Studien zur Konversion photochemischer und funktioneller Eigenschaften verschiedener Blaulichtrezeptoren

In BLUF domain containing proteins and DNA-photolyases, a noncovalently bound FAD chromophore interacts with blue-light and initiates a signalling cascade and DNA-repair, respectively. In both blue-light receptor classes, different subtype specific functional properties were observed. One part of this study was the conversion of the E. coli YcgF-(1-137) BLUF domain, a member of subtype II, into subtype I (e.g. SyPixD). To this purpose, various surface mutants of two amino acids that are not conserved in the E. coli YcgF-(1-137) BLUF domain (Met 23 and Ala 90) were characterized biophysically. Here, the successful conversion of the subtype II into the BLUF domain subtype I was shown by changed photochemical properties. Furthermore, on the basis of these photochemical variances was shown that the native amino acids Met 23 as well as Ala 90 are responsible for a major part of these E. coli YcgF BLUF domain specific characteristics. These changed characteristics include blue-shifted absorption maxima of the signalling state, an accelerated light-dark-conversion, the flavin triplet states and light-induced conformational alteration of the inserted Trp 90. Additionally, by producing the Y7F surface mutant it was shown, that this tyrosine residue, being conserved in all BLUF photoreceptors, is essential for the photo switch of the E. coli YcgF BLUF domain, too. Due to the lacking light induced red-shift and a long-living radical flavin species the Y7F mutant was presented as an intermediate of the BLUF photocycle that get arrested between the ground state and the formation of the radical pair. Based on these and previous determined results a possible blue-light induced photocycle and a signal transduction pathway was proposed for the E. coli YcgF BLUF domain. A further aim of this study was to convert the A. thaliana (6-4) photolyase into the CPD photolyases subtype. Accordingly, surface mutants were produced, which contain in their catalytic site amino acid residues mediating the binding and repair of the CPD lesion instead of the native amino acids required for the binding of the (6-4) lesion. For all purified A. thaliana (6-4) photolyases mutants, the catalytic active, completely reduced FADH- state was generated like in the wild type. In the case of the K246R, H364N_L365R and W408Y variants a slightly increased binding affinity to the CPD lesion was observed. Furthermore it could be pointed out that the inserted changes are not sufficient to induce CPD repair activity in the A. thaliana (6-4) enzyme. Hence, the conversion of the (6-4) photolyase into the CPD subtype was not successful, in contrast to the YcgF BLUF blue-light photoreceptor. The different binding behaviour was documented to be responsible for this negative observation Here, distinct varieties were shown in the manner of the flipping mechanism during DNA binding, in the degree of deformation and in the orientation of the bound duplex DNA. In one last subproject, the (6-4) photolyase from the halophilic eucaryote D. salina was investigated. This (6-4) photolyase possesses a very stable neutral semichinoide FAD state in vitro, which presents a possible signalling state similar to plant and animal cryptochromes. Additionally, structural changes in the C-terminal a-helical extension of the D. salina (6-4) photolyase induced by photo activation, that are potentially essential for the interaction between the half reduced enzyme and a signalling partner could be observed. On the basis of these results, an additional cryptochrome activity was proposed for the D. salina (6-4) photolyase, similar to the A. nidulans cryptochrome A, and a potential mechanism was presented that combines both functionalities. Thus, the D. salina (6-4) enzyme may represent a link between photolyases and cryptochromes.