|
|
Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
|
|
|
Study on conformational changes in proteins using mass spectrometry.
Rosůlek, Michal ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Some proteins and enzymes require presence of their specific ligand, cofaktor or prosthetic group for their activity. Binding of this specific molecule causes conformational changes, which permits to perform their function. In some occasions the identification of conformational changes is difficult. Using chemical cross-linking coupled with mass spectrometry perform complex tool for searching and low resolution visualization of this changes. The aim of this thesis is study of conformational changes induced by binding of calcium ion to calmodulin protein molecule. Calmodulin is a secondary intermediate messenger, which can interact with various proteins. This feature associates with wide dynamical range of calmodulin. Thus calmodulin is the suitable target for identifying conformational changes. After reaction of protein with chemical cross-linkers with different arm length (DSG and DSS) were products of reaction digested by trypsine. Formed linked peptides were separated by high-performance liquid chromatography and analysed followed mass spectrometry. Seven unique intramolecular cross-links were identified. Using isotope unlabeled cross-link reagents in the presence of Ca2+ in combination with using isotope labeled reagents in calcium free conditions we quantified formed lysine-lysine cross-links....
|
|
|
Characterization of cofactor influence on protein structure using mass spectrometry
Rosůlek, Michal ; Novák, Petr (advisor) ; Vaněk, Ondřej (referee)
Bacterial protein WrbA from E. coli is the founding member of a new family of FMN-dependent NAD(P)H oxidoreductases, forming a functional and structural bridge between bacterial flavodoxin and certain mammalian NAD(P)H:quinone oxidoreductase. For these reasons, protein WrbA is recently intensively studied using various analytical and computing methods. Protein WrbA participates in the protection of cells against oxidative stress, but precise function of the protein WrbA in vivo is still unknown. Protein WrbA forms multimers in solutions. In μM concentrations and at low temperature (4 řC) the protein is in the form of a dimer, with increasing temperature becomes tetrameric. Available three-dimensional crystal structure contains the information about the tetrameric form of the protein, the dimeric form has not been structurally characterized. This thesis was focused on the study of the dynamic behavior of protein WrbA in solution using methods of hydrogen-deuterium exchange and chemical cross-linking followed by mass spectrometric analysis with high resolution (FT-ICR). Behavior of the protein was monitored according to the presence of cofactor FMN. Effect of temperature and protein concentration was also studied. Hydrogen-deuterium exchange provided information about solvent accessibility and...
|
|
|
Study on conformational changes in proteins using mass spectrometry.
Rosůlek, Michal ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
Some proteins and enzymes require presence of their specific ligand, cofaktor or prosthetic group for their activity. Binding of this specific molecule causes conformational changes, which permits to perform their function. In some occasions the identification of conformational changes is difficult. Using chemical cross-linking coupled with mass spectrometry perform complex tool for searching and low resolution visualization of this changes. The aim of this thesis is study of conformational changes induced by binding of calcium ion to calmodulin protein molecule. Calmodulin is a secondary intermediate messenger, which can interact with various proteins. This feature associates with wide dynamical range of calmodulin. Thus calmodulin is the suitable target for identifying conformational changes. After reaction of protein with chemical cross-linkers with different arm length (DSG and DSS) were products of reaction digested by trypsine. Formed linked peptides were separated by high-performance liquid chromatography and analysed followed mass spectrometry. Seven unique intramolecular cross-links were identified. Using isotope unlabeled cross-link reagents in the presence of Ca2+ in combination with using isotope labeled reagents in calcium free conditions we quantified formed lysine-lysine cross-links....
|
guest ::
