Discovering a rotational barrier within a charge-transfer state of a photoexcited chromophore in solution.
ABSTRACT: Methylation occurs in a myriad of systems with protective and regulatory functions. 8-methoxypyrene-1,3,6-trisulfonate (MPTS), a methoxy derivative of a photoacid, serves as a model system to study effects of methylation on the excited state potential energy landscape. A suite of spectroscopic techniques including transient absorption, wavelength-tunable femtosecond stimulated Raman spectroscopy (FSRS), and fluorescence quantum yield measurements via steady-state electronic spectroscopy reveal the energy dissipation pathways of MPTS following photoexcitation. Various solvents enable a systematic characterization of the H-bonding interaction, viscosity, and dynamic solvation that influence the ensuing relaxation pathways. The formation of a charge-transfer state out of the Franck-Condon region occurs on the femtosecond-to-picosecond solvation timescale before encountering a rotational barrier. The rotational relaxation correlates with the H-bond donating strength of solvent, while the rotational time constant lengthens as solvent viscosity increases. Time-resolved excited-state FSRS, aided by quantum calculations, provides crucial structural dynamics knowledge and reveals the sulfonate groups playing a dominant role during solvation. Several prominent vibrational motions of the pyrene ring backbone help maneuver the population toward the more fluorescent state. These ultrafast correlated electronic and nuclear motions ultimately govern the fate of the photoexcited chromophore in solution. Overall, MPTS in water displays the highest probability to fluoresce, while the aprotic and more viscous dimethyl sulfoxide enhances the nonradiative pathways. These mechanistic insights may apply robustly to other photoexcited chromophores that do not undergo excited-state proton transfer or remain trapped in a broad electronic state and also provide design principles to control molecular optical responses with site-specific atomic substitution.
Project description:Photosynthesis in plants starts with the capture of photons by light-harvesting complexes (LHCs). Structural biology and spectroscopy approaches have led to a map of the architecture and energy transfer pathways between LHC pigments. Still, controversies remain regarding the role of specific carotenoids in light-harvesting and photoprotection, obligating the need for high-resolution techniques capable of identifying excited-state signatures and molecular identities of the various pigments in photosynthetic systems. Here we demonstrate the successful application of femtosecond stimulated Raman spectroscopy (FSRS) to a multichromophoric biological complex, trimers of LHCII. We demonstrate the application of global and target analysis (GTA) to FSRS data and utilize it to quantify excitation migration in LHCII trimers. This powerful combination of techniques allows us to obtain valuable insights into structural, electronic, and dynamic information from the carotenoids of LHCII trimers. We report spectral and dynamical information on ground- and excited-state vibrational modes of the different pigments, resolving the vibrational relaxation of the carotenoids and the pathways of energy transfer to chlorophylls. The lifetimes and spectral characteristics obtained for the S1 state confirm that lutein 2 has a distorted conformation in LHCII and that the lutein 2 S1 state does not transfer to chlorophylls, while lutein 1 is the only carotenoid whose S1 state plays a significant energy-harvesting role. No appreciable energy transfer takes place from lutein 1 to lutein 2, contradicting recent proposals regarding the functions of the various carotenoids (Son et al. Chem. 2019, 5 (3), 575-584). Also, our results demonstrate that FSRS can be used in combination with GTA to simultaneously study the electronic and vibrational landscapes in LHCs and pave the way for in-depth studies of photoprotective conformations in photosynthetic systems.
Project description: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.
