Since grant agencies take sometimes longer than expected to decide whether or not a certain project should get funded, I was running out of money. Prof. Dr. E. Bornberg-Bauer jumped in and funded me for half a year. During that time I helped teaching bioinformatics II and finish one of my old projects. It is now submitted - more to come :)
The Alexander von Humboldt grant allows german researchers to return to Germany and to settle down into a lab of choice for one year in order to establish ones own group/research profile and apply for grants. I choose the Lab of Prof. Dr. Michael Hippler in Münster. Here I started working on raw mass spec data analysis and a proof of principle that oxidative damage to proteins can be quantitatively estimated by ms. Additionally, I could work with Michael Specht a student of Michael Hippler to finish p3d, the python module for structural bioinformaticians. See [here].
During my PostDoc in Prof. Dr. Marylin Gunner's lab I am working on continuum electrostatics in Proteins and on a Heme-protein survey. The results can be found [here] and [here]. Files have been deposited [here]
To connect the stories of photosystem II and the bacterial reaction centre of Bl. viridis (the T4 mutant), a redox titration of QA in the presence of both herbicides was needed. This was also done by transient laser absorption spectroscopy with a 0.5 ns resolution. This faster time resolution was needed as I was monitoring the faster P+Pheo- charge recombination. This recombination has a half time of about 5 ns and appears if QA is in its reduced form. I could find that bromoxynil shifts the redox potential of QA towards a more negative potential, while DCMU lifts the redox potential compared to the "no addition" control sample. [read more]
The singlet oxygen project was finished by measuring with other spin-traps whether or not the herbicides also enhance the superoxid and/or hydroxyl radicals. Both herbicides produced the same amount of those radicals. Nevertheless I was given the chance to use the spin-trap technique during a co-operation project between Prof. Dr. Paulsen and PD Dr. Anja Krieger-Liszkay. This allowed me to investigate whether the water soluble chlorophyll binding protein (WSCP) efficiently hinders the bound chlorophyll in producing singlet oxygen. [read more]
Further investigation was needed. I therefore used site direct mutagenesis to obtain mutants in Themosynechococcus elongatus which have a modified QA pocket. I could isolate a mutant that shows an accelerated decay of the fluorescence yield and shifted thermoluminescence bands, both indicating that the protein environment was changed due to the mutation in a way that the properties of QA were influenced. [read more]
As a sidetrack I investigated the quinone acceptor side of photosystem II of Thermosynechococcus elongatus. This was possible because the PSII particles showed such a high QB activity. Therefore I could show that the EPR signals of QA(:Fe2+) and QB(:Fe2+) are distinguishable. Furthermore I investigated the temperature dependence of the electron transfer reactions between QA and QB. I could show that the first electron transfer reaction is already inhibited at temperatures at which the second electron transfer step is still functional. [read more]
During my diploma thesis I focused on two projects, both of them investigating the electron transfer reactions which the primary quinone, QA, is involved in.
In chromophores of Blastochloris viridis I investigated the charge recombination between P+QA- by transient laser absorption spectroscopy. This occurs if the donor side (cytochrome c) is oxidised and the acceptor side is inhibited by an herbicide. Now the bacterial reaction centre should only be blocked by bactericides, but the T4 mutant of I. Sinning was shown to be sensitive to herbicides such as DCMU or bromoxynil. I could show that the charge recombination is accelerated in the presence of bromoxynil but not in the presence DCMU. Surprisingly there was no difference in the activation energy within each of the two populations of reaction centres, but the difference induced by the herbicides was simply a shift in the equilibrium constant between the two populations. The two populations are visible as a two phasic recombination kinetic and both phases are associated to two populations that co-exist already in the dark as shown by Sebban and co-workers.
The second part of my thesis was done on enriched photosystem II membranes isolated from spinach. I investigated and compared the singlet oxygen production induced by blocking the reaction centre by using herbicides once more. I could show that, in comparison to DCMU, bromoxynil enhances singlet oxygen production as proposed by Krieger-Liszkay and Rutherford (see TIBS 26, 648-653).
Both projects were completed during my PhD thesis.