| Fluorescent proteins, whether intrinsically fluorescent or fluorescent through the incorporation of site-specific probes, serve as useful models and tools for the study of ultrafast processes such as chemical reactions and solvation response in proteins. In this thesis, the role of the protein matrix in mediating these processes is explored.; Green fluorescent protein, originally isolated from the jellyfish Aequorea victoria, has become a widely used probe of protein location in cell biology. Its fluorescence and absorbance properties can be modulated by mutation to further expand its utility. By using ultrafast time-resolved fluorescence and variable temperature fluorescence, the rate of non-radiative excited-state reactions in green fluorescent protein mutants (yellow fluorescent protein and dual-emission green fluorescent protein) were determined. Directed evolution has produced a far red emitting protein called mPlum. The cause of this far-red emission was shown by upconversion spectroscopy to be due to a reorganization of the protein environment surrounding the chromophore after excitation.; The dynamics of electrostatic interactions play an important role in the kinetics and energetics of fundamental biological reactions in proteins. The solvent response in the model protein, GB1, was studied using upconversion and time-correlated single photon counting by site specifically incorporating an environment sensitive probe at seven locations with diverse solvent accessibility. All sites showed an initial subpicosecond solvation that is characteristic of an inertial solvent response. Buried sites showed solvation throughout the entire fluorescent lifetime of the probe while more solvent exposed sites the solvation was concentrated at early times after excitation.; Somatic hypermutation is central to the immune system's response to foreign antigens. Using a combination of time-resolved fluorescence and three pulse photon echo peakshift, it was shown experimentally that antibodies evolved from a promiscuous and flexible germ line antibody to a mature antibody that is both rigid and specific to the antigen. |
