Redshifted and Near‐infrared Active Analog Pigments Based upon Archaerhodopsin‐3

Archaerhodopsin‐3 (AR3) is a member of the microbial rhodopsin family of hepta‐helical transmembrane proteins, containing a covalently bound molecule of all‐trans retinal as a chromophore. It displays an absorbance band in the visible region of the solar spectrum (λmax 556 nm) and functions as a light‐driven proton pump in the archaeon Halorubrum sodomense. AR3 and its mutants are widely used in neuroscience as optogenetic neural silencers and in particular as fluorescent indicators of transmembrane potential. In this study, we investigated the effect of analogs of the native ligand all‐trans retinal A1 on the spectral properties and proton‐pumping activity of AR3 and its single mutant AR3 (F229S). While, surprisingly, the 3‐methoxyretinal A2 analog did not redshift the absorbance maximum of AR3, the analogs retinal A2 and 3‐methylamino‐16‐nor‐1,2,3,4‐didehydroretinal (MMAR) did generate active redshifted AR3 pigments. The MMAR analog pigments could even be activated by near‐infrared light. Furthermore, the MMAR pigments showed strongly enhanced fluorescence with an emission band in the near‐infrared peaking around 815 nm. We anticipate that the AR3 pigments generated in this study have widespread potential for near‐infrared exploitation as fluorescent voltage‐gated sensors in optogenetics and artificial leafs and as proton pumps in bioenergy‐based applications.     

THE SUBSTRATE-DEPENDENT PHOTOINACTIVATION OF UROCANASE FROM RAT LIVER

Abstract— Rat liver urocanase was readily inactivated by near-UV light in the presence of the substrate. Irradiation of substrate or enzyme alone was ineffective. The purpose of this study was to examine the conditions which influenced this inactivation and to investigate the mechanism. The urocanate concentration needed for 50% of the maximum inactivation for a 15 min irradiation was 0.09 μ M . Temperatures from 0 to 30°C during irradiation had little influence. Inactivation occurred at -75°C, which indicated a photochemical reaction. The pH had little influence on inactivation. Photoinactivation was the same in nitrogen and air. Dialysis experiments showed that unbound small molecules were probably not involved. Inactivated enzyme did not inhibit active enzyme. Chelators, reducing agents, and pyridoxal phosphate did not affect the inactivation. Visible light was not effective. An action spectrum was established with the aid of a monochromator. The action spectrum had a peak at 280 nm and a shoulder extending from 300 to 340 nm which rules out flavins. pyridoxal phosphate, a simple protein, and free urocanate as the chromophore. The results suggest that this photochemical process is not photodynamic action. It appears that only substrate and enzyme are needed for this photoinactivation. The enzyme-substrate complex may be the chromophore.     

Excitations, optical absorption spectra, and optical excitonic gaps of heterofullerenes. I. C60, C59N+, and C48N12: theory and experiment

Low-energy excitations and optical absorption spectrum of C(60) are computed by using time-dependent (TD) Hartree-Fock, TD-density functional theory (TD-DFT), TD DFT-based tight-binding (TD-DFT-TB), and a semiempirical Zerner intermediate neglect of diatomic differential overlap method. A detailed comparison of experiment and theory for the excitation energies, optical gap, and absorption spectrum of C(60) is presented. It is found that electron correlations and correlation of excitations play important roles in accurately assigning the spectral features of C(60), and that the TD-DFT method with nonhybrid functionals or a local spin density approximation leads to more accurate excitation energies than with hybrid functionals. The level of agreement between theory and experiment for C(60) justifies similar calculations of the excitations and optical absorption spectrum of a monomeric azafullerene cation C(59)N(+), to serve as a spectroscopy reference for the characterization of carborane anion salts. Although it is an isoelectronic analogue to C(60), C(59)N(+) exhibits distinguishing spectral features different from C(60): (1) the first singlet is dipole-allowed and the optical gap is redshifted by 1.44 eV; (2) several weaker absorption maxima occur in the visible region; (3) the transient triplet-triplet absorption at 1.60 eV (775 nm) is much broader and the decay of the triplet state is much faster. The calculated spectra of C(59)N(+) characterize and explain well the measured ultraviolet-visible (UV-vis) and transient absorption spectra of the carborane anion salt [C(59)N][Ag(CB(11)H(6)Cl(6))(2)] [Kim et al., J. Am. Chem. Soc. 125, 4024 (2003)]. For the most stable isomer of C(48)N(12), we predict that the first singlet is dipole-allowed, the optical gap is redshifted by 1.22 eV relative to that of C(60), and optical absorption maxima occur at 585, 528, 443, 363, 340, 314, and 303 nm. We point out that the characterization of the UV-vis and transient absorption spectra of C(48)N(12) isomers is helpful in distinguishing the isomer structures required for applications in molecular electronics. For C(59)N(+) and C(48)N(12) as well as C(60), TD-DFT-TB yields reasonable agreement with TD-DFT calculations at a highly reduced cost. Our study suggests that C(60), C(59)N(+), and C(48)N(12), which differ in their optical gaps, have potential applications in polymer science, biology, and medicine as single-molecule fluorescent probes, in photovoltaics as the n-type emitter and/or p-type base of a p-n junction solar cell, and in nanoelectronics as fluorescence-based sensors and switches.     

