No abstract

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

Two new anil molecules exhibiting photochromism in the crystalline state, N-(4-hydroxy)-salicylideneamino-4-(methylbenzoate) (2) and N-(3,5-di-tert-butylsalicylidene)-4-aminopyridine (3), are obtained. Upon irradiation in the UV, the yellow crystals change color to red, owing to enol-keto intramolecular tautomerism. The red color disappears, when crystals are left in the dark or irradiated with visible light. 3 has the most stable keto form among all anil-type photochromic compounds (τ ) 460 days at room temperature). Both exhibit nonlinear optical (NLO) properties and show powder second harmonic generation (SHG) of respectively 10 and 3 times vs urea. X-ray diffraction shows acentric structures where molecules line up "head-to-tail" through hydrogen bonds for 2 (space group Pc), or form a chiral helix 3 (space group P3 2 ). Evidence of reversible structural change is given for 3, and we demonstrate the functionality of this crystal as an NLO switching material, as SHG can be photomodulated by about 30%.

The first systematic steady-state and time-resolved emission study of firefly oxyluciferin (emitter in firefly bioluminescence) and its analogues in aqueous buffers provided the individual emission spectra of all chemical forms of the emitter and the excited-state equilibrium constants in strongly polar environment with strong hydrogen bonding potential. The results confirmed the earlier hypothesis that excited-state proton transfer from the enol group is favored over proton transfer from the phenol group. In water, the phenol-keto form is the strongest photoacid among the isomers and its conjugate base (phenolate-keto) has the lowest emission energy (634 nm). Furthermore, for the first time we observed green emission (525 nm) from a neutral phenol-keto isomer constrained to the keto form by cyclopropyl substitution. The order of emission energies indicates that in aqueous solution a second deprotonation at the phenol group after the enol group had dissociated (that is, deprotonation of the phenol-enolate) does not occur in the first excited state. The pH-dependent emission spectra and the time-resolved fluorescence parameters revealed that the keto-enol tautomerism reaction, which can occur in a nonpolar environment (toluene) in the presence of a base, is not favored in water.

The mysterious flashes of light communicated by fireflies conceal a rich and exciting solution spectrochemistry that revolves around the chemiexcitation and photodecay of the fluorophore, oxyluciferin. A triple chemical equilibrium by double deprotonation and keto-enol tautomerism turns this simple molecule into an intricate case where the relative spectral contributions of six chemical species combine over a physiologically relevant pH range, rendering physical isolation and spectral characterization of most of the species unmanageable. To disentangle the individual spectral contributors, here we demonstrate the advantage of chemical oriented multivariate data analysis. We designed a set of specific oxyluciferin derivatives and applied a multivariate curve resolution-alternating least squares (MCR-ALS) procedure simultaneously to an extensive set of pH-dependent spectroscopic data for oxyluciferin and the target derivatives. The analysis provided, for the first time, the spectra of the pure individual components free of contributions from the other forms, their pH-dependent profiles and distributions, and the most accurate to date values for the three equilibrium constants.

Photodynamics of 2-hydroxybenzylideneaniline (photochromic salicylidene aniline SAOH) and N-(2-methoxybenzylidene)aniline (SAOMe) are studied by steady state and transient optical spectroscopy in solution and gas phase at different excitation wavelengths (266, 355 and 390 nm). Two competitive processes are observed from the enol* excited state: on one hand a rotation to get a twisted-enol, and on the other hand an excited state intramolecular proton transfer (ESIPT) followed by a cis-trans isomerisation to get the trans-keto photochromic product. For the first time both processes are characterized at an ultrashort time scale for salicylidene aniline. Resolution of the spectrokinetic data is achieved by multivariate curve resolution and attribution of the intermediate species recovered is performed in comparison with the results obtained for SAOMe, which can only undergo enol rotational isomerisation. It shows that ESIPT and rotation to the twisted-enol for SAOH occur within 100 fs, as predicted by recent quantum dynamical simulations, with an efficiency ratio dependent on the excitation wavelength. Therefore a general photoinduced mechanism for salicylidene aniline is drawn.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an Xray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final onstate. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the μs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

The single-molecule fluorescence blinking behavior of the organic dye Atto647N in various polymer matrixes such as Zeonex, PVK, and PVA as well as aqueous media was investigated. Fluorescence blinking with off-times in the millisecond to second time range is assigned to dye radical ions formed by photoinduced electron transfer reactions from or to the environment. In Zeonex and PVK, the measured off-time distributions show power law dependence, whereas, in PVA, no such dependence is observed. Rather, in this polymer, off-time distributions can be best fitted to monoexponential or stretched exponential functions. Furthermore, treatment of PVA samples to mild heating and low pressure greatly reduces the frequency of blinking events. We tentatively ascribe this to the removal of water pockets within the polymer film itself. Measurements of the dye immobilized in water in the presence of methylviologen, a strongly oxidizing agent, reveal simple exponential on- and off-time distributions. Thus, our data suggest that the blinking behavior of single organic molecules is sensitive to their immediate environment and, moreover, that fluorescence blinking on- and off-time distributions do not inherently and uniquely obey a power law.