Tracking Photoinduced Primary Events with Femtosecond Stimulated Raman Spectroscopy and Calculations Public Deposited

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  • Femtosecond stimulated Raman spectroscopy (FSRS) is a powerful ultrafast technique which can track photoinduced excited state structural events on femtosecond (fs) to picosecond (ps) timescales. In addition to high temporal and spectral resolutions, FSRS provides a broad spectral window from ca. 100—2000 cm-1 for detection, enabling the direct mapping of detailed structural evolution in the electronic excited state. With the aid of Gaussian calculations and molecular dynamics (MD) simulations, the structure-function relationships of a model photoacid pyranine (a.k.a. HPTS) and a GFP-based Ca2+ biosensor called GEM-GECO1 can be revealed. The first system I investigated is a widely used photoacid, pyranine (8-hydroxypyrene-1,3,6-trisulfonic acid, or HPTS), as a model system to uncover the previously hidden multidimensional photochemical reaction coordinates of excited state proton transfer (ESPT) in solution. In this work, we proposed the formation of a transient intermediate species, the HPTS-methanol S1 complex, with the shared proton shuttling between HPTS and adjacent solvent molecules. The anharmonic coupling between high- and low-frequency modes revealed the multi-staged pathway for HPTS to efficiently dissipate vibrational energy in the absence of ESPT. Next, we revealed the primary structural events before ESPT in the GEM-GECO1 calcium biosensor by analyzing three main skeletal motions in the low-frequency domain. Initial structural evolution following actinic photoexcitation involves small-scale proton motions on both ends of the chromophore two-ring system, manifesting as the excited-state Raman frequency shift of the 460 and 504 cm−1 modes on the sub-ps timescale. To further elucidate the role played by low-frequency vibrational modes, tunable FSRS was developed in our lab to achieve the desirable pre-resonance Raman enhancement conditions for both the excited state photoacid (PA*) and photobase (PB*) forms of HPTS during ESPT. An intermolecular H-bond stretching mode at ~180 cm-1 was observed in S0 and S1 for the first time and shown to play a functional role in facilitating the transient contact ion-pair formation in the electronic excited state. The anharmonic coupling between this H-bonding mode and a four-ring deformation mode of HPTS within the first 2 ps was also revealed by the unique time-frequency domain approach. Computational methods have also been extensively implemented with calculations performed in our work to aid the interpretation of ground/excited-state Raman spectra and the structural dynamics we experimental acquired. Quantum mechanics calculations were performed for small molecular systems, e.g., HPTS, the three-residue chromophore of a fluorescent protein, to mainly help the vibrational mode assignment at the chemical bond level. On a much larger length scale, molecular dynamics (MD) simulations were performed on proteins to shed light on the local environment around the chromophore, which is highly relevant for the structural evolution after electronic excitation. In particular, different geometries of the chromophore and its surrounding H-bonding network have been shown to correlate well with the ESPT capability and pathway (from proton donor to acceptor) which ultimately govern the fluorescence property of a photoacid (e.g., the embedded chromophore) in protein matrix. My future work will include studying the photoacid structural evolution under ultraviolet excitation and combining quantum/molecular mechanics calculations to better predict the chromophore structural constraints in protein pocket. These findings will further enrich our mechanistic understanding of the photoinduced primary events in condensed phase, which should guide efforts to control photoinduced proton motions for numerous applications in chemical and biological sciences.
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