ACTION SPECTRA FOR IN ACTIVATION AND MEMBRANE DAMAGE OF Saccharomyces cerevisiae CELLS IRRADIATED IN VACUUM BY MONOCHROMATIC SYNCHROTRON UV RADIATION (155-250 nm)

We investigated the wavelength dependence of inactivation and membrane damage in yeast cells ( Saccharomyces cerevisiae ) in the range from 170 to 200 nm. Action spectra constructed at wavelengths from 155 to 250 nm using published data were nearly the same for the two types of effects below 200 nm, but differed from the absorption spectrum of DNA, indicating that major lethal damage occurred in the membrane, not in DNA. This conclusion was strongly supported by the finding that far-UV-sensitive cells, which lack excision repair, showed no enhanced sensitivity below 200 nm.     

Experimental Correspondence between Spore Dosimetry and Spectral Photometry of Solar Ultraviolet Radiation

Abstract— The biologically effective dose of solar UV radiation was estimated from the inactivation of UV-sensitive Bacillus subtilis spores. Two types of independent measurements were carried out concurrently at the Aerological Observatory in Tsukuba: one was the direct measurement of colony-forming survival that provided the inactivation dose per minute (ID/min) and the other was the measurement of the spectral irradiance by a Brewer spectrophotometer. To obtain the effective spectrum, the irradiance for each 1 nm wavelength interval from 290 to 400 nm was multiplied with the efficiency for inactivation derived from the inactivation action spectrum of identically prepared spore samples. Integration of the effective spectrum provided the estimate for ID/min. The observed values of ID/min were closely concordant with the calculated values for the data obtained in four afternoons in 1993. The average ratio (±SD) between them was 1.24 (±0.16) for 14 data points showing high inactivation rates (<0.05 ID/min). Considering difficulties in the absolute dosimetry of UV radiation, the concordance was satisfactory and improved credibility of the two types of monitoring systems of biologically effective dose of solar UV radiation.     

An unusual case of photoreactivation observed in an insect egg (Smittia Spec., chironomidae, diptera).

Abstract— An action spectrum for inactivation by ultraviolet (UV) radiation of Smittia eggs during intravitelline cleavage was established, taking into account wavelength-dependent shielding of the effective targets. Under the assumption of a random distribution of the effective targets in the egg, the action spectrum displayed only one very distinct peak at 295 nm. The eggs were photoreactivable with an action spectrum similar but not identical to that found for direct photoreactivation (PR) in E. coli Indirect PR seems not involved because light was effective only after but not before UV. Temperature dependence and dose rate saturation could not be observed. The photoreactivable sector (PRS) was 0.75 after UV inactivation at 295 nm but only 0.32 after UV inactivation at 265 nm. Initial PR rates were highest after 295 nm and lowest after 265 nm. During migration of cleavage nuclei into the periplasm, when the shielding of nuclei by yolk material decreases by an order of magnitude, no corresponding increase in the sensitivity of the eggs to UV was observed. After inactivation at the blastoderm stage, when the nuclei are no longer shielded by yolk material, the PRS was also high (0.79) after UV of 295 nm but again lower (0.59) after 265 nm. These data are difficult to understand within the conceptual framework of light-dependent enzymatic splitting of UV-induced pyrimidine dimers in nucleic acids. Yet this type of PR seems to play a vital role in the survival of Smittia eggs under sunlight without need for pigmentation or shading.     

