Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed

Recently, it has been shown that the red fluorescent protein DsRed undergoes photoconversion on intense irradiation, but the mechanism of the conversion has not yet been elucidated. Upon irradiation with a nanosecond-pulsed laser at 532 nm, the chromophore of DsRed absorbing at 559 nm and emitting at 583 nm (R form) converts into a super red (SR) form absorbing at 574 nm and emitting at 595 nm. This conversion leads to a significant change in the fluorescence quantum yield from 0.7 to 0.01. Here we demonstrate that the photoconversion is the result of structural changes of the chromophore and one amino acid. Absorption, fluorescence, and vibrational spectroscopy as well as mass spectrometry suggest that a cis-to-trans isomerization of the chromophore and decarboxylation of a glutamate (E215) take place upon irradiation to form SR. At the same time, another photoproduct (B) with an absorption maximum at 386 nm appears upon irradiation. This species is assigned as a protonated form of the DsRed chromophore. It might be a mixture of several protonated DsRed forms as there is at least two ways of formation. Furthermore, the photoconversion of DsRed is proven to occur through a consecutive two-photon absorption process. Our results demonstrate the importance of the chromophore conformation in the ground state on the brightness of the protein as well as the importance of the photon flux to control/avoid the photoconversion process.     

THE BLUE LIGHT RESPONSE OF STOMATA: PULSE KINETICS AND SOME MECHANISTIC IMPLICATIONS*

Abstract— Intact leaves of Commelina communis irradiated with high fluence rates of red light, showed discrete increases in stomatal conductance in response to pulses (1-100 s) of blue light (250 μmol m^?2 s^?1). Red light pulses were ineffective, indicating that the conductance increases were not mediated by photosynthesis and that they constitute a specific stomatal response to blue light. The response peaked 15 min after the pulse and was completed within50–60 min. Conductance increases were proportional to pulse duration up to about 30 s and saturated at longer exposures. The relationship between stomatal responses and pulse duration approximately fitted an exponential function, with a t 9s. Pulse responses at two different fluences indicated that reciprocity held. Responses to two consecutive pulses varied with time between pulses. A saturating pulse applied immediately after a preceding one induced no additional response; two saturating pulses 50 min apart caused two identical, consecutive responses. Total increases in conductance induced by two pulses separated by intermediate time intervals increased with time between pulses with a = 9 min. These results point to a blue light-dependent photoconversion of a molecular form, with the activity of the photoconversion product decaying in a thermal reaction. Under continuous blue light, prevailing fluence rates and rates of the light and thermal reactions are postulated to determine steady-state activities of the photoconversion product and proportional increases in conductance levels. These findings have implications for the environmental and metabolic roles of the stomatal response to blue light.     

Photo-impulsive reactions in the electronic ground state without electronic excitation: non-photo, non-thermal chemical reactions

Allyl phenyl ether has an absorption band in the ultraviolet region (λ < 400 nm); therefore, irradiation with few-optical-cycle ultraviolet pulses (λ = 360-440 nm) causes a transition to the ultraviolet band, which leads to an electronic state and a photo-Claisen rearrangement (radical reaction) in the electronic excited state. However, the reaction scheme of allyl phenyl ether under irradiation with few-optical-cycle visible pulses (λ = 525-725 nm) was determined to be same as that of the thermal Claisen rearrangement ([3,3]-sigmatropic rearrangement), which is symmetry-allowed in the electronic ground state. Photo-excitation with few-optical cycle visible pulses below the absorption band induces a photo-impulsive reaction in the electronic ground state without electronic excitation, of which the trigger scheme is different from that of photoreaction or thermal-reaction. The photo-impulsive reaction in the electronic ground state is highly possible as a novel reaction scheme.     

