Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore.

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.

Why does a para-amino group make the green fluorescent protein chromophore non-fluorescent: coherent intramolecular charge transfer reduces the Z/E-photoisomerization barrier.

A para-N-phenyl-amino group significantly increases the fluorescence quantum yield of N-phenyl-4-aminostilbene by the "amino conjugation effect", but, in contrast, a para-amino group in the para-amino analogue (p-ABDI) of the green fluorescent protein (GFP) chromophore makes p-ABDI non-fluorescent because the coherent photo-induced intramolecular charge transfer reduces the barrier of the Z/E-photoisomerization.

Ultrafast trans-cis photoisomerization of the neutral chromophore in green fluorescent proteins: Surface-hopping dynamics simulation.

Reversible photoswitching fluorescent protein can reversibly switch between on-state (fluorescent) and off-state (dark). Anionic cis and neutral trans chromophores are the on- and off-states in green fluorescent proteins (GFPs), respectively. We investigated the ultrafast trans-cis photoisomerization mechanisms of the neutral GFP chromophore upon excitation to the S1 state by means of surface-hopping dynamics simulations based on the Zhu-Nakamura theory. Two trans isomers, located in the S0 state, were taken into consideration in dynamics simulation. After these two trans isomers are excited to the S1 state, the molecule moves to a excited-state minimum by increasing the imidazolinone-bridge bond length and decreasing the phenol-bridge bond length. The twist of imidazolinone-bridge bond drives the molecule toward a conical intersection, and internal conversion occurs. Then, a cis or trans conformer will be obtained in the S0 state. The torsion around the imidazolinone-bridge bond plays a key role in the ultrafast photoisomerization of a neutral chromophore. The torsional motion around the phenol-bridge bond is restricted in the S1 state, while it may occur in the S0 state. The isomerization reaction of this molecule is predicted to be not sensitive to solvent viscosity, and time-dependent density functional theory (TDDFT) calculations indicate that the fast excited-state decay from the Franck-Condon region of the trans isomer to the excited-state minimum was almost independent of solvent polarity.

Synthesis and properties of geometrical 4-diarylmethylene analogs of the green fluorescent protein chromophore.

We have developed a novel analog of the GFP chromophore: geo-DAIN. Since geo-DAIN is equipped with an E/Z-photoisomerizable geometrical diarylmethylene moiety instead of benzylidene of the GFP chromophore, different-colored reversible emissions are expected. We synthesized geo-DAIN by a condensation with methyl imidate and N-(diarylmethylene)glycinate. We found the emission from geo-DAIN to be different from that of benzylidene-type analogs; in the powder state, the E- and Z-isomers of geo-DAIN emitted different fluorescence colors.

A comparative kinetic and mechanistic study between tetrahydrozoline and naphazoline toward photogenerated reactive oxygen species.

Kinetic and mechanistic aspects of the vitamin B2 (riboflavin [Rf])-sensitized photo-oxidation of the imidazoline derivates (IDs) naphazoline (NPZ) and tetrahydrozoline (THZ) were investigated in aqueous solution. The process appears as important on biomedical grounds, considering that the vitamin is endogenously present in humans, and IDs are active components of ocular medicaments of topical application. Under aerobic visible light irradiation, a complex picture of competitive interactions between sensitizer, substrates and dissolved oxygen takes place: the singlet and triplet ((3)Rf*) excited states of Rf are quenched by the IDs: with IDs concentrations ca. 5.0 mM and 0.02 mM Rf, (3)Rf* is quenched by IDs, in a competitive fashion with dissolved ground state oxygen. Additionally, the reactive oxygen species: O(2)((1)Delta(g)), O(2)(*-), HO(*) and H(2)O(2), generated from (3)Rf* and Rf(*-), were detected with the employment of time-resolved methods or specific scavengers. Oxygen uptake experiments indicate that, for NPZ, only H(2)O(2) was involved in the photo-oxidation. In the case of THZ, O(2)(*-), HO(*) and H(2)O(2) were detected, whereas only HO(*) was unambiguously identified as THZ oxidative agents. Upon direct UV light irradiation NPZ and THZ generate O(2)((1)Delta(g)), with quantum yields of 0.2 (literature value, employed as a reference) and 0.08, respectively, in acetonitrile.

Deactivation mechanism of the green fluorescent chromophore.

We report time-resolved fluorescence data for the anion of p-hydroxybenzylidene dimethylimidazolinone (p-HBDI), a model chromophore of the green fluorescence protein, in viscous glycerol-water mixtures over a range of temperatures, T. The markedly nonexponential decay of the excited electronic state is interpreted with the aid of an inhomogeneous model possessing a Gaussian coordinate-dependent sink term. A nonlinear least-squares fitting routine enables us to achieve quantitative fits by adjusting a single activation parameter, which is found to depend linearly on 1/T. We derive an analytic expression for the absolute quantum yield, which is compared with the integrated steady-state fluorescence spectra. The microscopic origins of the model are discussed in terms of two-dimensional dynamics, coupling the phenyl-ring rotation to a swinging mode that brings this flexible molecule to the proximity of a conical intersection on its multidimensional potential energy surface.