THE ROLE OF HEMOGLOBIN IN THE PHOTOTAXIS OF THE NEMATODE Mermis nigrescens

Abstract A dense zone of crystalline hemoglobin in the head has been presumed to be involved in the photosensitivity of Mermis. With the aim of identifying its role, we have studied the wavelength dependence of the phototaxis. Measuring phototaxic efficiency at constant photon fluence rate (intensity), we find that the spectral response curve is approximately fiat from 350–540 nm and falls to an insignificant level by 580 nm. This is unlike the absorptance (fraction absorbed) spectrum of the hemoglobin pigmentation. Also, fluence-rate/response curves at 420 and 500 nm occur at the same fluence rates even though these wavelengths correspond to a maximum and a minimum of hemoglobin absorption. These results prove that the hemoglobin cannot be functioning as the visual pigment in phototaxis but, for reasons discussed, they neither confirm nor rule out a role as a shadowing pigment. The results are consistent with a shadowing role in the presence of contrast enhancement by the nervous system.
A steep fluence-rate dependence suggests that contrast enhancement does occur in Mermis phototaxis. The 420 or 500 nm fluence rate for half-maximal response is 6 times 10 photons s^-1 cm^-2 (about equivalent in effectiveness to pre-dawn twilight). The wide range of sensitivity, 350–560 nm, has interesting implications as to the nature of the visual pigment.
†NATO reaearch collaborator a n leave from the Department of Biophysics. Laboratorium voor Algemene Natuurkunde, Rijksuniversiteit Groningen, Westersin-gel 34. 9718 CM Groningen, The Netherlands.     

Glu 87 of Channelrhodopsin‐1 Causes pH‐dependent Color Tuning and Fast Photocurrent Inactivation†

Channelrhodopsins (ChR1 and ChR2) are directly light‐gated ion channels acting as sensory photoreceptors in the green alga Chlamydomonas reinhardtii. These channels open rapidly after light absorption and both become permeable for cations such as H⁺, Li⁺, Na⁺, K⁺ and Ca²⁺. K_m for Ca²⁺ is 16.6 mm in ChR1 and 18.3 mm in ChR2 whereas the K_m values for Na⁺ are higher than 100 mm for both ChRs. Action spectra of ChR1 peak between 470 and 500 nm depending on the pH conditions, whereas ChR2 peaks at 470 nm regardless of the pH value. Now we created two chimeric ChRs possessing helix 1–5 of ChR1 and 6, 7 of ChR2 (ChR1/2_5/2), or 1, 2 from ChR1 and 3–7 from ChR2 (ChR1/2_2/5). Both ChR‐chimera still showed pH‐dependent action spectra shifts. Finally, a mutant ChR1E87Q was generated that inactivated only slowly in the light and showed no spectral shift upon pH change. The results indicate that protonation/deprotonation of E87 in helix 1 alters the chromophore polarity, which shifts the absorption and modifies channel inactivation accordingly. We propose a trimodal counter ion complex for ChR1 but only a bimodal complex for ChR2.     

Ultrafast electrocyclic ring opening of 7-dehydrocholesterol in solution: the influence of solvent on excited state dynamics

Broadband UV-visible femtosecond transient absorption spectroscopy and steady-state integrated fluorescence were used to study the excited state dynamics of 7-dehydrocholesterol (provitamin D(3), DHC) in solution following excitation at 266 nm. The major results from these experiments are: (1) The excited state absorption spectrum is broad and structureless spanning the visible from 400 to 800 nm. (2) The state responsible for the excited state absorption is the initially excited state. Fluorescence from this state has a quantum yield of ～2.5 × 10(-4) in room temperature solution. (3) The decay of the excited state absorption is biexponential, with a fast component of ～0.4-0.65 ps and a slow component 1.0-1.8 ps depending on the solvent. The spectral profiles of the two components are similar, with the fast component redshifted with respect to the slow component. The relative amplitudes of the fast and slow components are influenced by the solvent. These data are discussed in the context of sequential and parallel models for the excited state internal conversion from the optically excited 1(1)B state. Although both models are possible, the more likely explanation is fast bifurcation between two excited state geometries leading to parallel decay channels. The relative yield of each conformation is dependent on details of the potential energy surface. Models for the temperature dependence of the excited state decay yield an intrinsic activation barrier of ～2 kJ/mol for internal conversion and ring opening. This model for the excited state behavior of DHC suggests new experiments to further understand the photochemistry and perhaps control the excited state pathways with optical pulse shaping.     

THE USE OF SHORT LIVED FLUORESCENT DYES TO CORRECT FOR ARTIFACTS IN THE MEASUREMENTS OF FLUORESCENCE LIFETIMES

Abstract— The ultrafast emission for fluorophores can be used as the effective excitation pulse for prompt response measurements by photo detectors in the same spectral region as that of unknown samples. This method corrects for such artifacts as wavelength and spatial dependence of the response function of the photodetector. It is shown that the emission from triphenylmethane dyes is an excellent effective pulse with relaxation time 2 ps in the red region of the spectrum. A microchannel plate photomultiplier has only a 35 ps increase in response or lag time between excitation at 420 nm and emission at 680 nm.     