From Graftable Biphotonic Chromophores to Water‐Soluble Organic Nanodots for Biophotonics: The Importance of Environmental Effects

The photophysical and two‐photon absorption (TPA) properties of biphotonic chromophores with one or two phenol pendant units were studied and compared with that of a model biphotonic quadrupolar chromophore. A water‐soluble dendritic structure was then synthesized by using the pendant moieties as starting points for the construction of dendritic branches. We show that the polarity of the environment significantly modulates both the fluorescence and the TPA responses of the different chromophoric derivatives. This extends to more subtle effects that involve phenol pendant moieties that were found to act as discrete solvating units and to modify both the photophysics and the TPA response of the chromophore. This demonstrates the high sensitivity of the TPA response of quadrupolar derivatives to minute alterations in the environment. Moreover, the dendritic branches were found to behave as a peculiar cybotactic environment that was able to tune the fluorescence and TPA response of the inner chromophore by creating a polar environment. This reveals a new direction for exploiting such effects by playing on the dendritic architecture (e.g., the nature and shape of the building blocks, the geometry and position of the chromophore) to modulate the TPA responses.     

A new emission band of Eu2+ and its efficient energy transfer to Mn2+ in Sr2Mg3P4O15:Mn2+, Eu2+

Eu(2+) singly and Eu(2+), Mn(2+) co-doped Sr(2)Mg(3)P(4)O(15) exhibit not only the well known blue emission band of Eu(2+) peaking at 448 nm but also a new band at 399 nm in violet. They are attributed to Eu(2+) on different Sr(2+) sites. The Eu(2+) for the violet band can transfer energy to the red emitting Mn(2+) more efficiently than Eu(2+) for the blue band. The new Eu(2+) band could enable Sr(2)Mg(3)P(4)O(15):Mn(2+), Eu(2+) to be a promising phosphor for enriching the red component of white LEDs.     

Star‐shaped polymer PFStODO by atom transfer radical polymerization: Its synthesis,characterization, and fluorescence property

A novel monomer bearing with pyrazoline chromophore, 3‐(4‐fluorophenyl)‐1‐phenyl‐5‐(4‐(4‐vinylbenzyloxy)phenyl‐4,5‐dihydro‐1H‐pyrazole (FStODO), was synthesized, and its atom transfer radical polymerization initiated by a tetrafunctional initiator, pentaerythritol tetrakis(2‐bromoisobutyrate) (4Bri‐Bu), was studied in detail and characterized by ¹H NMR, GPC, and TG‐DSC. The solution, film luminescence of monomer, and its polymer were studied in detail. Compared with that of monomer, both luminescence emission intensity and quantum yield of star‐shaped polymer PFStODO in DMF solution are decreased. However, the two‐photon absorption (TPA) spectra in DMF solution measured by a femtosecond laser show an entirely different result that the maxima TPA cross‐section value of polymer reaches to 203 GM, better than that of the monomer itself (13 GM). More interestingly, the film of polymer shows surprisingly white emission ranging from 400 to 700 nm assigned to the excimer formation. We attribute this formation of excimers to the ordered chromophore aggregates in film, which is further verified by X‐ray diffraction. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Synthesis of fluorescent diarylethenes having a 2,4,5-triphenylimidazole chromophore

Diarylethenes 1a-4a, having a fluorescent 2,4,5-triphenylimidazole chromophore in the aryl group, were synthesized. Upon excitation of the triphenylimidazole chromophore with 366 nm, 1a-4a underwent photocyclization reactions, and the solutions containing 1a-4a changed color from colorless to red-purple or to blue. The colors disappeared by irradiation with visible (lambda > 480 nm) light. The fluorescence intensity of the solutions also reversibly changed with the photochromic reactions. The fluorescence quantum yields of 1a, 2a, 3a, and 4a were determined to be 4.6, 7.7, 9.1, and 8.4%, respectively. The fluorescence quantum yields decreased with the increase in photocyclization quantum yields.     

