Exploring conformational dynamics of flavoenzymes with flavin fluorescence relaxation spectroscopy

Research described in this thesis was aimed at gaining more insight into the active-site dynamics of dimeric flavoproteins by means of fluorescence relaxation spectroscopy. Three flavoproteins for which crystallographic data have suggested different types of functionally important motions were chosen as central systems; E. coli glutathione reductase, which displays a local conformational change in the protein environment; E. coli thioredoxin reductase, for which a major domain rotation was proposed to be essential for catalysis; and P. fluorescens p- hydroxybenzoate hydroxylase, in which the isoalloxazine ring of the flavin cofactor itself is mobile during catalysis. For interpretation of fluorescence data in terms of dynamic events in the proteins, explicit attention was paid to the photophysical and dynamic characteristics of the flavin cofactor.Chapter 1 provides a general introduction into the enzyme systems and into the principles and mechanisms of conformational dynamics and fluorescence relaxation spectroscopy.In Chapter 2, the dynamic properties of wild-type E. coli glutathione reductase (GR) are studied in comparison with those of the mutant enzymes GR Y177F and GR Y177G. Emphasis is laid on the relations between fluorescence lifetime patterns, protein dynamics and the mechanisms for fluorescence quenching in proteins. Experimental evidence is provided for the multiple quenching sites model.The implications of the comparative study on the gluthatione reductase enzymes for the interpretation of time-resolved fluorescence anisotropy decays are described in Chapter 3 , where a new mechanism for flavin fluorescence depolarization is proposed.Chapter 4 focuses on the conformational dynamics of E. coli thioredoxin reductase (TrxR) and the mutant enzyme TrxR C138S. Two catalytically important conformational states of the enzyme are detected and characterized by (sub)picosecond time-resolved and spectrally resolved fluorescence techniques. Flavin fluorescence relaxation experiments are combined with steady-state optical techniques to gain insight into the dynamic properties of the enzyme and the conformational equilibrium. The importance of enlarging the time window for the fluorescence detection of dynamic events is discussed.The mobile flavin in p- hydroxybenzoate hydroxylase (PHBH) is subject of a time-resolved fluorescence investigation in Chapter 5 . Different binary (mutant) enzyme/substrate (analogue) complexes are used to direct the conformation of the cofactor. The chapter reflects on possibilities and limitations of ensemble fluorescence lifetime data for studying protein dynamics.In Chapter 6 , a link is created between time-resolved fluorescence data of ensembles of molecules and the molecular dynamics of single molecules as retrieved from molecular dynamics (MD) simulations. Hereto, the system of investigation is simplified to the FAD cofactor, which can exist in both 'open' and 'closed' conformations. MD simulations provide insight into the dynamic behaviour of the free cofactor and into pathways for conformational transitions.Chapter 7 describes the first steps into the world of single-molecule detection through natural flavin fluorescence. Fluorescence Correlation Spectroscopy studies on FAD, FMN and lipoamide dehydrogenase provide a first glance into the future perspectives of detecting single flavoproteins and give an understanding of the specific obstacles that need to be overcome.The thesis is concluded by a summarizing discussion reflecting on the research described in this thesis in relation to developments in the field

Universal Car-Board Unit with Pre-Ignition Control

Tato práce se zabývá návrhem univerzální palubní jednotky pro starší dvoutaktní automobily s řízením předstihu zapalování motoru. Jako doplňková funkce je zobrazování údajů spojených s provozem vozidla. Při návrhu byly zohledněny také požadavky majitelů vozidel, pro která je jednotka určena.This semestral thesis deal with designe of universal car-board unit for old two stroke engine, with pre-ignition control. Next function is displaying information conected with using vehicle. When the proposal to take into account also the requirements of owners of vehicles for which the unit is intended to.