Mechanism of resonant electron emission from the deprotonated GFP chromophore and its biomimetics.
ABSTRACT: The Green Fluorescent Protein (GFP), which is widely used in bioimaging, is known to undergo light-induced redox transformations. Electron transfer is thought to occur resonantly through excited states of its chromophore; however, a detailed understanding of the electron gateway states of the chromophore is still missing. Here, we use photoelectron spectroscopy and high-level quantum chemistry calculations to show that following UV excitation, the ultrafast electron dynamics in the chromophore anion proceeds via an excited shape resonance strongly coupled to the open continuum. The impact of this state is found across the entire 355-315 nm excitation range, from above the first bound-bound transition to below the opening of higher-lying continua. By disentangling the electron dynamics in the photodetachment channels, we provide an important reference for the adiabatic position of the electron gateway state, which is located at 348 nm, and discover the source of the curiously large widths of the photoelectron spectra that have been reported in the literature. By introducing chemical modifications to the GFP chromophore, we show that the detachment threshold and the position of the gateway state, and hence the underlying excited-state dynamics, can be changed systematically. This enables a fine tuning of the intrinsic electron emission properties of the GFP chromophore and has significant implications for its function, suggesting that the biomimetic GFP chromophores are more stable to photooxidation.
Project description:The reaction F + H2O ? HF + OH is a four-atom system that provides an important benchmark for reaction dynamics. Hydrogen atom transfer at the transition state for this reaction is expected to exhibit a strong dependence on reactant vibrational excitation. In the present study, the vibrational effects are examined by photodetachment of vibrationally excited F-(H2O) precursor anions using photoelectron-photofragment coincidence (PPC) spectroscopy and compared with full six-dimensional quantum dynamical calculations on ab initio potential energy surfaces. Prior to photodetachment at h?UV = 4.80 eV, the overtone of the ionic hydrogen bond mode in the precursor F-(H2O), 2?IHB at 2885 cm-1, was excited using a tunable IR laser. Experiment and theory show that vibrational energy in the anion can be effectively carried away by the photoelectron upon a Franck-Condon photodetachment, and also show evidence for an increase of branching into the F + H2O reactant channel. The experimental results suggest a greater role for product rotational excitation than theory. Improved potential energy surfaces and longer wavepacket propagation times would be helpful to further examine the nature of the discrepancy.
Project description:A dipolar molecule can weakly bind an electron in a diffuse orbital. However, the spin-orbit coupling between this weakly bound electron and the electrons in the molecular core is not known. Here we probe this coupling using the linear C2P- anion with the 3Σ+ ground state, which possesses dipole-bound excited states because neutral C2P (2Π) has a sufficiently large dipole moment. Photodetachment spectroscopy and resonant photoelectron spectroscopy are used to probe the nature of the dipole-bound states. Two dipole-bound excited states are observed with a binding energy of 37 cm-1, corresponding to the two spin-orbit states of neutral C2P (2Π1/2 and 2Π3/2). The current study demonstrates that the weakly bound electron in the dipole-bound excited states of C2P- is not spin-coupled to the electrons in the C2P core and can be considered as a quasi-free electron.
Project description:Deprotonated thymine can exist in two different forms, depending on which of its two N sites is deprotonated: N1[T-H]- or N3[T-H]-. Here we report a photodetachment study of the N1[T-H]- isomer cooled in a cryogenic ion trap and the observation of an excited dipole-bound state. Eighteen vibrational levels of the dipole-bound state are observed, and its vibrational ground state is found to be 238 ± 5 cm-1 below the detachment threshold of N1[T-H]-. The electron affinity of the deprotonated thymine radical (N1[T-H]?) is measured accurately to be 26?322 ± 5 cm-1 (3.2635 ± 0.0006 eV). By tuning the detachment laser to the sixteen vibrational levels of the dipole-bound state that are above the detachment threshold, highly non-Franck-Condon resonant-enhanced photoelectron spectra are obtained due to state- and mode-selective vibrational autodetachment. Much richer vibrational information is obtained for the deprotonated thymine radical from the photodetachment and resonant-enhanced photoelectron spectroscopy. Eleven fundamental vibrational frequencies in the low-frequency regime are obtained for the N1[T-H]? radical, including the two lowest-frequency internal rotational modes of the methyl group at 70 ± 8 cm-1 and 92 ± 5 cm-1.
