Fluorescent Protein Chromophore Research Articles

Bond alternation, polarizability, and resonance detuning in methine dyes

We derive structure-property relationships for methine ("Brooker") dyes relating the color of the dye and its symmetric parents to its bond alternation in the ground state and also to the dipole properties associated with its low-lying charge-resonance (or charge-transfer) transition. We calibrate and test these relationships on an array of different protonation states of the green fluorescent protein chromophore motif (an asymmetric halochromic methine dye) and its symmetric parent dyes. The relationships rely on the assumption that the diabatic states that define the Platt model for methine dye color [J. R. Platt, J. Chem. Phys. 25, 80 (1956)] can also be distinguished by their single-double bond alternation and by their charge localization character. These assumptions are independent of the primary constraint that defines the diabatic states in the Platt model--specifically, the Brooker deviation rule for methine dyes [L. G. S. Brooker, Rev. Mod. Phys. 14, 275 (1942)]. Taking these assumptions, we show that the Platt model offers an alternate route to known structure-property relationships between the bond length alternation and the quadratic nonlinear polarizability β. We show also that the Platt model can be parameterized without the need for synthesis of the symmetric parents of a given dye, using the dipole data obtained through spectroscopic measurements. This suggests that the Platt model parameters may be used as independent variables in free-energy relationships for chromophores whose symmetric parents cannot be synthesized or chromophores strongly bound to biomolecular environments. The latter category includes several recently characterized biomolecular probe constructs. We illustrate these concepts by an analysis of previously reported electroabsorption and second-harmonic generation experiments on green fluorescent proteins.

Relaxation of green fluorescent protein chromophore anion observed by photodissociation in an electrostatic storage ring

The gas-phase absorption properties of the green fluorescent protein chromophore anion were studied using an electrostatic storage ring. The time sequence of neutral particles produced by photodissociation was detected following laser irradiation. The lifetimes of the photo-absorbed ions depended on their storage time in an ion trap before injection into the storage ring. The lifetime increased with the storage time and saturated, indicating a change in the population of rovibrationally excited states with respect to the storage time. Photodissociation neutral spectrum of the relaxed ions measured as a function of the laser wavelength was characterized by a narrow asymmetric shape, which was in good agreement with the photo fragment ion action spectrum reported recently.

Fluorescence Turn On by Cholate Aggregates

Bile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side. By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules. Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns, we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems.

Comprehensive Studies on an Overall Proton Transfer Cycle of the ortho-Green Fluorescent Protein Chromophore

Initiated by excited-state intramolecular proton transfer (ESIPT) reaction, an overall reaction cycle of 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI), an analogue of the core chromophore of the green fluorescent protein (GFP), has been investigated. In contrast to the native GFP core, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (p-HBDI), which requires hydrogen-bonding relay to accomplish proton transfer in vivo, o-HBDI possesses a seven-membered-ring intramolecular hydrogen bond and thus provides an ideal system for mimicking an intrinsic proton-transfer reaction. Upon excitation, ESIPT takes place in o-HBDI, resulting in a ∼600 nm proton-transfer tautomer emission. The o-HBDI tautomer emission, resolved by fluorescence upconversion, is comprised of an instantaneous rise to a few hundred femtosecond oscillation in the early relaxation stage. Frequency analysis derived from ultrashort pulse gives two low-frequency vibrations at 115 and 236 cm(-1), corresponding to skeletal deformation motions associated with the hydrogen bond. The results further conclude that ESIPT in o-HBDI is essentially triggered by low-frequency motions and may be barrierless along the reaction coordinate. Femtosecond UV/vis transient absorption spectra also provide supplementary evidence for the structural evolution during the reaction. In CH(3)CN, an instant rise of a 530 nm transient is resolved, which then undergoes 7.8 ps decay, accompanied by the growth of a rather long-lived 580 nm transient species. It is thus concluded that following ESIPT the cis-proton transfer isomer undergoes cis-trans-isomerization. The results of viscosity-dependent dynamics are in favor of the one-bond-flip mechanism, which is in contrast to the volume-conserving isomerization behavior for cis-stilbene and p-HBDI. Further confirmation is given by the picosecond-femtosecond transient IR absorption spectra, where several new and long-lived IR bands in the range of 1400-1500 cm(-1) are assigned to the phenyl in-plane breathing motions of the trans-proton transfer tautomer. Monitored by the nanosecond transient absorption, the 580 nm transient undergoes a ∼7.7 μs decay constant, accompanied by the growth of a new ∼500 nm band. The latter is assigned to a deprotonated tautomer species, which then undergoes the ground-state reverse proton recombination to the original o-HBDI in ∼50 μs, achieving an overall, reversible proton transfer cycle. This assignment is unambiguously supported by pump-probe laser induced fluorescence studies. On these standpoints, a comparison of photophysical properties among o-HBDI, p-HBDI, and wild-type GFP is discussed in detail.

