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Expression and purification of kinase domain of ASK1 kinase.
Bártová, Hana ; Obšil, Tomáš (advisor) ; Gryčová, Lenka (referee)
The goal of this diploma thesis was to find optimal conditions for expression of ASK1 kinase in prokaryotic expression system and to optimize purification protocol which enables preparing of milligram amounts of stable and soluble protein. Different conditions of expression were tested in E. coli cells including temperature of expression, cultivation medium or the length of induction. Different methods of purification were tested during the development of the purification protocol. The final protocol is based on chelate chromatography followed by gel permeation chromatography. The result of the diploma thesis is a protocol that allows preparing 1 mg of pure ASK1 kinase from 1 liter of medium.
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Study of the relations between the structure and function of C - terminal vanilloid receptor TRPV1
Gryčová, Lenka ; Obšil, Tomáš (advisor) ; Heřman, Petr (referee) ; Urbánková, Eva (referee) ; Krůšek, Jan (referee)
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labeling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the Cterminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785...
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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Obšil, Tomáš (advisor) ; Gryčová, Lenka (referee)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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Study of structural differences among 14-3-3 protein isoforms.
Macáková, Eva ; Gryčová, Lenka (referee) ; Obšil, Tomáš (advisor)
The 14-3-3 proteins are a family of important regulatory proteins, found in all eukaryotes, which are involved in many cellular processes. In this diploma thesis, we studied structure/function relationships of 14-3-3 proteins, in this case it was the influence of the structure of H8-H9 loop on the binding affinity in barley isoform hv 14-3-3A and human isoform 14-3-3ζ. According to former results, hv 14-3-3A binds to a ligand with lowest affinity, which could be caused by present of a glycin in H8-H9 loop, while in other isoforms there is a serin on the same position. We measured the binding affinity in protein hv 14-3-3A WT and its mutant, which contained the serin instead of the glycin in H8-H9 loop. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ containing the serin in H8-H9 loop and its mutant, where the the serin was replaced by the glycin. Proteins were expressed in E. coli cells strain BL21(DE3) and then purified. The dissociation constant for the binding of peptide pRaf-259 labeled with fluorophores FITC and ATTO was measured using both the fluorescence correlated spectroscopy and the steady-state fluorescence intensity. Our results showed that in both isoforms the mutation of H8-H9 loop causes decrease in the binding affinity.
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Expression and purification of kinase domain of ASK1 kinase.
Bártová, Hana ; Gryčová, Lenka (referee) ; Obšil, Tomáš (advisor)
The goal of this diploma thesis was to find optimal conditions for expression of ASK1 kinase in prokaryotic expression system and to optimize purification protocol which enables preparing of milligram amounts of stable and soluble protein. Different conditions of expression were tested in E. coli cells including temperature of expression, cultivation medium or the length of induction. Different methods of purification were tested during the development of the purification protocol. The final protocol is based on chelate chromatography followed by gel permeation chromatography. The result of the diploma thesis is a protocol that allows preparing 1 mg of pure ASK1 kinase from 1 liter of medium.
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Study of the relations between the structure and function of C - terminal vanilloid receptor TRPV1
Gryčová, Lenka ; Obšil, Tomáš (advisor) ; Heřman, Petr (referee) ; Urbánková, Eva (referee) ; Krůšek, Jan (referee)
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labeling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. Calmodulin (CaM) is known to play an important role in the regulation of TRP channels activity. Although it has been reported that CaM binds to the Cterminus of TRPV1 (TRPV1-CT), no classic CaM-binding motif was found in this region. In this work, we explored this unusual TRPV1 CaM-binding motif in detail and found that five residues from a putative CaM-binding motif are important for TRPV1-CT's binding to CaM, with arginine R785...
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