Optical properties and photoreactivity of the Green Fluorescent Protein. A theoretical study.

This PhD thesis focuses on the theoretical analysis of Green Fluorescent Protein (GFP) photochemistry. GFP peculiar properties make it and its mutants ideal noninvasive markers in living cells, suited for numerous applications, like reporter of gene expression, cell lineage tracer, measure of protein-protein interactions, signaling and trafficking in cellular systems and useful in many fields, such as molecular biology, biotechnology or medicine. One of the most interesting aspects in the study of the GFPs is the analysis of their photophysics and photochemistry in order to improve the efficiency and efficacy of their applications. In this work we have performed an in-depth analysis of GFP absorption, combining DFT and TD-DFT with ONIOM method, in order to investigate the relation among structure, optical properties and GFP functionality. In addition, evaluating GFP photoinduced behaviour, we have focused our attention on the proton shuttle triggered by excitation and we have tried to shed light on this complex issue proposing an hypothesis for its mechanism. We have reached a fine control on GFPs absorption, a clear understanding of the photophysics and photochemistry changes induced by the molecular environment and a deep omprehension of the relation between GFP structure and properties. These results allow to achieve highly moduled design of fluorescence and to maximize the potentiality of these proteins and use them in a more effective way to improve bioimaging techniques and nanotechnology.