Non-Condon Effects in the One- and Two-Photon Absorption Spectra of the Green Fluorescent Protein

We report calculations of one and two photon absorption (OPA and TPA, respectively) spectra of the green fluorescent protein (GFP) chromophore using the double-harmonic parallel-mode approximation and including explicit dependence of the electronic transition moments on nuclear geometry. The non-Condon effects are found to be more significant for TPA resulting in a different shape of the spectra and a blue shift of 500 cm−1 of the TPA peak absorption relative to OPA. The computed shift is in excellent agreement with the experimentally observed 700 cm−1.

Absorption of the green fluorescent protein chromophore anion in the gas phase studied by a combination of FTICR mass spectrometry with laser-induced photodissociation spectroscopy

The optical absorption of the green fluorescent protein chromophore anion (HBDI −) in the gas phase has been addressed by a number of experimental and theoretical studies; however, there is no consensus on its spectral characteristics yet. In this report, the intrinsic absorption of HBDI − was probed by photo-induced dissociation (“action”) spectroscopy using discrete lines of a continuous-wave (CW) laser source. The observed spectral profile of dissociation efficiency revealed a pronounced dependence on the laser irradiance. Dissociation at high irradiance is governed by multiple-photon absorption processes whose efficiency peaks between 476 and 488 nm. At very low irradiance (<0.4 mW cm −2) dissociation of gas-phase HBDI − is mostly promoted by single-photon transitions and suggests an intrinsic absorption maximum of gas-phase HBDI − <476 nm, blue-shifted compared to earlier results obtained with pulsed lasers.

An OFF–ON fluorescent probe for Zn2+ based on a GFP-inspired imidazolone derivative attached to a 1,10-phenanthroline moiety

A green fluorescent protein chromophore inspired chemosenor for Zn(2+) was designed and synthesized. A Zn(2+) specific fluorescence enhancement was observed due to restricted rotation between the 1,10-phenanthroline and imidazolone moieties.

Steric and Electronic Effects in Capsule-Confined Green Fluorescent Protein Chromophores

The turn-on of emission in fluorescent protein chromophores sequestered in an "octaacid" capsule is controlled by stereoelectronic effects described by a linear free energy relationship. The stereochemical effects are governed by both the positions and volumes of the aryl substituents, while the electronic effects, including ortho effects, can be treated with Hammett σ parameters. The use of substituent volumes rather than A values reflects packing of the molecule within the confines of the capsule.

Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase and its response in particular to hydrogen bond interactions. The overall goal is to understand how the protein binding pocket influences the absorption behavior, and the current status of our ongoing efforts is presented.

Photoresponsive magnetization reversal in green fluorescent protein chromophore based diradicals

We present density functional theory based calculations on six diradicals in which imino nitroxide (IN) and IN or oxo- or phospha-verdazyl radical centres are linked by fragments of structures called cyan, blue and green fluorescent protein, respectively. In the latter two variants the indolyl fragment of cyan fluorescent protein (CFP) is replaced by phenyl and phenol moiety to obtain blue fluorescent protein Y66F GFP (BFPF) and green fluorescent protein (GFP). The photoinduced cis–trans isomerization of these diradical substituted chromophores is accompanied by changes from antiferro- to ferro-magnetic spin coupling on the radical centres. The calculations in this work provide predictions of the change of magnetic response to photo excitation. The estimated magnetic exchange coupling constants associated with cis–trans isomerization of the chromophores can be useful parameters in designing radical substituted isomeric fluorescent chromophores. Upon irradiation with light of appropriate wavelength, the dark trans diradicals turn into their fluorescent cis isomers. Therefore, photoinduced magnetic crossover from antiferromagnetic to ferromagnetic regime associated with the change in color would be noticed in all three cases. This is a novel observation in case of the systems with GFP chromophore and its variants. These diradicals are potentially very useful in different applications and biocompatibility of such systems makes them prospective in different in vivo treatments. Moreover, change in color associated with magnetic crossover for these diradicals increases their suitability as biological taggers.

