Phytochromes are photoreceptors in plants, bacteria and algae which are intensively studied for their use in optogenetics. They are located in the cytosol and consist of the apoprotein and a co-valently bound tetrapyrrole chromophore. The aim of this project is to characterise protonation dynamics of the chromophore and the protein binding pocket as well as to detect structural changes in the phytochrome Cph1 upon light activation. A combination of high field ultrafast magic angle spinning (MAS) and dynamic nuclear polarization (DNP) is used. The direct observation of protein structural changes during light activation is enabled by a unique probe-head design, equipped with a light beam. Adding dynamic information to the existing X-ray structures of phyto-chromes will help to complete the picture of the photo-switch process.
Depending on the chromophores π-conjugated electron system, we distinguish between far red (Pfr) and red (Pr) absorbing states of phytochromes, that are converted into one another during the photocycle. After the absorption of a photon in the Pr ground state, the chromophore under-goes a conformational change, flipping the D-ring which induces structural changes in the phyto-chrome binding pocket and the so-called tongue region, the part of the PHY domain that seals the binding pocket. The elucidation of this dynamic process is the major goal in this project.
Proton translocation across membranes is vital to all kingdoms of life. Mechanistically, it relies on characteristic proton flows and modifications of hydrogen bonding patterns, termed protonation dynamics, which can be directly observed by fast magic angle spinning (MAS) NMR. We demonstrated that reversible proton displacement in the active site of bacteriorhodopsin already takes place in its equilibrated dark-state, providing new information on the underlying hydrogen exchange processes.
In particular, MAS NMR reveals proton exchange at D85 and the retinal Schiff base, suggesting a tautomeric equilibrium and thus partial ionization of D85. We provide evidence for a proton cage and detect a preformed proton path between D85 and the proton shuttle R82. The protons at D96 and D85 exchange with water, in line with ab initio molecular dynamics simulations. We propose that retinal isomerization makes the observed proton exchange processes irreversible and delivers a proton towards the extracellular release site.
Reference: Friedrich D, Brüning FN, Nieuwkoop AJ, Netz RR, Hegemann P, Oschkinat H (2020) Collective exchange processes reveal an active site proton cage in bacteriorhodopsin. Commun Biol 3(1), 4. DOI: 10.1038/s42003-019-0733-7
Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen‐evolving center into the thylakoid lumen and stabilizes the catalytic Mn4CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. The pKa values of water‐accessible aspartate and glutamate side‐chain carboxylate groups have been determined by means of NMR‐monitored pH titration experiments. It was found that their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH‐dependent changes in backbone chemical shifts in the area of the lumen‐exposed loops are observed, indicating conformational changes. It was possible to deduce a site‐specific analysis of carboxylate group proton affinities in PsbO that provides a basis for further understanding of proton transport in photosynthesis.
Reference: Gerland L, Friedrich D, Hopf L, Donovan EJ, Wallmann A, Erdmann N, Diehl A, Bommer M, Buzar K, Ibrahim M, Schmieder P, Dobbek H, Zouni A, Bondar AN, Dau H, Oschkinat H (2020) pH-dependent protonation of surface carboxylate groups in PsbO enables local buffering and triggers structural changes. Chembiochem DOI: 10.1002/cbic.201900739
Research SectionBiological Projects