Project description:Photoexcited Nickel(II) tetramesitylporphyrin (NiTMP), like many open-shell metalloporphyrins, relaxes rapidly through multiple electronic states following an initial porphyrin-based excitation, some involving metal centered electronic configuration changes that could be harnessed catalytically before excited state relaxation. While a NiTMP excited state present at 100 ps was previously identified by X-ray transient absorption (XTA) spectroscopy at a synchrotron source as a relaxed (d,d) state, the lowest energy excited state (J. Am. Chem. Soc., 2007, 129, 9616 and Chem. Sci., 2010, 1, 642), structural dynamics before thermalization were not resolved due to the ?100 ps duration of the available X-ray probe pulse. Using the femtosecond (fs) X-ray pulses of the Linac Coherent Light Source (LCLS), the Ni center electronic configuration from the initial excited state to the relaxed (d,d) state has been obtained via ultrafast Ni K-edge XANES (X-ray absorption near edge structure) on a time scale from hundreds of femtoseconds to 100 ps. This enabled the identification of a short-lived Ni(I) species aided by time-dependent density functional theory (TDDFT) methods. Computed electronic and nuclear structure for critical excited electronic states in the relaxation pathway characterize the dependence of the complex's geometry on the electron occupation of the 3d orbitals. Calculated XANES transitions for these excited states assign a short-lived transient signal to the spectroscopic signature of the Ni(I) species, resulting from intramolecular charge transfer on a time scale that has eluded previous synchrotron studies. These combined results enable us to examine the excited state structural dynamics of NiTMP prior to thermal relaxation and to capture intermediates of potential photocatalytic significance.
Project description:Femtosecond time-resolved infrared spectroscopy was employed to study intramolecular charge transfer in triphenylmethane dyes, including malachite green (MG), malachite green carbinol base (MGCB), and leucomalachite green (LMG). A local excited state (LE) and a twisted intramolecular charge-transfer (TICT) state have been observed directly in MG. Furthermore, solvent-controlled TICT measurements in a series of linear alcohols indicate that the transition time (4-11 ps) from LE to TICT is strongly dependent on alcohol viscosity, which is due to rotational hindrance of dimethylaniline in high-viscosity solvents. For LMG, no TICT is observed due to steric hindrance caused by the sp(3)-hybridized central carbon atom. However, for MGCB, TICT is rescued by the addition of the electron-donating hydroxyl group to the bridge. These results for MG and its analogues provide new insight regarding the dynamics and mechanism of twisted intramolecular charge transfer (TICT) in triphenylmethane dyes.
Project description:The inclusion of explicit polarization in molecular dynamics simulation has gained increasing interest during the last several years. An understudied area is the role of polarizability in computer simulations of solvation dynamics around chromophores, particularly for the large solutes used in experimental studies. In this work, we present fully polarizable ground and excited state force fields for the common fluorophores N-methyl-6-oxyquinolium betaine and Coumarin 153. While analyzing the solvation responses in water, methanol, and the highly viscous ionic liquid 1-ethyl-3-methylimidazolium trifluoromethanesulfonate we found that the inclusion of solute polarizability considerably increases the agreement of the obtained Stokes shift relaxation functions with experimental data. Solute polarizability slows down the inertial solvation response in the femtosecond time regime and enables the chromophore to adapt its dipole moment to the environment. Furthermore, the developed chromophore force field reproduces the solute dipole moments in both the electronic ground and excited state in environments ranging from gas phase to highly polar media correctly. Based on these studies it is anticipated that polarizable models of chromophores will lead to an improved understanding of the relationship of their environment to their spectroscopic properties.
Project description:Fluorescent proteins (FPs) have played a pivotal role in bioimaging and advancing biomedicine. The versatile fluorescence from engineered, genetically encodable FP variants greatly enhances cellular imaging capabilities, which are dictated by excited-state structural dynamics of the embedded chromophore inside the protein pocket. Visualization of the molecular choreography of the photoexcited chromophore requires a spectroscopic technique capable of resolving atomic motions on the intrinsic timescale of femtosecond to picosecond. We use femtosecond stimulated Raman spectroscopy to study the excited-state conformational dynamics of a recently developed FP-calmodulin biosensor, GEM-GECO1, for calcium ion (Ca(2+)) sensing. This study reveals that, in the absence of Ca(2+), the dominant skeletal motion is a ? 170 cm(-1) phenol-ring in-plane rocking that facilitates excited-state proton transfer (ESPT) with a time constant of ? 30 ps (6 times slower than wild-type GFP) to reach the green fluorescent state. The functional relevance of the motion is corroborated by molecular dynamics simulations. Upon Ca(2+) binding, this in-plane rocking motion diminishes, and blue emission from a trapped photoexcited neutral chromophore dominates because ESPT is inhibited. Fluorescence properties of site-specific protein mutants lend further support to functional roles of key residues including proline 377 in modulating the H-bonding network and fluorescence outcome. These crucial structural dynamics insights will aid rational design in bioengineering to generate versatile, robust, and more sensitive optical sensors to detect Ca(2+) in physiologically relevant environments.