Fluorescence Response Profiling for Small Molecule Sensors Utilizing the Green Fluorescent Protein Chromophore and Its Derivatives

Using a fluorescence response profile, a systematic examination was performed for synthetic chromophores of the green fluorescent protein (GFP) to discover new small molecule sensors. A group of 41 benzylideneimidazolinone compounds (BDI) was prepared and screened toward 94 biologically relevant analytes to generate fluorescence response profiles. From the response pattern, compounds containing aminobenzyl and heteroaromatic cyclic substructures revealed a pH dependent emission decrease effect, and unlike other fluorescence scaffolds, most BDIs showed fluorescence quenching when mixed with proteins. On the basis of the primary response profile, we obtained three selective fluorescence turn-on sensors for pH, human serum albumin (HSA), and total ribonucleic acid (RNA). Following analysis, a fluorescence response profile testing four nucleic acids revealed the alkyloxy (Ph-OR) functional group in the para position of benzyl analogues contributes to RNA selectivity. Among the primary hit compounds, BDI 2 showed outstanding selectivity toward total RNA with 5-fold emission enhancement. Finally, BDI 24 showed selective fluorescence increase to HSA (K(d) = 3.57 μM) with a blue-shifted emission max wavelength (Δλ(em) = 15 nm). These examples of fluorescence sensor discovery by large-scale fluorescence response profiling demonstrate the general applicability of this approach and the usefulness of the response profiles.

Quantum localization and protein-assisted vibrational energy flow in cofactors

Quantum effects influence vibrational dynamics and energy flow in biomolecules, which play a central role in biomolecule function, including control of reaction kinetics. Lifetimes of many vibrational modes of proteins and their temperature dependence, as determined by quantum golden-rule-based calculations, exhibit trends consistent with experimental observation and distinct from estimates based on classical modeling. Particularly notable are quantum coherence effects that give rise to localization of vibrational states of sizable organic molecules in the gas phase. Even when such a molecule, for instance a cofactor, is embedded in a protein, remnants of quantum localization survive that influence vibrational energy flow and its dependence on temperature. We discuss these effects on the mode-damping rates of a cofactor embedded in a protein, using the green fluorescent protein chromophore as a specific example. We find that for cofactors of this size embedded in their protein and solvent environment at room temperature a golden-rule calculation often overestimates the mode-damping rate.

Potential Energy Landscape of the Electronic States of the GFP Chromophore in Different Protonation Forms: Electronic Transition Energies and Conical Intersections.

We present the results of quantum chemical calculations of the transition energies and conical intersection points for the two lowest singlet electronic states of the green fluorescent protein chromophore, 4'-hydroxybenzylidene-2,3-dimethylimidazolinone, in the vicinity of its cis conformation in the gas phase. Four protonation states of the chromophore, i.e., anionic, neutral, cationic, and zwitterionic, were considered. Energy differences were computed by the perturbatively corrected complete active space self-consistent field (CASSCF)-based approaches at the corresponding potential energy minima optimized by density functional theory and CASSCF (for the ground and excited states, respectively). We also report the EOM-CCSD and SOS-CIS(D) results for the excitation energies. The minimum energy S0/S1 conical intersection points were located using analytic state-specific CASSCF gradients. The results reproduce essential features of previous ab initio calculations of the anionic form of the chromophore and provide an extension for the neutral, cationic, and zwitterionic forms, which are important in the protein environment. The S1 PES of the anion is fairly flat, and the barrier separating the planar bright conformation from the dark twisted one as well as the conical intersection point with the S0 surface is very small (less than 2 kcal/mol). On the cationic surface, the barrier is considerably higher (∼13 kcal/mol). The PES of the S1 state of the zwitterionic form does not have a planar minimum in the Franck-Condon region. The S1 surface of the neutral form possesses a bright planar minimum; the energy barrier of about 9 kcal/mol separates it from the dark twisted conformation as well as from the conical intersection point leading to the cis-trans chromophore isomerization.

The N-Arylamino Conjugation Effect in the Photochemistry of Fluorescent Protein Chromophores and Aminostilbenes

To understand the nonradiative decay mechanism of fluorescent protein chromophores in solutions, a systematic comparison of a series of (Z)-4-(N-arylamino)benzylidene-2,3-imidazolinones (ABDIs: 2 P, 2 PP, 2 OM, and 2 OMB) and the corresponding trans-4-(N-arylamino)-4'-cyanostilbenes (ACSs: 1 P, 1 PP, 1 OM, and 1 OMB) was performed. We have previously shown that the parameter Phi(f)+2 Phi(tc), in which Phi(f) and Phi(tc) are the quantum yields of fluorescence and trans-->cis photoisomerization, respectively, is an effective probe for evaluating the contribution of twisted intramolecular charge transfer (TICT) states in the excited decays of trans-aminostilbenes, including the push-pull ACSs. One of the criteria for postulating the presence of a TICT state is Phi(f)+2 Phi(tc)<<1.0, because its formation is decoupled with the C=C bond (tau) torsion pathway and its decay is generally nonradiative. Our results show that the same concept also applies to ABDIs 2 with the parameter Phi(f)+2 Phi(ZE) in which Phi(ZE) is the quantum yield of Z-->E photoisomerization. We conclude that the tau torsion rather than the C-C bond (phi) torsion is responsible for the nonradiative decays of ABDIs 2 in aprotic solvents (hexane, THF, acetonitrile). The phenyl-arylamino C--N bond (omega) torsion that leads to a nonradiative TICT state is important only for 2 OM in THF and acetonitrile. If the solvent is protic (methanol and 10-20 % H(2)O in THF), a new nonradiative decay channel is present for ABDIs 2, but not for ACSs 1. It is attributed to internal conversion (IC) induced by solvent (donor)-solute (acceptor) hydrogen-bonding (HB) interactions. The possible HB modes and the concept of tau torsion-coupled proton transfer are also discussed.

