a switch to control protein function. As a proof of principle, we selected two model systems, the yeast ABC transporter Pdr5 and the isolated NBD of the E. coli ABC Transporter HlyB. Both systems were expressed in S. cerevisiae, the organism, in which the orthogonal tRNA / aminoacyl tRNA synthetase pair responsible for the amber stop codon mediated incorporation of DMNB-L-Ser, was operational. After the expression of Pdr5 was optimized and the expression of the HlyB-NBD was established in S. cerevisiae, the incorporation of the nonnatural amino acid was demonstrated in both systems. However, any functional studies of the importance of phosphorylation states of Pdr5 or the influence of the serine residue of the C-loop of Pdr5 could not be analyzed in vitro due to the extremely low expression levels of Pdr5. Selection of those cells expressing the corresponding DMNB-L-Ser mutants of Pdr5 by single cell analysis or FACS was not successful. Thus, functional studies could not be conducted. For the HlyB-NBD, a mutational analysis of serine and threonine residues of conserved motifs identified certain residues that were selected for the incorporation of the non-natural amino acid. In the end, only one system (S634 located in the D-loop) of the NBD could be analyzed. It turned out that biologically non significant times of irradiation (22 minutes) were required to restore ATPase activity. Irradiation of only one minute resulted in an active protein, but the kinetic parameters suggested that the function of the protein was severely modified. In summary one has to state that an efficient incorporation of DMNB-L-Ser into Pdr5 or HlyB-NBD was achieved, but that functional studies with the two systems suggested in thie grant application was not possible. Either due to the expression level (Pdr5) or the time required to de-cage the protein (HlyB-NBD). This is in clear contrast to published studies by the Schultz group. However, in the case of a transcription factor, not all of the corresponding proteins need to be activated, a clear requirement in the case of Pdr5 of the HlyB-NBD.
