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Clinically relevant protein-protein interactions participating in process of bacterial pathogenesis B. pertusis
Málek, Albert ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Whooping cought in human population was strongly suppressed during the 20th century. But in the past few years, the incidence of whooping cough began to rise. The origin of this disease is a pathogenic gram-negative bacteria Bordetella pertussis, which is becoming resistang to currecently used antibiotics or vaccination. B. pertussis attacks human respiratory system. One of it's virulent factors is adenylate cyclase toxin (ACT). It's secreted extracellularly from bacteria and binds to cytoplasmic membrane of host cells and translocates adenylatecyclase domain (dAC) to cytosol. This enzymatic domain is activated by non- covalent interaction with eukaryotic Calmodulin (CaM). After it's activation, dAC in high concentrations synthesizes cAMP, triggering host cell's apoptosis. We studied protein interaction of dAC with CaM by the PIXL method (Photo Induced Cross Linking) and mass spectrometry (MS). Mutant of dAC, with photo-methionine (pM), incorporated in position of Leucine 240 (dACL240pM) was expressed in transformed E.coli cells B834 in mineral medium containing pM. Expressed protein was isolated by affinite chromatography and characterized by MS (determined incorporation of pM was approximately 50 % in the final protein preparation). We performed a photochemical cross-linking with isolated...
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Clinically relevant protein-protein interactions participating in process of bacterial pathogenesis B. pertusis
Málek, Albert ; Šulc, Miroslav (advisor) ; Černá, Věra (referee)
Whooping cought in human population was strongly suppressed during the 20th century. But in the past few years, the incidence of whooping cough began to rise. The origin of this disease is a pathogenic gram-negative bacteria Bordetella pertussis, which is becoming resistang to currecently used antibiotics or vaccination. B. pertussis attacks human respiratory system. One of it's virulent factors is adenylate cyclase toxin (ACT). It's secreted extracellularly from bacteria and binds to cytoplasmic membrane of host cells and translocates adenylatecyclase domain (dAC) to cytosol. This enzymatic domain is activated by non- covalent interaction with eukaryotic Calmodulin (CaM). After it's activation, dAC in high concentrations synthesizes cAMP, triggering host cell's apoptosis. We studied protein interaction of dAC with CaM by the PIXL method (Photo Induced Cross Linking) and mass spectrometry (MS). Mutant of dAC, with photo-methionine (pM), incorporated in position of Leucine 240 (dACL240pM) was expressed in transformed E.coli cells B834 in mineral medium containing pM. Expressed protein was isolated by affinite chromatography and characterized by MS (determined incorporation of pM was approximately 50 % in the final protein preparation). We performed a photochemical cross-linking with isolated...
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Structure-functional study of electrotransport protein systems
Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kukačka, Zdeněk (referee)
Electron transport processes are an extremely important field of study in modern biochemistry and structural/functional proteomics. Azurin is one of the most utilised model systems for study of redox and electron transport processes in proteins. We have utilised photo-induced crosslinking (PIXL) to study oligomerization of azurin in solution and the effect of L-2-amino-5,5-azi-hexanoic acid (photo-Met) - a structural photoinducible analogue of canonical amino acid Met - on electron transport processes in azurin. The optimisation of expression conditions of recombinant azurin in auxotrophic E. coli B834 cells was done to maximise photo-Met incorporation percentage in azurin sequence (70% incorporation was measured via MALDI-TOF mass spectrometry). Through the optimisation of purification protocol (example: cell disintegration, acid precipitation of proteins, adding metallic ligand during cell sonication) we have increased the purity and yield of final product and reduced the purification time. Final preparations (wild-type azurin (WT) with Met, WT with photo-Met and "All-Phe" mutant (all Trp/Tyr replaced by Phe) with photo-Met) were exposed to intense UV-light (PIXL) and evaluated via UV-VIS spectroscopy and SDS-PAGE. During PIXL experiment some photo-Mets incorporated into azurin were able to: (i)...
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