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 t...
Saved in:
Published in | Physical chemistry chemical physics : PCCP Vol. 21; no. 19; pp. 9728 - 9739 |
---|---|
Main Authors | , , , , , |
Format | Journal Article |
Language | English |
Published |
England
15.05.2019
|
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | 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.
Upon photoexcitation, a delayed structural bending motion facilitates the "dark" GFP chromophore ring-twisting isomerization in reaching a conical intersection in solution. |
---|---|
Bibliography: | 1 marker band, FSRS spectra at late time delay points, coherent residual plot for 2D-FSRS with the control sample in 50% (v/v) glycerol-water solution, ESI Tables S1-S3 on the transient absorption dynamics, excited state Raman peak intensity dynamics, and frequency shift trend of Raman marker bands the anionic HBDI chromophore ring twisting angles, and ESI references. See DOI versus Electronic supplementary information (ESI) available: Additional discussions of the motivation and vibrational mode assignment, ESI Fig. S1-S9 on the steady-state electronic spectra of anionic HBDI in aqueous solution, ground state FSRS and Raman mode assignment, transient absorption and probe-dependent dynamics, global analysis and time constants, semi-automatic FSRS baselines, early-time dynamics of the 866 cm 10.1039/c9cp01077g ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 1463-9076 1463-9084 |
DOI: | 10.1039/c9cp01077g |