Dynamics of guests bound to biomolecules




Pace, Tamara Catherine Selina

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Supramolecular systems are held together by non-covalent forces, and include systems involving the interaction of small molecules with biomolecules such as DNA and proteins. The inherent reversibility of supramolecular systems means that dynamic processes are important for many of the functions achieved. The first objective of this work was to develop methodology to study the dynamics and binding mechanism of small molecules with DNA. Though there is a great deal known about the thermodynamics of molecules binding to DNA there is much less known about the binding dynamics. Aminoxanthones were chosen as appropriate guest molecules, and their photophysics were examined in a number of solvents, showing that both the singlet and triplet excited states are strongly affected by solvent polarity, with the excited state energies decreasing in polar solvents. Laser flash photolysis experiments for quenching of the triplet excited state by nitrite anions in the absence and presence of DNA allowed a residence time of microseconds to be estimated for these guests. These experiments also showed that this methodology is not widely applicable when studying the binding dynamics of small molecules with DNA, and that other fast kinetic techniques are necessary. Laser temperature jump experiments allow measurement of dynamics in supramolecular systems, while avoiding the problems encountered in laser flash photolysis experiments. A custom-built system was developed as an adaptation of systems described in the literature, with the main difference being a laser optimized for the excitation of water, and the ability to operate across a wide dynamic range. The laser and the detection systems for absorbance and fluorescence were successfully implemented and a number of artifacts were eliminated. The expected temperature jump was obtained and signals were detected by both fluorescence and absorption; the signal-to-noise ratio still needs improvement before systematic studies can be carried out. The second objective of this work was to study bimolecular reactions in proteins. Using biomolecules to effectively modulate reactivity in bimolecular reactions requires knowledge of the reaction mechanism. When the dimerization of 2-anthracenecarboxylate (AC) takes place in serum albumin proteins enantiomeric excess (ee) is obtained for the chiral products. There are a number of binding sites for AC in these proteins and it has been shown that a balance between strength of binding and mobility of the reactants is essential to achieve high ee's. In human serum albumin remarkable ee's of more than 80% are achieved. It is important to differentiate between reactant molecules bound to different binding sites so that the binding sites where reaction preferentially forms one enantiomer can be identified. Steady-state and time-resolved fluorescence studies identified two types of 2-anthracenecarboxylate bound to HSA: one that has a short lifetime and is very protected from the bulk aqueous solution, and one that has a longer lifetime, but is exposed to the bulk solution. Further work using binding site inhibitors allowed identification of a third type of molecule that has a longer lifetime, but is very protected from the bulk solution, which is likely the species responsible for the majority of the observed ee.



Molecules, DNA, Supramolecular, Proteins