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Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA)
Beranová, Lenka ; Hof, Martin (advisor) ; Svoboda, Petr (referee) ; Večeř, Jaroslav (referee)
The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dual-focus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be "all or non" for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids - a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in...
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Study of associated anisotropy decay curves of tryptophan fluorescence of proteins
Pultarová, Martina ; Večeř, Jaroslav (advisor) ; Heřman, Petr (referee)
This work focuses on simulation of anisotropy decay curves of a model system where protein P1 with one tryptophan interacts with regulating protein P2 without tryptophan to form a complex P1P2 with simultaneous change of tryptophan fluorescence lifetime of the complex. Under these conditions, the heterogeneous solution of proteins P1 and P1P2 exhibits non- exponential fluorescence anisotropy decay which is deduced also from equations presented in this work. Such curves analytically simulated with and without Poisson noise are the main results of this work. The heterogeneous curves were analyzed using computer programs developed earlier for homogeneous systems. As expected such analysis yields correct values of fluorescence lifetimes but does not recover anisotropy parameters used in simulations. Despite of high intensities of simulated data, anisotropy decay curves of homogeneous solutions are very noisy and can be used only on a relatively short time interval of their decay.
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Advanced fluorescence techniques applied on biomolecules (lipid membranes and DNA)
Beranová, Lenka ; Hof, Martin (advisor) ; Svoboda, Petr (referee) ; Večeř, Jaroslav (referee)
The thesis describes time dependent fluorescence shift method and fluorescence correlation spectroscopy method (FCS) with its extensions FLCS, Z-scan FCS and dual-focus FCS applied on specific problems in DNA and lipid research. Compaction mechanism of a DNA molecule smaller than a resolution of a confocal microscope was elucidated. The process was revealed to be "all or non" for a polycation spermine as a condenser in contrast with the gradual compaction caused by a cationic surfactant. Biophysical properties of a phospholipid bilayer influenced by presence of oxidized phospholipids with truncated sn-2 chain were explored. The dynamics of hydrated functional groups in the headgroup region was proved to get faster while the hydration of the headgroup region increased. These effects are in relation with the reorientation of the short sn-2 chains observed in molecular dynamics simulations. Presence of oxidized species may also influence the lateral diffusion of the lipids - a slight increase of the diffusion coefficient was observed. Decrease of hydration and mobility in the headgroup region was found as an influence of heavy water on the phospholipid membrane. These finding are in line with molecular dynamics simulations which show longer lifetimes of hydrogen bonds between water and lipid molecules in...
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