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Modelling of tick borne enecephalitis virus (TBEV) proteins and their complexes
HALBEISEN, Marco
In this bachelor thesis the focus was on the study of non-structural proteins of the tick-borne encephalitis virus involved in the replicatory process of the virus. Non-structural protein number 3 (NS3) and number 5 (NS5) contain a helicase and an RNA-dependent RNA polymerase domain, respectively, and play a crucial role in the life cycle of the virus. Therefore, they are of interest to develop a potential antiviral drug inhibiting the replication of the pathogen. It has been found that these two proteins form a dimer during the replication of the viral RNA. The aim of the thesis was to identify the exact interaction of the two proteins with existing hydrogen-deuterium mass spectroscopy data as well as modelling the RNA into the complexes. The research was conducted by using computational biology tools including homology modelling using MODELLER, manual modelling in PyMOL and Chimera as well as Molecular Dynamics Simulations in GROMACS.
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Action at a Distance in Arginine Repressor and the Store-operated Calcium Channel Orai: Molecular Modeling and Simulations
PANDEY, Saurabh Kumar
In this thesis molecular modeling tools have been applied to investigate the phenomenon of allostery in two systems: Arginine repressor and human Orai channel. Arginine repressor protein binds to DNA in response to the intracellular concentration of L-arginine and controls the arginine metabolism in bacteria. Using molecular dynamics simulations sampling of the conformational space of arginine repressor and the allosteric effects of L- arginine binding on structure and dynamics of systems were studied. Orai channels are responsible for entry of calcium ion in the cell in response to Ca 2+ depletion in the endoplasmic reticulum. Homology modeling was applied to prepare a structural model of human Orai3. Molecular dynamics simulations of the earlier published Orai1 model and the Orai3 model described here were performed. Results pointed to differences in the structures and dynamics of the two Orai isoforms, Orai1 and Orai3 and consequent effects on the channel which are probably responsible for the different behavior of Orai isoforms. A putative cholesterol binding site was identified using in silico docking approach and possible effects of cholesterol binding on the Orai1 channel structure and function were reported. Allosteric effects of mutations on a distant cholesterol- binding pocket was investigated.
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New regulatory mechanisms of microtubule nucleation
Černohorská, Markéta ; Dráber, Pavel (advisor) ; Binarová, Pavla (referee) ; Hašek, Jiří (referee)
MT nucleation from γ-tubulin complexes, located at centrosome, is an essential step in the formation of MT cytoskeleton. In mammalian cells, -tubulin is encoded by two genes. We functionally characterized two γ-tubulin proteins and have found that both are functionally equivalent. γ-Tubulin 2 is able to substitute for γ-tubulin 1 in MT nucleation. However, we revealed that unlike TUBG1, TUBG2 expression is downregulated in mouse preimplantation development. Mast cells represent effectors of the allergy reaction. Their activation by antigen induces number of cellular processes such as degranulation, proliferation and cytoskeleton rearrangements. The regulatory mechanisms of MT reorganization during mast cell activation are unknown. We identified new signaling proteins, GIT1 and PIX that interact with - tubulin. Depletion of GIT1 or PIX leads to changes in MT nucleation. GIT1 is phosphorylated on tyrosine and associates with γ-tubulin in a Ca2+ -dependent manner. Our data suggested a novel signaling pathway for MT rearrangement in mast cells where tyrosine kinase-activated GIT1 and βPIX work in concert with Ca2+ signaling to regulate MT nucleation. We tested the capability of GIT1 and PIX to influence -tubulin function in more cell types. We found out that GIT1/βPIX signaling proteins together...
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Simulation of the interaction of steroid allosteric modulators of NMDA receptors with membrane
Riedlová, Kamila ; Konopásek, Ivo (advisor) ; Novák, Josef (referee)
Molecular dynamics (MD) method allows the real-time monitoring of the system composed of molecules and atoms, such as phospholipid bilayer or biomolecule. Applications of MD are very common in drug design where the real experimental procedures could be much more financially- and time-consuming or even impossible. The aim of this project is to explain the applications and advantages of MD method in case of studies of lipid membranes, with a special emphasis on a study od neurosteriod behaviour in lipid bilayer. Properly designed and synthesized neurosteroids could be used for the treatment of the serious neurological diseases. This work also included the experimental data obtained by MD simulations for two neurosteroids - pregnanolone glutamate and pregnanolone sulphate. Behaviour of this molecules in model membranes was observed and analyzed by MD simulations. Key words: molecular dynamics simulation, model membrane, lipid bilayer, NMDA receptor, neurosteroids, pregnanolone glutamate, pregnanolone sulfate
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Membrane protein interactions studied on single molecular level by force spectroscopy, optical spectroscopy and methods of computational biochemistry
MATĚNOVÁ, Martina
I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).
