An official website of the United States government
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.
more »
« less
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.
more »
« less
- PAR ID:
- 10159264
- Date Published:
- Journal Name:
- Molecules
- Volume:
- 23
- Issue:
- 9
- ISSN:
- 1420-3049
- Page Range / eLocation ID:
- 2226
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca2+-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca2+-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading to a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.more » « less
-
Some diarylethene molecular switches have a low quantum yield for cycloreversion when excited by a single photon, but react more efficiently following sequential two-photon excitation. The increase in reaction efficiency depends on both the relative time delay and the wavelength of the second photon. This paper examines the wavelength-dependent mechanism for sequential excitation using excited-state resonance Raman spectroscopy to probe the ultrafast (sub-30 fs) dynamics on the upper electronic state following secondary excitation. The approach uses femtosecond stimulated Raman scattering (FSRS) to measure the time-gated, excited-state resonance Raman spectrum in resonance with two different excited-state absorption bands. The relative intensities of the Raman bands reveal the initial dynamics in the higher-lying states, Sn, by providing information on the relative gradients of the potential energy surfaces that are accessed via secondary excitation. The excited-state resonance Raman spectra reveal specific modes that become enhanced depending on the Raman excitation wavelength, 750 or 400 nm. Many of the modes that become enhanced in the 750 nm FSRS spectrum are assigned as vibrational motions localized on the central cyclohexadiene ring. Many of the modes that become enhanced in the 400 nm FSRS spectrum are assigned as motions along the conjugated backbone and peripheral phenyl rings. These observations are consistent with earlier measurements that showed higher efficiency following secondary excitation into the lower excited-state absorption band and illustrate a powerful new way to probe the ultrafast dynamics of higher-lying excited states immediately following sequential two-photon excitation.more » « less
-
Proton transfer processes of organic molecules are key to charge transport and photoprotection in biological systems. Among them, excited-state intramolecular proton transfer (ESIPT) reactions are characterized by quick and efficient charge transfer within a molecule, resulting in ultrafast proton motions. The ESIPT-facilitated interconversion between two tautomers (PS and PA) comprising the tree fungal pigment Draconin Red in solution was investigated using a combination of targeted femtosecond transient absorption (fs-TA) and excited-state femtosecond stimulated Raman spectroscopy (ES-FSRS) measurements. Transient intensity (population and polarizability) and frequency (structural and cooling) dynamics of –COH rocking and –C=C, –C=O stretching modes following directed stimulation of each tautomer elucidate the excitation-dependent relaxation pathways, particularly the bidirectional ESIPT progression out of the Franck–Condon region to the lower-lying excited state, of the intrinsically heterogeneous chromophore in dichloromethane solvent. A characteristic overall excited-state PS-to-PA transition on the picosecond timescale leads to a unique “W”-shaped excited-state Raman intensity pattern due to dynamic resonance enhancement with the Raman pump–probe pulse pair. The ability to utilize quantum mechanics calculations in conjunction with steady-state electronic absorption and emission spectra to induce disparate excited-state populations in an inhomogeneous mixture of similar tautomers has broad implications for the modeling of potential energy surfaces and delineation of reaction mechanisms in naturally occurring chromophores. Such fundamental insights afforded by in-depth analysis of ultrafast spectroscopic datasets are also beneficial for future development of sustainable materials and optoelectronics.more » « less
-
Abstract Large Stokes shift (LSS) red fluorescent proteins (RFPs) are highly desirable for bioimaging advances. The RFP mKeima, with coexistingcis‐andtrans‐isomers, holds significance as an archetypal system for LSS emission due to excited‐state proton transfer (ESPT), yet the mechanisms remain elusive. We implemented femtosecond stimulated Raman spectroscopy (FSRS) and various time‐resolved electronic spectroscopies, aided by quantum calculations, to dissect thecis‐ andtrans‐mKeima photocycle from ESPT, isomerization, to ground‐state proton transfer in solution. This work manifests the power of FSRS with global analysis to resolve Raman fingerprints of intermediate states. Importantly, the deprotonatedtrans‐isomer governs LSS emission at 620 nm, while the deprotonatedcis‐isomer's 520 nm emission is weak due to an ultrafastcis‐to‐transisomerization. Complementary spectroscopic techniques as a table‐top toolset are thus essential to study photochemistry in physiological environments.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/molecules23092226
