Photoacid Behaviour in a Fluorinated Green Fluorescent Protein Chromophore: Ultrafast Formation of Anion and Zwitterion States.†.
ABSTRACT: The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction. In contrast the photophysics of the same chromophore in solution are dominated by radiationless decay, and photoacid behaviour is not observed. Here we show that modification of the pKa of the chromophore by fluorination leads to an excited state proton transfer on an extremely fast (50 fs) time scale. Such a fast rate suggests a barrierless proton transfer and the existence of a pre-formed acceptor site in the aqueous solution, which is supported by solvent and deuterium isotope effects. In addition, at lower pH, photochemical formation of the elusive zwitterion of the GFP chromophore is observed by means of an equally fast excited state proton transfer from the cation. The significance of these results for understanding and modifying the properties of fluorescent proteins are discussed.
Project description:Proton transfer is critical in many important biochemical reactions. The unique three-step excited-state proton transfer in avGFP allows observations of protein proton transport in real-time. In this work we exploit femtosecond to microsecond transient IR spectroscopy to record, in D2 O, the complete proton transfer photocycle of avGFP, and two mutants (T203V and S205V) which modify the structure of the proton wire. Striking differences and similarities are observed among the three mutants yielding novel information on proton transfer mechanism, rates, isotope effects, H-bond strength and proton wire stability. These data provide a detailed picture of the dynamics of long-range proton transfer in a protein against which calculations may be compared.
Project description:Steady-state and time-resolved fluorescence techniques as well as quantum-mechanical calculations were used to study the photophysics and photochemistry of a newly synthesized photoacid-the phenol cyanine picolinium salt. We found that the nonradiative rate constant <i>k</i> <sub>nr</sub> of the excited protonated form of the photoacid is larger than that of the excited-state proton transfer (ESPT) to the solvent, <i>k</i> <sub>ESPT</sub>. We estimate that the quantum efficiency of the ESPT process is about 0.16. The nonradiative process is explained by a partial trans-cis isomerization reaction, which leads to the formation of a "dark" excited state that can cross to the ground state by nonadiabatic coupling. Moreover, the ESPT process is coupled to the photo-isomerization reaction, as this latter reaction enhances the photoacidity of the studied compound, as a result of photoinduced charge transfer. To prevent trans-cis isomerization of the cyanine bridge, we conducted experiments of PCyP adsorbed on cellulose in the presence of water. We found that the steady-state fluorescence intensity increased by about a factor of 50 and the lifetime of the ROH band increased by the same factor. The fluorescence intensity of the RO<sup>-</sup> band with respect to that of the ROH band was the same as in aqueous solution. This explains why inhibiting the photo-isomerization reaction by adsorbing the PCyP on cellulose does not lead to a higher ESPT rate.
Project description:mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (lambda(em)(max) approximately 535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of approximately 4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (lambda(ex)(max) approximately 565 nm, lambda(em)(max) approximately 620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.
Project description:The chemical locking of the central single bond in core chromophores of green fluorescent proteins (GFPs) influences their excited-state behavior in a distinct manner. Experimentally, it significantly enhances the fluorescence quantum yield of GFP chromophores with an ortho-hydroxyl group, while it has almost no effect on the photophysics of GFP chromophores with a para-hydroxyl group. To unravel the underlying physical reasons for this different behavior, we report static electronic structure calculations and nonadiabatic dynamics simulations on excited-state intramolecular proton transfer, cis-trans isomerization, and excited-state deactivation in a locked ortho-substituted GFP model chromophore (o-LHBI). On the basis of our previous and present results, we find that the S1 keto species is responsible for the fluorescence emission of the unlocked o-HBI and the locked o-LHBI species. Chemical locking does not change the parts of the S1 and S0 potential energy surfaces relevant to enol-keto tautomerization; hence, in both chromophores, there is an ultrafast excited-state intramolecular proton transfer that takes only 35 fs on average. However, the locking effectively hinders the S1 keto species from approaching the keto S1/S0 conical intersections so that most of trajectories are trapped in the S1 keto region for the entire 2 ps simulation time. Therefore, the fluorescence quantum yield of o-LHBI is enhanced compared with that of unlocked o-HBI, in which the S1 excited-state decay is efficient and ultrafast. In the case of the para-substituted GFP model chromophores p-HBI and p-LHBI, chemical locking hardly affects their efficient excited-state deactivation via cis-trans isomerization; thus, the fluorescence quantum yields in these chromophores remain very low. The insights gained from the present work may help to guide the design of new GFP chromophores with improved fluorescence emission and brightness.
