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Interaction of Cytochromes P450 with Flavodoxin: a theoretical study
Culka, Martin ; Martínek, Václav (advisor) ; Chmelík, Josef (referee)
Cytochromes P450 are diverse group of heme enzymes found in most species on Earth. In humans they are involved in metabolism of foreign compounds or steroids, bacteria employ cytochromes P450 for utilization of various hydrophobic substrates. General reaction catalyzed by cytochromes P450 is monooxygenation, when one atom of oxygen molecule is introduced into the substrate, while the other is reduced producing water. NADPH:cytochrome P450 oxidoreductase or cytochrome b5 usually serves as an electron donor providing electrons needed for activation of oxygen in eukaryotic organisms, in bacteria small FeS proteins or flavoproteins are these electron donors. It was shown earlier that bacterial electron donor flavodoxin could also interact with human cytochromes P450 in vitro. This thesis employs molecular modeling techniques to support a hypothesis that flavodoxin is responsible for reduction of human (1A2, 2A6, 2A13, 2C9, 2C19, 3A4) and bacterial (101A1 a 176A1) cytochromes P450 heterologously expressed in Escherichia coli. An initial guess of possible mutual orientations of cytochrome P450 and flavodoxin was predicted using information-driven protein-protein docking. The stability of these complexes was examined by directed dissociation method. The most stable orientation for each cytochrome P450 was further...
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Protein fractionation and relative quantitation using PF 2D and ITRAQ for biomarker quest
Gadher, S. J. ; Skalníková, Helena ; Halada, Petr ; Řehulka, Pavel ; Chmelík, Josef ; Kovářová, Hana
ProteomeLab PF 2D System - Protein Fractionation in 2 Dimensions (Beckman Coulter, Fullerton, CA, USA) has been developed to fractionate complex protein mixtures by chromatofocusing in the first dimension followed by high-resolution non-porous silica reversed phase chromatography (RP LC) in the second dimension. Despite the high-resolution power of ProteomeLab PF 2D, UV-based quantitation could be compromised due to possible co-elution of several proteins into one fraction. Hence, we present an optimized protocol for application of isobaric tags for relative and absolute quantitation (iTRAQ) and MALDI-TOF/TOF mass spectrometry to obtain quantitative data from peptides derived by tryptic digestions of intact proteins fractionated by ProteomeLab PF 2D technique. To demonstrate the feasibility of such an approach, protein expression patterns obtained from the ProteomeLab PF 2D fractionation of human T-lymphoblastic leukemia CEM cell line were utilised.
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Influence of the laser power and analyte concentration on the results in MALDI-TOF MS of oligosaccharides
Čáslavský, Josef ; Chmelík, Josef
MALDI-TOF MS has been successfully applied for the analysis of a wide range of organic biopolymers including oligosaccharides. We focus our attention to the group of oligosaccharides occuring in beer and in malting and mashing products, which are mainly oligosaccharides containing from 3 to more than 30 glucose units in their molecules. We tried to evaluate various factors influencing the obtained results. We found that the shape of the spectrum depends significantly on the energy of ionising laser pulse and also on the amount of the analyte on the MALDI target. These effects were studied with standard mixtures as well as with real samples.
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