Graduate Thesis Or Dissertation
 

Tracking Excited State Proton Transfer and Inhibition with Ultrafast Raman and Transient Absorption Spectroscopy

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  • In this dissertation, excited state proton transfer (ESPT) and its inhibition in solution and protein environments are revealed using both femtosecond transient absorption (fs-TA) spectroscopy and femtosecond stimulated Raman spectroscopy (FSRS). Using a tunable Raman pump to enhance transient vibrational features of the photoacidic chromophore HPTS in methanol and methanol acetate solution, the Raman modes from ca. 125−1700 cm-1 were collected through the first 600 ps following actinic photoexcitation. In pure methanol, ESPT from the weak photoacid is inhibited and solvation dynamics on a ~10 ps timescale account for the main energy dissipation pathways in the first excited state. Compared to a previous FSRS study performed on HPTS in methanol using an 800 nm Raman pump, the resonance enhancement afforded by a tunable Raman pump resulted in a significant increase in signal intensity allowing detailed analysis of solvation dynamics. To further explore the roles of solvation and vibrational cooling in the absence of ESPT, both Stokes and anti-Stokes FSRS spectra were collected with a series of Raman pump wavelengths spanning the excited state absorption of HTPS in methanol. Dramatic changes in the anti-Stokes lineshape were observed when the anti-Stokes pump and probe photons approach electronic resonance and both contribute to the four-wave mixing schemes that lead to the generation of the stimulated Raman scattering photons from a reactive potential energy surface. In the presence of the 1 M acetate ions, however, bimolecular diffusioncontrolled proton transfer from HPTS to acetate proceeds on a 30 ps timescale in methanol, which is confirmed by the intensity dynamics of vibrational modes assigned to the photoacid and the photobase as well as global analysis of the relevant electronic dynamics. The ESPT reaction onset was also found to be modulated by a low-frequency intermolecular hydrogen-bond stretching mode between the photoacid and the solvent. Analogously in proteins, local environment impacts the ability of the embedded chromophore to undergo ESPT. The dual-emission GEM-GECO1 biosensor consists of green fluorescent protein (GFP), calmodulin (CaM), and light chain kinase subunits, while allosteric binding of the Ca2+ ions changes the hydrogen bonding network around the hydroxy group of the GFP chromophore. In the absence of Ca2+, ESPT modulated by a low-frequency mode occurs on a ~30 ps timescale, much like HTPS in a methanol acetate solution (with green emission). When Ca2+ is bound, the chromophore relaxes within the first excited state over the course of hundreds of ps without undergoing ESPT, reminiscent of HPTS in pure methanol (with blue emission). Increased hydrophobicity and disruption of the H-bonding network around the chromophore in the protein pocket inhibits the proton transfer event. FSRS, especially tunable FSRS, has shown its power and versatility to follow both photoacid and photobase structural dynamics in the excited state and to elucidate contribution of processes like solvation, vibrational cooling, and proton transfer to the overall photochemical reaction.
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