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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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Preparation and characterization of human cellular cofactors of retroviral integration.
Čermáková, Kateřina ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular binding partner of Human Immunodeficiency Virus type 1 (HIV-1) integrase. It is a human nuclear protein, which has been implicated in transcriptional regulation and cell survival. The role of LEDGF/p75 in HIV integration is well characterized, the HIV integrase binding domain (IBD) was identified and structural studies, which provide detail information about this interaction, were done. However, very little is known about its physiological function. As a transcriptional co-activator, LEDGF/p75 is implicated not only in HIV replication, but also in human cancer and autoimmunity. Key feature for both, the viral and cellular role of this protein, is its ability to act as a molecular adaptor tethering proteins to the chromatin fiber. Recently, PogZ (Pogo transposable element derived protein with zinc finger domain) was identified and validated as a new cellular interaction partner of LEDGF/p75. It was shown, that their interaction is mediated by IBD of LEDGF/p75 and the C-terminal domain of PogZ. To gain more insight in this interaction, we have initiated structural studies of their complex. Structural information is crucial for understanding the LEDGF/p75 biological role and might help in design of inhibitors selectively blocking...
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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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