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Application of chiroptical techniques for exploration of inhomogeneous systems
Jungwirth, Jakub ; Bouř, Petr (advisor) ; Burda, Jaroslav (referee)
Master's Thesis Abstract Jakub Jungwirth Understanding molecular structure of biochemically relevant molecules is of funda- mental interest for these molecules ultimately determine all functions of living organisms. Raman optical activity (ROA) is a chiroptical spectroscopic technique highly sensitive to molecular structure. This thesis presents an introduction to important concepts of ROA and two independent projects aiming to extend the possibilities of ROA, both from the- oretical and experimental points of view. The first project is a conformational analysis of dialanine, an important model peptide. A combined quantum mechanics / molecu- lar dynamics approach was used in spectral simulations and resulted in spectra with an unprecedented agreement with experiment. To obtain information about conformer equi- libria, a decomposition procedure of an experimental spectrum into calculated individual conformer spectra was coded and tested, and proved to be a viable approach. The sec- ond project was an attempt to carry out pioneering ROA measurements of amyloid fibrils, which are difficult to measure due to their inhomogeneous nature (insolubility, birefrin- gence). Within this project, the preparation protocol for such samples was improved. The performance of an all new rotational cuvette was examined and found...
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Characterization of proteins of 2'-5' oligoadenylate pathway by means of vibrational spectroscopy
Víšová, Ivana ; Kopecký, Vladimír (advisor) ; Bednárová, Lucie (referee)
The work concerns to structural characterization of two important proteins of 2'-5' oligoadenylate pathway participating in an immune response of organism to a viral infection. Studied proteins were ankyrin domain of mouse RNase L, the C-terminal part of human phosphodiesterase 12 and the complete human phosphodiesterase 12. The proteins were characterized by Raman spectroscopy, infrared spectroscopy, electronic circular dichroism, dynamic light scattering and in addition by two non-spectroscopic methods- differential calorimetry and electrophoresis. For each protein the secondary structures, thermal stability, weight of oligomers and generally a basic characterization by above mentioned methods were provided.
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Therapeutic use of alternative protein binders targeting tumor biomarkers in clinical testing of oncology patients
Tauš, Petr ; Drbal, Karel (advisor) ; Lepšík, Martin (referee)
Almost until the end of the last century, antibodies (aka immunoglobulins) were considered the only class of specific binding proteins. The discovery of hybridoma technology in 1975 had enabled the production of monoclonal antibodies and after twenty years some of them have entered clinical practice. Meanwhile, the first non-immunoglobulin protein scaffold, in which new specific binding sites could be introduced was discovered. To date, many different alternative scaffolds have been described, but only a few of them are being further developed for diagnostics, therapeutics or tools in basic research. Since these structures are overcoming the drawbacks of immunoglobulin structure, which are big size, expensive production and difficult rational design, they have potential to replace and exceed them. In this bachelor's thesis all the alternative scaffolds in development are summarized. Moreover, their advancements in clinical trials are described and compared with approved therapeutics based on immunoglobulin structure.
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Analysis of substrate specificity and mechanism of GlpG, an intramembrane protease of the rhomboid family.
Peclinovská, Lucie ; Stříšovský, Kvido (advisor) ; Konvalinka, Jan (referee)
Membrane proteins of the rhomboid-family are evolutionarily widely conserved and include rhomboid intramembrane serine proteases and rhomboid-like proteins. The latter have lost their catalytic activity in evolution but retained the ability to bind transmembrane helices. Rhomboid-family proteins play important roles in intercellular signalling, membrane protein quality control and trafficking, mitochondrial dynamics, parasite invasion and wound healing. Their medical potential is steeply increasing, but in contrast to that, their mechanistic and structural understanding lags behind. Rhomboid protease GlpG from E.coli has become the main model rhomboid-family protein and the main model intramembrane protease - it was the first one whose X-ray structure was solved. GlpG cleaves single-pass transmembrane proteins in their transmembrane helix, but how substrates bind to GlpG and how is substrate specificity achieved is still poorly understood. This thesis investigates the importance of the transmembrane helix of the substrate in its recognition by GlpG using mainly enzyme kinetics and site-directed mutagenesis. We find that the transmembrane helix of the substrate contributes significantly to the binding affinity to the enzyme, hence to cleavage efficiency, but it also plays a role in cleavage site...
