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Bioinformatic analysis of protein/DNA interactions
Božíková, Paulína ; Schneider, Bohdan (advisor) ; Hašek, Jindřich (referee)
In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.
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Recombinant Fragments of Antibodies
Král, Vlastimil ; Sedláček, Juraj (advisor) ; Pěknicová, Jana (referee) ; Hašek, Jindřich (referee)
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Recombinant Fragments of Antibodies
Král, Vlastimil ; Sedláček, Juraj (advisor) ; Pěknicová, Jana (referee) ; Hašek, Jindřich (referee)
6. CoNct usloN The aim of the thesis was to establish, in the national conditions, technology of preparation of antibody reconrbinant fragrnents and to verífo the cornplete procedure using several model monoclonal antibodies with a potential diagnostic and therapeutic use. For tfuee antibodies' mAb TU-20, M75 and MEM97, recombinant |Ťagments in various formats (scFv fragments monovalent and bivalent, i.e. diabody, and intrabody for intracellular expression) were constructed and for their heterologous expression, vectors allowing expression in E. coli (as cytoplasmic inclusions, periplasmic inclusions and in soluble form) and in Drosophila 32 cells (expression of glycosylated forms of scFv fragments into the medium) were used. In case of proteins expressed in irrsoluble form, especially scFv F|1.2.32, renaturation procedures to obtain active scFv fragments were developed and optimized. The efnect of the length of the linker -(GlyrSer).- (where x is I to 4) connecting the variable domains of the light and heavy chain on the formation of different multimeric forms of scFv rvas studied. For obtaining solell monomeric scFv fragment, the length of 20 amino acid residues tumsd out optimal. Fragnrents rvith a linker l5 residues long. formed a mixture of monomers, dimers and trimers. the proportion of rvhich rvas...
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Bioinformatic analysis of protein/DNA interactions
Božíková, Paulína ; Schneider, Bohdan (advisor) ; Hašek, Jindřich (referee)
In this thesis, we focused on local structural features of the DNA backbone in protein-complexed DNA and non-complexed (naked) DNA, and its dependence on types of a base pairing in DNA, and on the base sequence. To reach this goal we analyzed about 1,400 crystal structures of DNA in complexes with proteins and more than 400 crystal structures of naked DNA. DNA local conformations were structurally classified into 38 dinucleotide conformers ntCs, which were described previously (Svozil et al. Nucleic Acids Res. 2008). The ntC were further clustered into 16 structural alphabet classes ntA to reduce the number of analyzed variables. We assembled base-paired dinucleotides from double helical DNA structures accord- ing to their assigned structural alphabet classes into so called Association matrices. Three basic Association matrices were analyzed; two compare ntA/ntA associations between dinucleotides forming only Watson-Crick base pairs in protein/DNA com- plexes and in naked DNA, respectively; the third one ntA/ntA associations between dinucleotides base-paired also by non-Watson-Crick pairs. We also analyzed As- sociation matrices of dinucleotides as a function of their sequences. The analyzes revealed differences in structural behavior of various ntA and their dependence on dinucleotide sequences.
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Difrakční měření na zdrojích synchrotronového záření
Hašek, Jindřich
The paper summarizes the present state of art in the use of different sources of synchrotron radiation in protein structure determination and discusses possibility of the future development. Current experiences with the newly developed "small intensive sources of radiation" MIRROCLE and LYNCEAN indicate that the practical usage of these sources needs really long and difficult development. The three XFEL instruments presently under construction in Japan, USA and Germany will open new areas for science. They are complementary to current synchrotron equipment used for protein crystallography but cannot substitute it and the current sources of synchrotron radiation of the third generation equipped by undulators will remain dominant at least in the next decade.
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