Tracking vibrational motions during a photochemical or photophysical process has gained momentum, due to its sensitivity to the progression of reaction and change of environment. In this work, we implemented an advanced ultrafast vibrational technique, femtosecond-stimulated Raman spectroscopy (FSRS), to monitor the excited state structural evolution of an engineered green fluorescent protein (GFP) single-site mutant S205V. This mutation alters the original excited state proton transfer (ESPT) chain. By strategically tuning the Raman pump to different wavelengths (i.e., 801, 539, and 504 nm) to achieve pre-resonance with transient excited state electronic bands, the characteristic Raman modes of the excited protonated (A*) chromophore species and intermediate deprotonated (I*) species can be selectively monitored. The inhomogeneous distribution/population of A* species go through ESPT with a similar ~300 ps time constant, confirming that bridging a water molecule to protein residue T203 in the ESPT chain is the rate-limiting step. Some A* species undergo vibrational cooling through high-frequency motions on the ~190 ps time scale. At early times, a portion of the largely protonated A* species could also undergo vibrational cooling or return to the ground state with a ~80 ps time constant. On the photoproduct side, a ~1330 cm−1 delocalized motion is observed, with dispersive line shapes in both the Stokes and anti-Stokes FSRS with a pre-resonance Raman pump, which indicates strong vibronic coupling, as the mode could facilitate the I* species to reach a relatively stable state (e.g., the main fluorescent state) after conversion from A*. Our findings disentangle the contributions of various vibrational motions active during the ESPT reaction, and offer new structural dynamics insights into the fluorescence mechanisms of engineered GFPs and other analogous autofluorescent proteins.
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Panoramic portrait of primary molecular events preceding excited state proton transfer in water
Photochemistry powers numerous processes from luminescence and human vision, to light harvesting. However, the elucidation of multidimensional photochemical reaction coordinates on molecular timescales remains challenging. We developed wavelength-tunable femtosecond stimulated Raman spectroscopy to simultaneously achieve pre-resonance enhancement for transient reactant and product species of the widely used photoacid pyranine undergoing excited-state proton transfer (ESPT) reaction in solution. In the low-frequency region, the 280 cm −1 ring deformation mode following 400 nm photoexcitation exhibits pronounced intensity oscillations on the sub-picosecond timescale due to anharmonic vibrational coupling to the 180 cm −1 hydrogen-bond stretching mode only in ESPT-capable solvents, indicating a primary event of functional relevance. This leads to the contact ion pair formation on the 3 ps timescale before diffusion-controlled separation. The intermolecular 180 cm −1 mode also reveals vibrational cooling time constants, ∼500 fs and 45 ps in both H 2 O and D 2 O, which differ from ESPT time constants of ∼3/8 and 90/250 ps in H 2 O/D 2 O, respectively. Spectral results using H 2 18 O further substantiate the functional role of the intermolecular 180 cm −1 mode in modulating the distance between proton donor and acceptor and forming the transient ion pair. The direct observation of molecular structural evolution across a wide spectral region during photochemical reactions enriches our fundamental understanding of potential energy surface and holds the key to advancing energy and biological sciences with exceptional atomic and temporal precision.
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- Award ID(s):
- 1455353
- NSF-PAR ID:
- 10159269
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 7
- Issue:
- 8
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 5484 to 5494
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/C6SC00672H