Project description:The femtosecond transient absorption technique was used to study the relaxation of excited electronic states created by absorption of 267-nm light in all of the naturally occurring pyrimidine DNA and RNA bases in aqueous solution. The results reveal a surprising bifurcation of the initial excited-state population in <1 ps to two nonradiative decay channels within the manifold of singlet states. The first is the subpicosecond internal conversion channel first characterized in 2000. The second channel involves passage through a dark intermediate state assigned to a lowest-energy (1)npi* state. Approximately 10-50% of all photoexcited pyrimidine bases decay via the (1)npi* state, which has a lifetime of 10-150 ps. Three- to 6-fold-longer lifetimes are seen for pyrimidine nucleotides and nucleosides than for the corresponding free bases, revealing an unprecedented effect of ribosyl substitution on electronic energy relaxation. A small fraction of the (1)npi* population is proposed to undergo intersystem crossing to the lowest triplet state in competition with vibrational cooling, explaining the higher triplet yields observed for pyrimidine versus purine bases at room temperature. Some simple correlations exist between yields of the (1)npi* state and yields of some pyrimidine photoproducts, but more work is needed before the photochemical consequences of this state can be definitively determined. These findings lead to a dramatically different picture of electronic energy relaxation in single pyrimidine bases with important ramifications for understanding DNA photostability.
Project description:The relaxation of photoexcited nanosystems is a fundamental process of light-matter interaction. Depending on the couplings of the internal degrees of freedom, relaxation can be ultrafast, converting electronic energy in a few fs, or slow, if the energy is trapped in a metastable state that decouples from its environment. Here, we study helium nanodroplets excited resonantly by femtosecond extreme-ultraviolet (XUV) pulses from a seeded free-electron laser. Despite their superfluid nature, we find that helium nanodroplets in the lowest electronically excited states undergo ultrafast relaxation. By comparing experimental photoelectron spectra with time-dependent density functional theory simulations, we unravel the full relaxation pathway: Following an ultrafast interband transition, a void nanometer-sized bubble forms around the localized excitation (He[Formula: see text]) within 1 ps. Subsequently, the bubble collapses and releases metastable He[Formula: see text] at the droplet surface. This study highlights the high level of detail achievable in probing the photodynamics of nanosystems using tunable XUV pulses.
Project description:The observation of chemical reactions on the time scale of the motion of electrons and nuclei has been made possible by lasers with ever shortened pulse lengths. Superfluid helium represents a special solvent that permits the synthesis of novel classes of molecules that have eluded dynamical studies so far. However, photoexcitation inside this quantum solvent triggers a pronounced response of the solvation shell, which is not well understood. Here, we present a mechanistic description of the solvent response to photoexcitation of indium (In) dopant atoms inside helium nanodroplets (HeN), obtained from femtosecond pump-probe spectroscopy and time-dependent density functional theory simulations. For the In-HeN system, part of the excited state electronic energy leads to expansion of the solvation shell within 600?fs, initiating a collective shell oscillation with a period of about 30?ps. These coupled electronic and nuclear dynamics will be superimposed on intrinsic photoinduced processes of molecular systems inside helium droplets.
Project description:Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the ortho site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm-1 in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.