Resonance Raman Structural Evidence that the Cis-to-Trans Isomerization in Rhodopsin Occurs in Femtoseconds

Picosecond time-resolved resonance Raman spectroscopy is used to probe the structural changes of rhodopsin's retinal chromophore as the cis-to-trans isomerization reaction occurs that initiates vision. Room-temperature resonance Raman spectra of rhodopsin's photoproduct with time delays from -0.7 to 20.8 ps are measured using 2.2 ps, 480 nm pump and 1.5 ps, 600 nm probe pulses. Hydrogen-out-of-plane (HOOP) modes at 852, 871, and 919 cm(-1), fingerprint peaks at 1272, 1236, 1211, and 1166 cm(-1), and a broad red-shifted ethylenic band at 1530 cm(-1) are present at the earliest positive pump-probe time delay of 0.8 ps, indicating that the chromophore is already in a strained, all-trans configuration. Kinetic analyses of both the HOOP and ethylenic regions of the photoproduct spectra reveal that these features grow in with fast ( approximately 200 fs) and slow ( approximately 2-3 ps) components. These data provide the first structural evidence that photorhodopsin has a thermally unrelaxed, torsionally strained all-trans chromophore within approximately 1 ps, and possibly within 200 fs, of photon absorption. Following this ultrafast product formation, the all-trans chromophore cools and conformationally relaxes within a few picoseconds to form bathorhodopsin. This cooling process is revealed as an ethylenic frequency blue-shift of 6 cm(-1) (tau approximately 3.5 ps) as well as an ethylenic width narrowing (tau approximately 2 ps). The ultrafast production of photorhodopsin is likely accompanied by an impulsively driven, localized protein response. More delocalized protein modes are unable to relax on this ultrafast time scale enabling the chromophore-protein complex to store the large amounts of photon energy (30-35 kcal/mol) that are subsequently used to drive activating protein conformational changes.     

Dansyl-anthracene dyads for ratiometric fluorescence recognition of Cu2+

Dansyl-anthracene dyads 1 and 2 in CH(3)CN-H(2)O (7:3) selectively recognize Cu(2+) ions amongst alkali, alkaline earth and other heavy metal ions using both absorbance and fluorescence spectroscopy. In absorbance, the addition of Cu(2+) to the solution of dyads 1 or 2 results in appearance of broad absorption band from 200 nm to 725 nm for dyad 1 and from 200 nm to 520 nm for dyad 2. This is associated with color change from colorless to blue (for 1) and fluorescent green (for 2). This bathochromic shift of the spectrum could be assigned to internal charge transfer from sulfonamide nitrogen to anthracene moiety. In fluorescence, under similar conditions dyads 1 and 2 on addition of Cu(2+) selectively quench fluorescence due to dansyl moiety between 520-570 nm (for 1)/555-650 nm (for 2) with simultaneous fluorescence enhancement at 470 nm and 505 nm for dyads 1 and 2, respectively. Hence these dyads provide opportunity for ratiometric analysis of 1-50 μM Cu(2+). The other metal ions viz. Fe(3+), Co(2+), Ni(2+), Cd(2+), Zn(2+), Hg(2+), Ag(+), Pb(2+), Li(+), Na(+), K(+), Mg(2+), Ca(2+), Ba(2+) do not interfere in the estimation of Cu(2+) except Cr(3+) in case of dyad 1. The coordination of dimethylamino group of dansyl unit with Cu(2+) causes quenching of fluorescence due to dansyl moiety between 520-600 nm and also restricts the photoinduced electron transfer from dimethylamino to anthracene moiety to release fluorescence between 450-510 nm. This simultaneous quenching and release of fluorescence respectively due to dansyl and anthracene moieties emulates into Cu(2+) induced ratiometric change.     