Project description:Wild type green fluorescent protein (wt-GFP) and the variant S65T/H148D each exhibit two absorption bands, A and B, which are associated with the protonated and deprotonated chromophores, respectively. Excitation of either band leads to green emission. In wt-GFP, excitation of band A ( approximately 395 nm) leads to green emission with a rise time of 10-15 ps, due to excited-state proton transfer (ESPT) from the chromophore hydroxyl group to an acceptor. This process produces an anionic excited-state intermediate I* that subsequently emits a green photon. In the variant S65T/H148D, the A band absorbance maximum is red-shifted to approximately 415 nm, and as detailed in the accompanying papers, when the A band is excited, green fluorescence appears with a rise time shorter than the instrument time resolution ( approximately 170 fs). On the basis of the steady-state spectroscopy and high-resolution crystal structures of several variants described herein, it is proposed that in S65T/H148D, the red shift of absorption band A and the ultrafast appearance of green fluorescence upon excitation of band A are due to a very short (<or=2.4 A), and possibly low-barrier, hydrogen bond between the chromophore hydroxyl and introduced Asp148.
Project description:The fluorescence properties of GFP are strongly influenced by the protonation states of its chromophore and nearby amino acid side chains. In the ground state, the GFP chromophore is neutral and absorbs in the near UV. Upon excitation, the chromophore is deprotonated, and the resulting anionic chromophore emits its green fluorescence. So far, only excited-state intermediates have been observed in the GFP photocycle. We have used ultrafast multipulse control spectroscopy to prepare and directly observe GFP's hidden anionic ground-state intermediates as an integral part of the photocycle. Combined with dispersed multichannel detection and advanced global analysis techniques, the existence of two distinct anionic ground-state intermediates, I(1) and I(2), has been unveiled. I(1) and I(2) absorb at 500 and 497 nm, respectively, and interconvert on a picosecond timescale. The I(2) intermediate has a lifetime of 400 ps, corresponding to a proton back-transfer process that regenerates the neutral ground state. Hydrogen/deuterium exchange of the protein leads to a significant increase of the I(1) and I(2) lifetimes, indicating that proton motion underlies their dynamics. We thus have assessed the complete chain of reaction intermediates and associated timescales that constitute the photocycle of GFP. Many elementary processes in biology rely on proton transfers that are limited by slow diffusional events, which seriously precludes their characterization. We have resolved the true reaction rate of a proton transfer in the molecular ground state of GFP, and our results may thus aid in the development of a generic understanding of proton transfer in biology.
Project description:The excited state relaxation pathways of isoxazole and oxazole upon excitation with UV-light were investigated by nonadiabatic ab initio dynamics simulations and time-resolved photoelectron spectroscopy. Excitation of the bright ??*-state of isoxazole predominantly leads to ring-opening dynamics. Both the initially excited ??*-state and the dissociative ??*-state offer a combined barrier-free reaction pathway, such that ring-opening, defined as a distance of more than 2 Å between two neighboring atoms, occurs within 45 fs. For oxazole, in contrast, the excited state dynamics is about twice as slow (85 fs) and the quantum yield for ring-opening is lower. This is caused by a small barrier between the ??*-state and the ??*-state along the reaction path, which suppresses direct ring-opening. Theoretical findings are consistent with the measured time-resolved photoelectron spectra, confirming the timescales and the quantum yields for the ring-opening channel. The results indicate that a combination of time-resolved photoelectron spectroscopy and excited state dynamics simulations can explain the dominant reaction pathways for this class of molecules. As a general rule, we suggest that the antibonding ?*-orbital located between the oxygen atom and a neighboring atom of a five-membered heterocyclic system provides a driving force for ring-opening reactions, which is modified by the presence and position of additional nitrogen atoms.
