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Study of interactions between protein kinase CaMKK2 and calmodulin using fluorescence spectroscopy.
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Ca2+ /calmodulin-dependent kinases are members of CaMK family, which is involved in CaMK cascade. One of CaMK family members is Ca2+ /calmodulin-dependent kinase kinase 2 (CaMKK2), which is activated by Ca2+ /CaM-binding. There are some structural differences between CaMKK2 and other protein kinases, one of them is a structure near αE-helix and autoinhibitory domain. Due to the overlap of autoinhibitory domain and Ca2+ /CaM-binding domain it can be supposed that Ca2+ /CaM-binding induces structural changes near autoinhibitory do- main and thus can affect the accessibility of this region. CaMKK2 W445F mutant, which contains only one tryptophane residue Trp374 close to the αE-helix, was expressed and purified. Structural changes in this region were monitored using tryptophan fluorescence intensity quenching experiments, which can provide information about the accessibility of region surrounding tryptophan residue. The fluorescence of Trp374 was quenched using acrylamide. Comparison of fluorescence quenching experiments performed in the presence and absence of calmodulin suggests that the complex formation induces structural change in the region surrounding Trp374 . 1
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Structural study of the complex between the 14-3-3 protein, CaMKK1 and CaMKK1:Ca2+/CaM
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...
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Structural study of the complex between the 14-3-3 protein, CaMKK1 and CaMKK1:Ca2+/CaM
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
The Ca2+ -signaling pathway is an important mechanism of cell signaling. Ca2+ /Cal- modulin (CaM)-dependent protein kinases (CaMKs) are members of Ser/Thr protein kinase family. CaMKs are regulated by Ca2+ /CaM binding in response to increase in intracellular level of Ca2+ . An important member of this protein family is Ca2+ /CaM- dependent protein kinase kinase (CaMKK), which is an upstream activator of CaMKI and CaMKIV. There are two isoforms of CaMKK, CaMKK1 and CaMKK2. CaMKK1 is regulated not only by Ca2+ /CaM-binding, but also by phosphorylation by cAMP-dependent protein kinase A (PKA). PKA phosphorylation induces inter- action with the 14-3-3 proteins. Previous studies of interaction between CaMKK1 and 14-3-3 proteins suggested, that the interaction with 14-3-3 proteins keeps CaMKK1 in the PKA-induced inhibited state and blocks its active site. However, the exact mecha- nism of this inhibition is still unclear mainly due to the absence of structural data. Main aim of this diploma thesis was to characterize the protein complexes between CaMKK1, Ca2+ /CaM and 14-3-3γ using analytical ultracentrifugation, small angle X-ray scattering, and chemical cross-linking coupled to mass spectrometry. Analytical ultracentrifugation revealed concentration-dependent dimerization of CaMKK1, which is...
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Study of interactions between protein kinase CaMKK2 and calmodulin using fluorescence spectroscopy.
Mikulů, Martina ; Obšil, Tomáš (advisor) ; Pavlíček, Jiří (referee)
Ca2+ /calmodulin-dependent kinases are members of CaMK family, which is involved in CaMK cascade. One of CaMK family members is Ca2+ /calmodulin-dependent kinase kinase 2 (CaMKK2), which is activated by Ca2+ /CaM-binding. There are some structural differences between CaMKK2 and other protein kinases, one of them is a structure near αE-helix and autoinhibitory domain. Due to the overlap of autoinhibitory domain and Ca2+ /CaM-binding domain it can be supposed that Ca2+ /CaM-binding induces structural changes near autoinhibitory do- main and thus can affect the accessibility of this region. CaMKK2 W445F mutant, which contains only one tryptophane residue Trp374 close to the αE-helix, was expressed and purified. Structural changes in this region were monitored using tryptophan fluorescence intensity quenching experiments, which can provide information about the accessibility of region surrounding tryptophan residue. The fluorescence of Trp374 was quenched using acrylamide. Comparison of fluorescence quenching experiments performed in the presence and absence of calmodulin suggests that the complex formation induces structural change in the region surrounding Trp374 . 1
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