Crystal Growth,Structure, and Spectral Properties of Cr~(4+):Ca_2(Al_(1.8)Ga_(0.2))SiO_7

A new crystal, Ca_2(Al_(1.8)Ga_(0.2))SiO_7, was obtained by substituting Ga~(3+) ions for some Al~(3+) ions in Ca_2Al_2SiO_7 crystal. The growth, structure and optical spectroscopic properties of Cr~(4+)-doped Ca_2(Al_(1.8)Ga_(0.2))SiO_7 were studied. It shows strong absorption at 693 and 762 nm and a broad emission band with peak wavelength at 1223 nm. Both the absorption and emission peaks of Cr_(4+)-doped Ca_2(Al_(1.8)Ga_(0.2))SiO_7 crystal are red-shifted in comparison with that of Cr~(4+)-doped Ca_2Al_2SiO_7 crystal due to its weaker lattice field. The investigation results show that there is only one kind of tetrahedral site for Cr~(4+) occupation in the lattice of Ca_2(Al_(1.8)Ga_(0.2))SiO_7 crystal.     

RESONANCE RAMAN SPECTRA OF BACTERIORHODOPSIN MUTANTS WITH SUBSTITUTIONS AT ASP-85, ASP-96, AND ARG-82

Detergent solubilized bacteriorhodopsin (BR) proteins which contain alterations made by site-directed mutagenesis (Asp-96----Asn, D96N; Asp-85----Asn, D85N; and Arg-82----Gln, R82Q) have been studied with resonance Raman spectroscopy. Raman spectra of the light-adapted (BRLA) and M species in D96N are identical to those of native BR, indicating that this residue is not located near the chromophore. The BRLA states of D85N and especially R82Q contain more of the 13-cis, C = N syn (BR555) species under ambient illumination compared to solubilized native BR. Replacement of Asp-85 with Asn causes a 25 nm red-shift of the absorption maximum and a frequency decrease in both the ethylenic (-7 cm-1) and the Schiff base C = NH+ (-3 cm-1) stretching modes of BRLA. These changes indicate that Asp-85 is located close to the protonated retinal Schiff base. The BRLA spectrum of R82Q exhibits a slight perturbation of the C = NH+ band, but its M spectrum is unperturbed. The Raman spectra and the absorption properties of D85N and R82Q suggest that the protein counterion environment involves the residues Asp-85-, Arg-82+ and presumably Asp-212-. These data are consistent with a model where the strength of the protein-chromophore interaction and hence the absorption maximum depends on the overall charge of the Schiff base counterion environment.     

Dual Illumination Enhances Transformation of an Engineered Green-to-Red Photoconvertible Fluorescent Protein

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species.     

Sugar-induced blue membrane: release of divalent cations during phase transition of purple membranes observed in sugar-derived glasses

The formation of blue membrane from purple membranes (PM) has been observed in glassy films made from PM and various sugars. The phase transition of PM at about 70 degrees C causes the complexation of divalent cations to be weakened. The vicinal diol structures in sugars are capable to complex divalent cations and delocalize them throughout the matrix as long as its glass transition temperature is lower than the phase transition temperature of PM. The loss of divalent cations from bacteriorhodopsin (BR), the only protein in PM, causes the formation of blue membrane (BM), which is accompanied by a loss of beta-sheet structure observable in the infrared spectrum. Glassy sugars are particular useful to observe this transition, as sugar entrapment does not restrict conformational changes of BR but rather retards them. The material obtained was named sugar-induced blue membrane (SIBM). The formation of SIBM is inhibited by the addition of divalent cations. Furthermore, SIBM is reverted immediately to PM by addition of water. A characteristic time dependence of the thermal reversion of SIBM to PM proves that the phase transition of PM triggers the release and uptake of divalent cations and the corresponding color change.     

Single-molecule fluorescence lifetime and anisotropy measurements of the red fluorescent protein,DsRed, in solution