Project description:Cyclovalone is a synthetic curcumin derivative in which the keto-enolic system is replaced by a cyclohexanone ring. This modification of the chemical structure might in principle result in an excited state that is more stable than that of curcumin, which in turn should produce an enhanced phototoxicity. Indeed, although curcumin exhibits photosensitized antibacterial activity, this compound is characterized by very fast excited-state dynamics which limit its efficacy as a photosensitizer. In previous works we showed that the main non-radiative decay pathway of keto-enolic curcuminoids is through excited-state transfer of the enolic proton to the keto-oxygen. Another effective deactivation pathway involves an intermolecular charge transfer mechanism occurring at the phenyl rings, made possible by intramolecular H-bonding between the methoxy and the hydroxyl substituent. In this paper we present UV-Vis and IR absorption spectra data with the aim of elucidating the intramolecular charge distribution of this compound and its solvation patterns in different environments, with particular focus on solute-solvent H-bonding features. Moreover, we discuss steady state and time-resolved fluorescence data that aim at characterizing the excited-state dynamics of cyclovalone, and we compare its decay photophysics to that of curcumin. Finally, because during the characterization procedures we found evidence of very fast photodegradation of cyclovalone, its photostability in four organic solvents was studied by HPLC and the corresponding relative degradation rates were calculated.
Project description:Proton transfer plays an important role in the optical properties of green fluorescent protein (GFP). While much is known about excited-state proton transfer reactions (ESPT) in GFP occurring on ultrafast time scales, comparatively little is understood about the factors governing the rates and pathways of ground-state proton transfer. We have utilized a specific isotopic labeling strategy in combination with one-dimensional (13)C nuclear magnetic resonance (NMR) spectroscopy to install and monitor a (13)C directly adjacent to the GFP chromophore ionization site. The chemical shift of this probe is highly sensitive to the protonation state of the chromophore, and the resulting spectra reflect the thermodynamics and kinetics of the proton transfer in the NMR line shapes. This information is complemented by time-resolved NMR, fluorescence correlation spectroscopy, and steady-state absorbance and fluorescence measurements to provide a picture of chromophore ionization reactions spanning a wide time domain. Our findings indicate that proton transfer in GFP is described well by a two-site model in which the chromophore is energetically coupled to a secondary site, likely the terminal proton acceptor of ESPT, Glu222. Additionally, experiments on a selection of GFP circular permutants suggest an important role played by the structural dynamics of the seventh ?-strand in gating proton transfer from bulk solution to the buried chromophore.
Project description:A series of artificial metalloenzymes containing a ruthenium chromophore and [NiII(cyclam)]2+, both incorporated site-selectively, have been constructed within an azurin protein scaffold. These light-driven, semisynthetic enzymes do not evolve hydrogen, thus displaying complete selectivity for CO2 reduction to CO. Electrostatic effects rather than direct excited-state electron transfer dominate the ruthenium photophysics, suggesting that intramolecular electron transfer from photogenerated RuI to [NiII(cyclam)]2+ represents the first step in catalysis. Stern-Volmer analyses rationalize the observation that ascorbate is the only sacrificial electron donor that supports turnover. Collectively, these results highlight the important interplay of elements that must be considered when developing and characterizing molecular catalysts.
Project description:Phytochromes are multi-domain red light photosensor proteins, which convert red light photons to biological activity utilizing the multitude of structural and chemical reactions. The steady increase in structural information obtained from various bacteriophytochromes has increased understanding about the functional mechanism of the photochemical processes of the phytochromes. Furthermore, a number of spectroscopic studies have revealed kinetic information about the light-induced reactions. The spectroscopic changes are, however, challenging to connect with the structural changes of the chromophore and the protein environment, as the excited state properties of the chromophores are very sensitive to the small structural and chemical changes of their environment. In this article, we concentrate on the results of ultra-fast spectroscopic experiments which reveal information about the important initial steps of the photoreactions of the phytochromes. We survey the excited state properties obtained during the last few decades. The differences in kinetics between different research laboratories are traditionally related to the differences of the studied species. However, we notice that the variation in the excited state properties depends on the subunit composition of the protein as well. This observation illustrates a feedback mechanism from the other domains to the chromophore. We propose that two feedback routes exist in phytochromes between the chromophore and the remotely located effector domain. The well-known connection between the subunits is the so-called tongue region, which changes its secondary structure while changing the light-activated state of the system. The other feedback route which we suggest is less obvious, it is made up of several water molecules ranging from the dimer interface to the vicinity of the chromophore, allowing even proton transfer reactions nearby the chromophore.
Project description:Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at approximately 395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place approximately 30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.
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.