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