Efficient Synthesis of New 4-Arylideneimidazolin-5-ones Related to the GFP Chromophore by 2+3 Cyclocondensation of Arylideneimines with Imidate Ylides

A 2+3 condensation of a wide assortment of Schiff bases, prepared from aromatic aldehydes and primary amines, with methyl 2-(1-ethoxyethylideneamino)acetate allows convenient access to an extensive family of substituted 4-arylideneimidazolin-5-one analogues of the green fluorescent protein (GFP) chromophore.

ChemInform Abstract: Synthesis and Properties of Chromophores of Fluorescent Proteins

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Structural Evidence for a Dehydrated Intermediate in Green Fluorescent Protein Chromophore Biosynthesis

The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C(alpha) and C(beta) atoms in the sp(3) hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C(alpha)=C(beta) bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.

A dark excited state of fluorescent protein chromophores, considered as Brooker dyes

The green fluorescent protein (GFP) chromophore is a dihydroxyarylmethine (oxonol) dye. Resonance and molecular orbital theories predict two low-lying excitations with distinct oscillator strength and polarization in these systems. Using a variational quantum-chemical model that reflects both theories, we estimate the energy of both states in resonant anionic and cationic forms of the chromophore. The energy of the higher, dark state in these forms is in the same range as the bright transition of the non-resonant neutral form, estimated by similar methods. This suggests interesting roles for the dark state in GFP photophysics.

A role for molecular compression in the post-translational formation of the Green Fluorescent Protein chromophore

Spontaneous chromophore formation is probably the key feature for the remarkable success of GFPs (Green Fluorescent Proteins) and related proteins in fluorescence microscopy. Though a quantitative analysis of the involved energetics still remains elusive, substantial progress has been made in identifying the steps of chromophore biosynthesis and the contribution of individual residues and surrounding protein matrix. The latter clearly enforces a peculiar configuration of the pre-cyclized chromophore-forming tripeptide. However, it is debated whether a mechanical compression is also at play in triggering backbone cyclization. Here, by molecular dynamics and potential of mean force calculations, we estimate the contribution of the protein scaffold in promoting the proximity of reacting atoms- and hence backbone cyclization - by a sort of compression mechanism. Comparing several mutants we highlight the role of some surrounding residues. Finally, we analyze the case of HAL (Histidine Ammonia-Lyase) active site, which undergoes an analogous cyclization reaction.

A Modified Resonance-Theoretic Framework for Structure−Property Relationships in a Halochromic Oxonol Dye

I demonstrate that a modification of the resonance color theory (in its form advocated by Brooker (Rev. Mod. Phys. 1942, 14, 275) and by Platt (J. Chem. Phys. 1956, 25, 80)) provides an accurate framework for rationalizing the ab initio excitation energies of the protonation states of the green fluorescent protein (GFP) chromophore (an asymmetric oxonol dye). I suggest that the original model space used in the resonance theory (specifically, a pair of Lewis structures) is formally inconsistent with a core aspect of the theory (specifically, a relationship between excitation energies and group-specific basicities (Brooker basicities) of the terminal rings). I argue that a more appropriate model space would consist of a complete active space ansatz based on group-localized orbitals. I then show that there is a solution to the state-averaged complete active space self consistent field (SA-CASSCF) problem with exactly this form. This family of SA-CASSCF solutions provides an objectively rigorous foundation for the resonance color theory. The solutions can be expressed in a localized set of active space orbitals, which display the same transferability pattern implied by the Brooker basicity scale. Using Platt’s model Hamiltonian formulation of the resonance theory, I show that the accuracy of the set of excitation energies calculated with these solutions can be accurately reproduced using only two parameters per dye in the set. One of these parameters is the isoenergetic energy of the dye—the harmonic mean of the excitation energies of its symmetric parent dyes. The other parameter is a local basicity index (Brooker basicity), which is specific to each terminal ring and independent of the ring to which it is conjugated in a given dye. I proceed to show that the Brooker basicities, defined by differences between many-electron states, are also basicities in the usual (one-electron) sense and, finally, that Platt’s construction of the color theory is an approximation to a ab initio effective Hamiltonian obtained by a minimum-norm block diagonalization procedure. What emerges is a powerful, simple, and accurate conceptual framework for thinking generally about color in monomethine dyes, and specifically about color tuning in the chromophore of green fluorescent proteins.