Fluorescence lifetime and anisotropy measurements were made on the red fluorescent protein (DsRed) from tropical coral of the Discosoma genus, both at single-molecule and bulk concentrations. As expected from previous work, the fluorescence lifetime of DsRed in solution is dependent on laser power, decreasing from an average fluorescence lifetime in the beam of about 3.3 ns at low power (3.5 ns if one extrapolates to zero power) to about 2.1 ns at 28 kW/cm2. At the single-molecule level, exciting with 532 nm, 10 ps laser pulses at 80 MHz repetition rate, DsRed particles entering the laser beam initially have a lifetime of about 3.6 ns and convert to a form having a lifetime of about 3.0 ns with a quantum yield of photoconversion on the order of 10(-3) (calculated in terms of photons per DsRed tetramer). The particles then undergo additional photoconversion with a quantum yield of roughly 10(-5), generating a form with an average lifetime of 1.6 ns. These results may be explained by rapid photoconversion of one DsRed monomer in a tetramer, which acts as an energy transfer sink, resulting in a lower quantum yield for photoconversion of subsequent monomers. Multiparameter correlation and selective averaging can be used to identify DsRed in a mixture of fluorophores, in part exploiting the fact that fluorescent lifetime of DsRed changes as a function of excitation intensity.     

EDXRF quantitative analysis of chromophore chemical elements in corundum samples

Corundum is a crystalline form of aluminum oxide (Al₂O₃) and is one of the rock-forming minerals. When aluminum oxide is pure, the mineral is colorless, but the presence of trace amounts of other elements such as iron, titanium, and chromium in the crystal lattice gives the typical colors (including blue, red, violet, pink, green, yellow, orange, gray, white, colorless, and black) of gemstone varieties. The starting point for our work is the quantitative evaluation of the concentration of chromophore chemical elements with a precision as good as possible to match the data obtained by different techniques as such as optical absorption photoluminescence. The aim is to give an interpretation of the absorption bands present in the NIR and visible ranges which do not involve intervalence charge transfer transitions (Fe²⁺ → Fe³⁺ and Fe²⁺ → Ti⁴⁺), commonly considered responsible of the important features of the blue sapphire absorption spectra. So, we developed a method to evaluate as accurately as possible the autoabsorption effects and the secondary excitation effects which frequently are sources of relevant errors in the quantitative EDXRF analysis.     

Intramolecular charge resonance in dimer radical anions of di-, tri-, tetra-, and pentaphenylalkanes

Intramolecular dimer radical anions of di-, tri-, tetra-, and pentaphenylalkanes were investigated on the basis of absorption spectral measurements during γ-radiolysis in 2-methyltetrahydrofuran (MTHF) glassy matrix at 77 K and theoretical calculations. The absorption spectrum of 1,1,2,2-tetraphenylethane (1,1,2,2-Ph(4)E) radical anion showed two bands in the near-infrared (NIR) region (900-2600 nm). One band observed at shorter wavelength than 2000 nm is assigned to the intramolecular charge resonance (CR) band between two phenyl groups of the 1,1-diphenylmethyl chromophore (1,1-dimer radical anion). The intramolecular CR band of the 1,1-dimer radical anion was observed for various alkanes having 1,1-diphenylmethyl chromophore such as 1,1,1-triphenylmethane (1,1,1-Ph(3)M), 1,1,1,1-tetraphenylmethane (1,1,1,1-Ph(4)M), and so on. The other intramolecular CR band observed at longer wavelength than 2200 nm is assigned to intramolecular dimer radical anion between two phenyl groups of the 1,2-diphenylethyl chromophore (1,2-dimer radical anion). The intramolecular CR band of the 1,2-dimer radical anion was observed for various alkanes having a 1,2-diphenylethyl chromophore, such as 1,1,2-triphenylethane (1,1,2-Ph(3)E), 1,1,2,2-Ph(4)E, and 1,1,1,2,2-pentaphenylethane (1,1,1,2,2-Ph(5)E) and so on. No dimer radical anion was observed for 1,n-diphenylalkanes (n > 2) without 1,1-diphenylmethyl chromophore. The relationship between the structure and negative charge delocalization over two phenyl groups connected by an sp(3) carbon is discussed.     