Project description:Green fluorescent protein (GFP) and GFP-like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore. According to quantum mechanical calculations, the excited state of chromophore model systems has significant dihedral freedom, which may lead to fluorescence quenching intersystem crossing. Molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green, yellow, and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state. Most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield. According to our calculations, the dihedral freedom of the systems studied (BFP > A5 > YFP > GFP) increases in the inverse order to the quantum yield. In all simulations, the chromophore undergoes a negatively correlated hula twist (also known as a bottom hula twist mechanism).
Project description:To better understand the diverse mechanisms of spectral tuning operational in fluorescent proteins (FPs), we determined the 2.1-A X-ray structure of dsFP483 from the reef-building coral Discosoma. This protein is a member of the cyan class of Anthozoa FPs and exhibits broad, double-humped excitation and absorbance bands, with a maximum at 437-440 nm and a shoulder at 453 nm. Although these features support a heterogeneous ground state for the protein-intrinsic chromophore, peak fluorescence occurs at 483 nm for all excitation wavelengths, suggesting a common emissive state. Optical properties are insensitive to changes in pH over the entire range of protein stability. The refined crystal structure of the biological tetramer (space group C2) demonstrates that all protomers bear a cis-coplanar chromophore chemically identical with that in green fluorescent protein (GFP). To test the roles of specific residues in color modulation, we investigated the optical properties of the H163Q and K70M variants. Although absorbance bands remain broad, peak excitation maxima are red shifted to 455 and 460 nm, emitting cyan light and green light, respectively. To probe chromophore ground-state features, we collected Raman spectra using 752-nm excitation. Surprisingly, the positions of key Raman bands of wild-type dsFP483 are most similar to those of the neutral GFP chromophore, whereas the K70M spectra are more closely aligned with the anionic form. The Raman data provide further evidence of a mixed ground state with chromophore populations that are modulated by mutation. Possible internal protonation equilibria, structural heterogeneity in the binding sites, and excited-state proton transfer mechanisms are discussed. Structural alignments of dsFP483 with the homologs DsRed, amFP486, and zFP538-K66M suggest that natural selection for cyan is an exquisitely fine-tuned and highly cooperative process involving a network of electrostatic interactions that may vary substantially in composition and arrangement.
Project description:The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferred for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found that the color of the neutral chromophore can be more sensitive to protein electrostatics than can the anionic counterpart. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model as compared to that of the anionic state, which requires only two forms ( J. Am. Chem. Soc. 2019, 141, 15250-15265). Specifically, an underlying zwitterionic charge-transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to excited-state proton transfer (ESPT) of the protonated chromophore, we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of the two-form model for donor-acceptor systems with localized ground-state electronic distributions.
Project description:The simple structure of the chromophore of the green fluorescent protein (GFP), a phenol and an imidazolone ring linked by a methyne bridge, supports an exceptionally diverse range of excited state phenomena. Here we describe experimentally and theoretically the photochemistry of a novel sterically crowded nonplanar derivative of the GFP chromophore. It undergoes an excited state isomerization reaction accompanied by an exceptionally fast (sub 100 fs) excited state decay. The decay dynamics are essentially independent of solvent polarity and viscosity. Excited state structural dynamics are probed by high level quantum chemical calculations revealing that the fast decay is due to a conical intersection characterized by a twist of the rings and pyramidalization of the methyne bridge carbon. The intersection can be accessed without a barrier from the pre-twisted Franck-Condon structure, and the lack of viscosity dependence is due to the fact that the rings twist in the same direction, giving rise to a volume-conserving decay coordinate. Moreover, the rotation of the phenyl, methyl and imidazolone groups is coupled in the sterically crowded structure, with the methyl group translating the rotation of one ring to the next. As a consequence, the excited state dynamics can be viewed as a torsional couple, where the absorbed photon energy leads to conversion of the out-of-plane orientation from one ring to the other in a volume conserving fashion. A similar modification of the range of methyne dyes may provide a new family of devices for molecular machines, specifically torsional couples.