Delayed vibrational modulation of the solvated GFP chromophore into a conical intersection.

Green fluorescent protein (GFP) has revolutionized bioimaging and life sciences. Its successes have inspired modification of the chromophore structure and environment to tune emission properties, but outside the protein cage, the chromophore is essentially non-fluorescent. In this study, we employ the tunable femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption (TA) to map the energy dissipation pathways of GFP model chromophore (HBDI) in basic aqueous solution. Strategic tuning of the Raman pump to 550 nm exploits the stimulated emission band to enhance excited state vibrational motions as HBDI navigates the non-equilibrium potential energy landscape to pass through a conical intersection. The time-resolved FSRS uncovers prominent anharmonic couplings between a global out-of-plane bending mode of ∼227 cm-1 and two modes at ∼866 and 1572 cm-1 before HBDI reaches the twisted intramolecular charge transfer (TICT) state on the ∼3 ps time scale. Remarkably, the wavelet transform analysis reveals a ∼500 fs delayed onset of the coupling peaks, in correlation with the emergence of an intermediate charge-separated state en route to the TICT state. This mechanism is corroborated by the altered coupling matrix for the HBDI Raman modes in the 50% (v/v) water-glycerol mixture, and a notable lengthening of the picosecond time constant. The real-time molecular "movie" of the general rotor-like HBDI isomerization reaction following photoexcitation represents a significant advance in comprehending the photochemical reaction pathways of the solvated GFP chromophore, therefore providing a crucial foundation to enable rational design of diverse nanomachines from efficient molecular rotors to bright fluorescent probes.

Excited State Structural Evolution of a GFP Single-Site Mutant Tracked by Tunable Femtosecond-Stimulated Raman Spectroscopy.

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.