The project is dedicated to elucidate the mechanisms of the photoinduced structural changes in various phytochromes that (de)activate the biological function via coupling photoisomerization of the tetrapyrrole cofactor with a functional conformational transition of the protein.
The investigations of canonical phytochromes are carried out within a joint project with the group of P. Scheerer (Charité Berlin). The methodical concept is based on combining protein engineering, X-ray crystallography, and vibrational spectroscopy. Protein X-ray crystallography aims at high-resolution structures of the various states of the photoconversion processes using time-resolved free-electron laser techniques and/or conventional synchrotron radiation combined with cryogenic trapping. In addition, we employ cryo-electron microscopy to determine the structure of functionally complete (“full-length”) phytochromes. Cryogenic static and freeze-quench resonance Raman spectroscopy, carried out with frozen “glassy” samples and single crystals, as well as IR difference spectroscopy provide complementary information about the chromophore and protein structural changes including proton transfer reactions. Using prototypical and bathy bacteriophytochromes as well as fungal and plant phytochromes, the structural and spectroscopic studies aim at identifying differences and similarities in the proton-coupled mechanistic patterns of the Pr/Pfr and Pfr/Pr transitions and the specific roles of protonation dynamics for the different modes of photosensor activation in eukaryotic and prokaryotic phytochromes. To elucidate cause-effect relationships ruling the coupling between photoinduced chromophore structural changes, proton translocation, and protein conformational transitions, we determine local electric field changes by exploiting the vibrational Stark effect of nitrile-labelled amino acids. The project is part of the CRC1078 and thus benefits from the mutual interactions with other researchers in the consortium. Additional funding is obtained from the Cluster of Excellence UniSysCat.
In addition, we aim at analyzing the molecular parameters controlling the photoconversion of cyanobacteriochromes. Here, special emphasis is also laid on the implementation of artificial amino acids including nitrile groups that monitor local electric fields. The project is funded by the Einstein Foundation Berlin (Einstein Visiting Fellow Professor Katrina Forest) and the CRC1078.
Collaborations: TU Berlin: M. A. Mroginski; FU Berlin: U. Alexiev, C. Clementi, K. Heyne, R. Schlesinger HU Berlin: F. Bartl; Charité Berlin: P. Scheerer; Univ. Madison, USA: K. Forest; Univ. Gießen: J. Hughes; Hebrew Univ. Israel: I. Schapiro; FMP Berlin: H. Oschkinat.