We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing... Show moreWe developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule fluorescence correlation spectroscopy (FCS) of the fluorescently labeled Copper azurin in solution reveals how the position of the label in the 3-D structure of the protein affects the redox kinetics of the redox center as well as the label. Under certain redox conditions, we have been able to observe a microsecond dynamics for intramolecular ET reaction between the label and the metal center in azurin. Our results show that this FRET technique can be profitably used to study the enzyme activity of dye-labeled oxidoreductases. Show less
The work described in this thesis was aimed at the study of the functional properties of (isolated and purified) biomolecular systems at the single-molecule level. Two prerequisites are essential... Show moreThe work described in this thesis was aimed at the study of the functional properties of (isolated and purified) biomolecular systems at the single-molecule level. Two prerequisites are essential for successfully achieving this goal. First of all, single biomolecules should be observable, which means that they should be natively fluorescent or they should be rendered fluorescent by suitable biochemical or biomolecular 12 engineering. The other challenge is to engineer the system in such a way that the fluorescence intensity reports the actual, functional state of the biomolecule. Show less
The investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool... Show moreThe investigation of electron-transfer (ET) processes, as well as redox reactions is important to understand a whole series of biochemical processes. Single-molecule techniques are a precious tool whose diffusion and technical evolution made them available for the study of biologically relevant reactions. In this thesis an approach to the study of electron-transfer and redox reaction by means of single-molecule techniques is presented. Two proteins were investigated: azurin, from Pseudomonas aeruginosa, which contains a type-1 copper center, and the blue nitrite reductase (bNiR), from Alcaligenes xylosoxidans, which contains a type-1 and a type-2 copper center. By means of a resonant energy transfer (FRET) __ based approach it was possible to use a fluorescent label attached to the surface of the proteins to obtain information about their redox state. By using fluorescence correlation spectroscopy in solution it was possible to study ET processes between azurin and the label and between two covalently linked azurin monomers. Nitrite reductase was immobilized in an agarose matrix and single enzyme molecules were investigated by using scanning confocal microscopy: new details about the internal ET between the two copper centers of bNiR were revealed and new light was shed on the catalytic cycle of the enzyme. Show less
Spin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in... Show moreSpin labeling and electron paramagnetic resonance (EPR) have been employed to study structure and dynamics of proteins. The surface polarity of four single cysteine mutants of the Zn-azurin in frozen solution were studied using 275 GHz EPR (J-band), with the advantage compared to 9 GHz (X-band) and 95 GHz (W-band) to allow differentiating between sites having small differences in polarity. The polarity/proticity profiles of the four mutants have been obtained and compared with data already reported in literature of spin labels in different solvents. In order to detect distances at the nm scale, azurin was used as a known model system for double electron-electron spin resonance. The conformation of the spin label linker was modelled. The model was found to be in good agreement with experimental results and can be extended to other proteins. The dynamics of the transient complex of Nostoc sp. PCC 7119 cytochrome f __ plastocyanin was investigated by NMR using the paramagnetic relaxation enhancements. The experimental distance restraints, used in docking calculations, are best interpreted by the presence of a dynamic ensemble of protein-protein orientations within the complex, rather than by a single, well-defined structure, and can be described with an encounter complex model. Show less
To be able to perform Molecular Dynamics (MD) simulations of proteins, a model is needed to describe the interactions between the atoms witin the protein. The interactions between a metal-ion, in... Show moreTo be able to perform Molecular Dynamics (MD) simulations of proteins, a model is needed to describe the interactions between the atoms witin the protein. The interactions between a metal-ion, in this case copper, and a ligand can vary strongly so that a standard set can not be used. This thesis describes the development of a non-bonded and a covalently bonded force field for the copper-site by using an empirically resp. quantum chemically derived charge distribution. The force fields were applied on several instances. 1) It appears that the possible involvement of a mobile loop after substrate binding to the copper-containing quercetinase may help keeping the substrate in place. 2) It turns out that the thermodynamic integration method only allows qualitative predictions of the variation in redox potential observed for azurin variants. 3) The dynamics of the hydrophobic core of azurin variants were analysed in light of previous time dependent phosphorescence experiments. They stress the importance of charge and dipole variations of the surroundings of the tryptophane in the structure for its phosphorescence properties. 4) Finally MD simulations show that a loop, carrying an engineered cysteine, is able to undergo a large conformational change enabling to co-ordinate the copper site of azurin. Show less
