Forschung
Keywords
- Photosynthetic Light reactions
- Structure and Function of Photosystem II
- Stability of Chloroplast Proteins
- Plastidic Proteases
Summary Photosynthesis is a basic process on earth supplying all organisms with carbohydrates and oxygen. In this process light is used as an energy source to fix carbondioxide into carbohydrates and to split water into oxygen and protons. In higher plants and algae, this complex set of reactions takes place in the chloroplast, a specialized cell organell with its own genome. Within the chloroplast so-called thylakoid membranes harbor four major protein complexes catalyzing light-dependent electron transport from water to NADP and the synthesis of ATP. One of these complexes is photosystem II, which drives the oxidationof water and the reduction of plastoquinone. The complex consists of more than 20 subunits, one of which is the D1 protein. This protein has several interesting properties: together with its "sister" protein D2 it binds cofactors like e.g. chlorophylls, iron, manganese and plastoquinone, it is synthesized as a precursor protein and processed at its C-terminus to yield the mature protein, and it has an unusual high turnover in the light. Using the model organism Chlamydomonas reinhardtii (http://www.botany.duke.edu/chlamy ), we are investigating the structure, function, stability and assembly of this protein using biochemical and genetic tools. Of crucial importance for our projects is the generation of mutants: with the help of a specific transformation device (particle gun) it is possible to introduce site directed mutations into the chloroplast or nuclear genome and study their effects on the mutant organism. We are currently also using these techniques to investigate proteases in the chloroplast.
Techniken
Molekularbiologische Techniken:
Klonierung, Sequenzierung, Gerichtete Mutagenese (Austausch von Aminosäuren, Epitop-Insertionen), "random" Mutagenese, Polymerase-Kettenreaktion (PCR), DNA- und RNA-Blotting (Southern- und Northern-Blots), Expression von Fremdproteinen in Bakterien, Transformation von Chloroplasten- und Kerngenomen
Proteinchemische Techniken:
Immunochemische Techniken (Western-Blots, Immunopräzipitationen), Proteinreinigungen (FPLC, Affinitätschromatographie), Polyacrylamid-Gelelektrophorese
Physiologische Techniken:
Messung der Sauerstoffentwicklung (Sauerstoffelektrode), Messung der Fluoreszenz, Proteinsynthese in ganzen Zellen und isolierten Chloroplasten, Pulse- und Pulse-Chase Experimente
Mikrobiologische Techniken:
Algenanzucht, Bakterienanzucht