Valadan, Mohammadhassan (2015) Revealing Biomolecules Dynamics by UV Ultrafast Spectroscopy. [Tesi di dottorato]

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Establishing a stable covalent bond between proteins and nucleic acids, in molecular biology usually referred to as crosslinking, is a fundamental tool for the identification of the partners in the DNA-protein interaction, the latter being a vital biological process. Crosslinking with fs-UV lasers has been presented in the literature as a revolutionary technique to increase the otherwise low process yield of conventional methods based on chemical catalysts, conventional UV sources, or longer UV pulses. It is known that crosslinking induced in cells by ultrashort laser pulses has a twofold advantage over conventional methods: (i) it binds only species that are in proximity ("zero length" covalent bond) of the absorbed photons rather than favoring unspecific bonds amongst many possible species in the cell and (ii) it only takes place until the radiation is incident on the sample, thus paving the way for time-resolved studies of transient interactions. UV-based cross-link relies upon the large absorption cross section of DNA base in the UV region, although, good for our health, the probability for these photo-induced changes is not as big as the excitation rate since many of the photo-excited species will relax into the ground state in an ultrafast time scale, preventing any change or damage. Using femtosecond lasers is a real step ahead to make the biomolecules photo-react in a reasonable amount, but they also offer the unique possibility for time-resolved measurements thereby allowing the investigation of the basic mechanisms following the photo-activation. While irradiation with a relatively high intensity UV laser greatly increases the efficiency of protein-nucleic acid cross-linking, it is difficult, however, to investigate cross-linking in the presence of very complex molecules, namely DNA and protein, themselves. Therefore, in a reductionist approach, to mimic the bond formation between the DNA base and the nearby proteins the photocyclization in 5-Benzyluracil (5BU) has been proposed as a model system of crosslink reactions, in view of the simultaneous presence of the Uracil and Benzene playing the role of the DNA base and the aromatic residue of a protein, respectively. In order to design an experiment aimed at the measurement of the dynamics of photocyclization, we studied the steady-state absorption and emission (fluorescence) properties of 5BU and 5,6-benzyluracil (5,6BU), the latter being produced by exposing 5BU to UV ultrashort laser pulses. We found that after some time we can assume that all 5BU is completely transformed into 5,6BU and modifications in the absorption and fluorescence spectrum, fluorescence anisotropy, fluorescence quantum yields, and excited state lifetimes are observed when 5BU is photocyclized thus becoming 5,6BU. The high value of anisotropy in 5BU indicates that a large fraction of its fluorescence signal has a very short lifetime, in the range of few picoseconds; whereas in the case of 5,6BU, the main part of fluorescence has a much longer lifetime in the range of; few nanoseconds. The role of solvent is also studied in the photocyclization process and, in particular, we show that using water as the solvent, photocyclization rate is larger than that observed when methanol is used. Such a finding can be explained in terms of different configuration 5BU molecules assume in the solution, thereby providing hints on the paths leading to 5,6BU formation. Time-resolved 5BU fluorescence has been measured in nanosecond and femtosecond regimes by "Time Correlated Single Photon Counting" and "Fluorescence up-conversion" techniques, respectively, finding a very good agreement with a theoretical model (Molecular dynamics modeling). The results described so far allows one to design a possible experiment in which in a pump-probe scheme the photocyclization process can be followed in time. In such a setup an ultrashort UV laser pulse has to be split into two replica: One is used to trigger the interaction (excite the sample and start the photocyclization, for instance); the second pulse is then properly delayed to probe an optical observable like absorption or fluorescence, and eventually monitor the reaction. Of course, laser pulse properties, especially the temporal characteristics, are then important in determining the time resolution of such experiments. A common technique to measure the temporal duration of ultrashort pulses is based on nonlinear phenomena such as second-harmonic-generation; one of the most used experimental schemes is based on noncollinear autocorrelation. Unfortunately, such an approach is not feasible in the UV frequency range because of no crystal allows light propagation in the deep UV. Thus, we used an alternative nonlinear process in autocorrelation measurements: an autocorrelator is set up based on Two-Photon Absorption (TPA). The TPA signal has been measured and the information on the pulse duration has been retrieved form the FWHM of the fitted function. A deeper knowledge of the processes occurring in biomolecules and biological samples, as those reported in this thesis, might enable us to engineer them by preventing the undesirable ones or increasing the rate for the preferred routes.

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