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Analysis of the mechanism of action of metallacarborane inhibitors of HIV PR
Svoboda, Michal ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
English Abstract Shortly after the identification of HIV as a causative agent of AIDS, an aspartic protease was identified in the viral genetic information. The very same time protease has become one of the dominant therapeutical targets in AIDS therapy. The introduction of protease inhibitors into the antiretroviral therapy has led to a significant improvement in the quality and length of life of HIV patients. However, the virus is still able to effectively prevent the impact of an inhibitor via generating inhibitor-resistant mutated protease variants. Thus, there is a constant need for novel types of inhibitors that would be capable of effectively blocking these resistant variants and simultaneously not supporting the development of novel resistant viral strains. One way to identify such inhibitors could be searching for compounds interacting with the enzyme at different sites than the active cavity, via the mechanisms of noncompetitive or uncompetitive inhibition. The group of compounds called metallacarboranes - inorganic compounds consisting of carbon, boron, hydrogen and metall ion - were shown to exhibit such an activity against HIV-1 protease. However, for further optimization of these inhibitors, detailed biophysical investigation of the enzyme-inhibitor complex is needed. This work focuses on the...
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Mechanism of action of non-peptide inhibitors of HIV protease
Began, Jakub ; Konvalinka, Jan (advisor) ; Bořek Dohalská, Lucie (referee)
The inhibition of HIV-1 protease plays an important role in combating HIV. Nine HIV-1 protease inhibitors have been succesfully marketed for the treatment since 1995. However, their efficiencies decrease due to the resistance development. More potent compounds with novel structural motifs and mechanisms of action are therefore still needed. Several inhibitory compounds have been reported to bind to the protease at the loci different from the active site. Interestingly, darunavir, which is the last approved inhibitor with supposedly competitive mode of action, was also suggested to bind to the flap region of the protease. Two studies discussed this alternative binding mode based on the X-ray structural and kinetic analysis, respectively. Nevertheless, it is questionable, if such a mechanism is relevant also in physiological conditions or if it is only an artifact of crystallization. Another study provided a strong evidence for the alternative binding of darunavir to highly mutated HIV-1 protease. Based on thermodynamic analysis, it was shown that two molecules of darunavir bind to the protease dimer. Surprisingly, this observation was not confirmed by the X-ray structure analysis since the inhibitor was bound only within the active site. However, this protease variant was employed in further...
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Protein Domains Prediction
Valenta, Martin ; Martínek, Tomáš (referee) ; Burgetová, Ivana (advisor)
The work is focused on the area of the proteins and their domains. It also briefly describes gathering methods of the protein´s structure at the various levels of the hierarchy. This is followed by examining of existing tools for protein´s domains prediction and databases consisting of domain´s information. In the next part of the work selected representatives of prediction methods are introduced. These methods work with the information about the internal structure of the molecule or the amino acid sequence. The appropriate chapter outlines applied procedure of domains´ boundaries prediction. The prediction is derived from the primary structure of the protein, using a neural network The implemented procedure and its possibility of further development in the related thesis are introduced at the conclusion of this work.
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Structure-modelling studies of the haloalkane dehalogenase LinB
ŘEŽÁBEK, Josef
The main aim of this bachelor thesis was structure-dynamics study of LinB86 mutant variant protein isolated from the soil bacterium Sphingobium japonicum UT26. Standard and advanced protein crystallization techniques, basis of solving protein structures from diffraction data and study of enzyme structure and dynamics with the use of standard SW for molecular modelling were applied.
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