PICOSECOND KINETICS and A MODEL FOR THE PRIMARY EVENTS OF BACTERIORHODOPSIN

The picosecond absorption kinetics of light adapted bacteriorhodops in (BR) are reported for water and glycerol-water suspensions of purple membrane from Halobacieriwn hulobium at different temperatures (77-300 K) and pH (5-10). It is shown that a photoproduct of BR, called P-BR (pseudo-bacteriorhodopsin). is responsible for the 16 ± 2 ps relaxation lifetime observed under steady state irradiation (i.e. with a train of ps pulses) at room temperature. At 77 K the absorption recovery lifetime is 62 ± 5 ps in good agreement with previous fluorescence lifetime studies. The observed signal is very sensitive to the sample flow rate and at the highest flow rates a fast absorbance increase (≤ 2 ps) is observed at λ > 620 nm. while at 576 nm a similarly fast absorption recovery alter bleaching is observed. These results imply that the reaction BR → K-BR occurs within 2 ps. In order to explain the simultaneous formation of P-BR and K-BR at 77 K. a model for the primary events including a traus-cis isomerization and a protein relaxation about the chromophore is suggested. The same model can also be used in explaining the simultaneous formation of batho- and hypsorhodopsin in some rhodopsins. Finally, a scheme for the last steps in the photocycle of bacteriorhodopsin including protonation, protein relaxation and cis-trans isomerization is proposed     

Promising oxonitridosilicate phosphor host Sr3Si2O4N2: synthesis, structure, and luminescence properties activated by Eu2+ and Ce3+/Li+ for pc-LEDs

A novel oxonitridosilicate phosphor host Sr(3)Si(2)O(4)N(2) was synthesized in N(2)/H(2) (6%) atmosphere by solid state reaction at high temperature using SrCO(3), SiO(2), and Si(3)N(4) as starting materials. The crystal structure was determined by a Rietveld analysis on powder X-ray and neutron diffraction data. Sr(3)Si(2)O(4)N(2) crystallizes in cubic symmetry with space group Pa ?3, Z = 24, and cell parameter a = 15.6593(1) ?. The structure of Sr(3)Si(2)O(4)N(2) is constructed by isolated and highly corrugated 12 rings which are composed of 12 vertex-sharing [SiO(2)N(2)] tetrahedra with bridging N and terminal O to form three-dimensional tunnels to accommodate the Sr(2+) ions. The calculated band structure shows that Sr(3)Si(2)O(4)N(2) is an indirect semiconductor with a band gap ≈ 2.84 eV, which is close to the experimental value ≈ 2.71 eV from linear extrapolation of the diffuse reflection spectrum. Sr(3-x)Si(2)O(4)N(2):xEu(2+) shows a typical emission band peaking at ~600 nm under 460 nm excitation, which perfectly matches the emission of blue InGaN light-emitting diodes. For Ce(3+)/Li(+)-codoped Sr(3)Si(2)O(4)N(2), one excitation band is in the UV range (280-350 nm) and the other in the UV blue range (380-420 nm), which matches emission of near-UV light-emitting diodes. Emission of Sr(3-2x)Si(2)O(4)N(2):xCe(3+),xLi(+) shows a asymmetric broad band peaking at ~520 nm. The long-wavelength excitation and emission of Eu(2+) and Ce(3+)/Li(+)-doped Sr(3)Si(2)O(4)N(2) make them attractive for applications in phosphor-converted white light-emitting diodes.     

Dual Illumination Enhances Transformation of an Engineered Green‐to‐Red Photoconvertible Fluorescent Protein

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light‐induced processes. We implemented time‐resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP‐like protein LEA, involving semi‐trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual‐illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge‐H bending motions enables rational design of functional proteins. With the improved H‐bonding network and structural motions, the photoexcited chromophore could increase the photoswitching‐aided photoconversion while reducing trapped species.     

Formation of high concentrations of BrO(2) in acidic bromate solutions

A new procedure to produce the BrO(2) transient species allowed time-resolved UV-vis spectra that show a structured band (lambda(max) = 502 nm) in dichloromethane to be obtained. In water, because of the increase of the dielectric constant, the lambda(max) presents a blue shift to 474 nm and the species decomposes much faster. The time-resolved spectra show evidence for its equilibrium with a nonidentified colorless form. This route opens new possibilities to the study this species in solution.