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Phytochromes

Phytochromes are photosensory proteins which regulate various biological processes in plants, bacteria, and fungi. The chromophore of phytochrome is a linear tetrapyrrole which upon absorption of red or far-red light undergoes a photoisomerization and subsequent relaxation processes that are eventually coupled to large-scale conformational changes of the protein.

Resonance Raman (RR) spectroscopy allows observing subtle changes of the chromophore structure due to the (pre-)resonance enhancement of the Raman bands of the chromophore over the bands of the protein matrix. The structural changes of the chromophore during the photocycle result in different RR spectra of the various states of phytochromes. The RR spectra of protein variants with isotopically labeled PCB chromophores show characteristic band shifts which help with identifying individual vibrational modes. Comparison of RR spectra of wild-type proteins with those of samples with site-specific mutations allows elucidating details of the mechanism of the reaction cycle of this photoreceptor.

To complement the experimental RR spectra and to facilitate their analysis, Raman spectra were calculated based on in-vacuo chromophores as well as for quantum-mechanical/quantum-chemical (QM/MM) hybrid model systems. In addition, the underlying molecular dynamics simulations of different phytochromes allow evaluating various details of the protein function, such as amino acid contacts, motion of internal water molecules, or side chain orientations.


Hydrogenases

Elucidation of the three dimensional structure of MBH: before the crystal structure of the MBH became available, we constructed a homology model of the O2-tolerant enzyme using five template structure of standard Hase. The structure of the active site and its immediate environment was validated by comparison of experimental (Zebger / Hildebrandt) and QM/MM calculated vibrational frequencies of the inorganic ligands of the Ni-Fe center.

Elucidation of the geometric arrangement of the inorganic ligands at the active site of NiFe Hase: Since CO and CN are isoelectronic, despite the high resolution of the crystallographic data (1.5 Å for MBH), a definite assignment by means of X-ray diffraction is not possible. A conscientious analysis of the IR spectra (Zebger / Hildebrandt) together with accurate QM/MM calculations of the active site, allowed us to determine the arrangement of the inorganic ligands at the Fe site in [NiFe] Hases. In contrast to the structures published so far, we suggest an arrangement of the inorganic ligands in which the CO resides in the pocket containing the L533, trans to the substrate binding site.

Simulation of enzyme adsorption onto coated electrodes.

The interaction and immobilization of [NiFe] hydrogenases on functionalized surfaces is an important field in bioenergetics and the development of biofuel cells. Therefore, we investigated the adsorption behaviour of the standard [NiFe] hydrogenase of D. gigas on amino-terminated alkanethiol SAMs with different levels of protonation using atom MD simulations. These calculations were in both cases supported by SEIRA measurements (Zebger / Hildebrandt) which showed a clear dependence of the adsorption dynamics and strength on the level of ionization of the surface.


Protein Adsorption on surfaces

In order to obtain a microscopical picture of the dynamical behaviour of proteins adsorbed on surfaces we perform classical molecular dynamics simulations. Detail static information of the interaction at the protein- material surface interface can be achieved with hybrid QM/MM approaches.
Our research is focused in understandig at a molecular level the adsorption of Bone Morphogenetic Protein on surfaces of relevance for implant technology, or the adsorption/immobilization of enzymes, such as Sulfite Oxidase and Hydrogenase, on coated electrode surface. These studies are of relevant for biotechnological applications.

Computation of spectroscopical properties of protein cofactors

Our research is mainly focussed on the calculation of vibrational and electronic properties of protein cofactors. Special emphasis is done on the prediction of Raman spectra. These calculations constitute a powerfull tool for the interpretation of experimental resonance Raman measurments (AK-Hildebrandt) and the extraction of valuable structural information from them. This combined theoretical / experimental approach has been succesfully applied to the elucidation of the structural changes undergone by the phytochromobilin chromophore of Phytochrome A from plant during the photoinduced cycle (JACS 126,16734, 2004).Phytochrome A photocycle.

In order to asses the effect of the protein environment on the RR spectra of photoreceptors, we established a quantum mechanics (QM) /molecular mechanics (MM) for calculating the Raman spectra of protein fragments. The potential of this methods has been tested on the a-subunit of the C-Phycocyanin antenna pigment.

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