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National Repository of Grey Literature 2 records found  Search took 0.01 seconds. 
The novel combinations of experimental approaches: mass spectrometry (MS) and photo-induced surface labelling, electron release (PIER), or cross-linking (PIXL)
 Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...
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The novel combinations of experimental approaches: mass spectrometry (MS) and photo-induced surface labelling, electron release (PIER), or cross-linking (PIXL)
 Tuzhilkin, Roman ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
Countless electron transport/transfer (ET) processes occur in living organisms every day. Therefore, their study is a crucial field of modern structural and functional proteomics. In many cases model proteins like azurin from P. aeruginosa are utilised in experiments. This blue copper protein is favoured due to a characteristic absorbance maximum at 630 nm in Cu(II) redox state of the central Cu atom. During its oxidation to Cu(I) state the A630 value decreases allowing UV-Vis detection of ET reaction progress. We have introduced a structural photoinducible analogue of canonical amino acid Met - L-2-amino-5,5-azihexanoic acid (photo-Met) - into azurin structure to study oligomerization in solution via photo-induced cross-linking (PIXL). Using previously optimised protocols for recombinant expression in E. coli B834 we have inserted photo-Met into azurin moieties: wild type azurin and Az2W mutant where two adjacent W residues with confirmed role in electron hopping across protein-protein interface are present. The incorporation percentage of photo-Met in analysed samples was determined after SDS-PAGE and in-gel protease digestion via MALDI-TOF MS. PIXL was employed to study azurin-azurin interaction and oligomerization under different total concentrations of protein (in range of 15-300 µM). The...
Detailed record

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