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Mechanisms involved in sodium uptake activation by the Tumor Necrosis Factor-derived TIP peptide
DULEBO, Alexander
The Tumor Necrosis Factor derived-TIP peptide is a small 17 amino acids cyclic peptide with lectin-like activity, that possesses several therapeutically relevant biological activities, among which is activation of alveolar liquid clearance in both healthy and injured lungs in vivo. Accumulation of fluid in the lungs? alveoli and interstitial spaces is a life-threatening condition called pulmonary edema. The mortality rate due permeability pulmonary edema, accompanied by a dysfunction of the alveolar/capillary barrier, is high because no effective treatment lacking side effects exists nowadays. It is known that the TIP peptide is able to activate vectorial Na+ transport ? which mediates lung liquid clearance. However, the mechanism of action of remains elusive. The aim of this thesis was to investigate the initial steps of interaction between the TIP peptide and airway epithelial cells. Numerous novel methods and single-molecule techniques were used to unravel: (i) how the TIP peptide interacts with the molecules on the apical side of the lung epithelial cells; (ii) whether the TIP peptide need to be internalized inside of the cells to trigger its effects; (iii) the nature of the interaction between the TIP peptide and its putative receptor(s); (iv) the putative receptor(s) for the TIP peptide on the apical surface of the lung epithelial cells.
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Software for molecular dynamics
ŠILHAVÁ, Kristýna
The software for molecular dynamics provides the ability of simulating systems that consist of millions of particles, just for a few nanoseconds. This method allows for the carrying out of virtual experiments without real laboratory equipment and only with computer technology. Molecular dynamics play an important role in understanding the structure and function of biological, organic and inorganic systems. The objective of this thesis was to introduce the basics of molecular dynamics, solve model tasks in Amber software and Gromacs and to compare their capabilities. I have worked on the Hermes Computer Cluster that belongs to the Faculty of Science at the University of South Bohemia and this was part of the MetaCentrum Project. I have worked with both programs using the command line of the Linux Operating System. This thesis describes all of the file formats that are needed for working with these programs, and how they are to be used. I have built up several systems in Gromacs and Amber: 1) water 2) water and ions 3) solvated lysozyme. Based upon these sample systems, I have compared the basic utilities for preparation, the execution and the analysis of these simulations. This thesis should serve as a quick introduction and familiarization with Amber software and Gromacs.
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Membrane protein interactions studied on single molecular level by force spectroscopy, optical spectroscopy and methods of computational biochemistry
MATĚNOVÁ, Martina
I have set for a challenging study that combined experimental and theoretical approaches in an attempt to resolve a role of small aminoacids in intermolecular interactions. First, I have proposed a hypothesis that described the interaction among individual aminoacids forming D helices of D1 and D2 proteins based on molecular dynamic simulations of a simplified model representing the reaction centre of photosystem II. Stability of the putative interhelical hydrogen bond network connecting D1 and D2 proteins was investigated experimentally with dynamic force spectroscopy using atomic force microscope. The results of both methods are in a full agreement with each other and reveal the key role of D1-Gly208 aminoacid in stability and functionality of photosystem II by providing milieu for weak interactions among three contact points at the cross of D helices: D1-Gly208 (O) and D2-Cys211 (O?), D1-Ser209 (O?) and D2-Ile204 (O), D1-Ser212 (O?) and D2-Gly207 (O). Mutation of the D1-Gly208 led to the increase in probability of the binding among the aforementioned aminoacids, undesirably strengthening the overall interactions among the proteins compromising photosynthetic capacity (D1-Ser208) or disabling of autotrophic growth (D1-Val208).
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Large model simulations in molecular dynamics problems
Pelikán, Vladimír ; Hora, Petr ; Machová, Anna
Molecular dynamics simulation is a well-established techniquefor modelling complex many-particle systems in diverse areas of physics andchemistry. The computational requirements of simulations of large systems,especially when taking into account long-range interactions, are enormous.Two basic tasks in molecular dynamics simulation are surface relaxation andheating. Both of these tasks are studied in this contribution.
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