Amphibious sensor of temperature and refractive index based on D-shaped photonic crystal fibre filled with liquid crystal

ABSTRACT

A D-shaped photonic crystal fibre filled with liquid crystal was demonstrated as an amphibious sensor for detection of both temperature and refractive index, when combined with plasma materials. Specifically, the optical component is implanted into a complete optical system ensuring modulation of the external electric field. When the refractive index of the external solution changes from 1.0 to 1.6, the y-polarised mode has a loss spectrum with a wavelength sensitivity of up to 2275 nm/RIU, and the corresponding amplitude sensitivity is ?88.2RIU^?1. When the perceived temperature changes from 15°C to 50°C, the temperature of the sensor is correspondingly expressed as the maximum wavelength sensitivity of 9.09 nm/°C and the amplitude sensitivity of ?0.311°C^?1. In addition, the actual micro-operation processes have been studied in detail, such as polishing depth, coating thickness and coating method. This provides practical ideas for real-time sensing analysis that requires harsh environments.     

Oxygen-independent inactivation of Haemophilus influenzae transforming DNA by monochromatic radiation: action spectrum, effect of histitine and repair.

Abstract— The action spectrum for the oxygen-independent inactivation of native transforming DNA from Haemophilus influenzae with near-UV radiation revealed a shoulder beginning at 334 and extending to 460 nm. The presence of 0.2 M histidine during irradiation produced a small increase in inactivation at 254, 290 and 313 nm, a large increase at 334 nm and a decrease in inactivation at 365, 405 and 460 nm. Photoreactivation did not reverse the DNA damage produced at pH 7.0 at 334, 365, 405 and 460 nm, but did reactivate the DNA after irradiation at 254, 290 and 313 nm. The inactivation of DNA irradiated at 254, 290 and 313 nm was considerably greater when the transforming ability was assayed in an excision-defective mutant compared with the wild type, although DNA irradiated at 334, 365, 405 and 460 nm showed smaller differences. These results suggest that the oxygen-independent inactivation of H. influenzae DNA at pH 7 by irradiation at 334, 365, 405 and 460 nm is caused by lesions other than pyrimidine dimers.     

THE WAVELENGTH DEPENDENCE OF HERPES SIMPLEX VIRUS INACTIVATION BY ULTRAVIOLET RADIATION

The effect of different wavelengths of ultraviolet (UV) radiation on Herpes simplex virus when assayed on mammalian cells (measured by plaque forming ability) was investigated. The wavelength dependence of viral inactivation was obtained for 11 different wavelengths over the region 238–297 nm. The resulting action spectrum does not closely follow the absorption spectrum of either nucleic acid or protein. The most effective wavelengths for viral inactivation are over the region 260–280 nm.     

STUDIES OF THE ROLE OF THE COAT PROTEIN IN THE U.V. PHOTOINACTIVATION OF U(1) AND U(2) STRAINS OF TMV*

Abstract— Two properties of the u.v. inactivation process in the u.v. sensitive U(2) strain have been investigated: (1) The increased binding of protein to RNA induced by irradiation of the virus at 254 nm; (2) The action spectrum for u.v. inactivation of U(2) between 250 nm and 285 nm. The extent of the u.v. induced binding of protein to RNA is similar to that previously found in the resistant U(1) strain, thereby eliminating the possibility that the capacity for this binding phenomenon bears any correlation to the difference in u.v. sensitivities of these two viruses at 254 nm. The results indicate that the radiation induced interaction of protein and RNA in U(1) and U(2) are probably similar. The action spectrum for U(2) resembles the absorption spectrum of the RNA between 250 nm and 285 nm implicating the RNA as the primary absorber leading to inactivation of the virus in this region of the spectrum. Quantum yields calculated for U(2) virus and free TMV-RNA irradiated at 254 nm reveal that the irradiated free RNA may be as much as 1–4 times more sensitive to inactivation at this wavelength than RNA in the intact virus. It is concluded that the coat protein of U(2) probably offers some protection to the enclosed RNA against u.v. damage at 254 nm, therefore, the difference in u.v. sensitivity between U(1) and U(2) TMV at this wavelength is a consequence of a difference in the degree of protection offered by the respective coat proteins to the enclosed RNA.     

Optical excitation and absorption spectra of C50Cl10

C50Cl10 [S. Y. Xie et al., Science 304, 699 (2004)] has been synthesized in large quantities enabling the capture of the labile fullerene C50. In this Communication, we report ab initio calculations on the optical excitation and absorption spectra of C50Cl10. We successfully explain and assign the measured UV-visible absorption spectrum of C50Cl10. The first singlet excitation for C50Cl10 is optically forbidden, and its optical absorption gap is redshifted by 0.6 eV (110 nm) relative to that of C60. We demonstrate that passivating C50 with 10 hydrogen atoms and replacing one Cl in C50Cl10 by one methoxy group lead to 100 nm blueshift and 90 nm redshift of the optical gap predicted for C50Cl10, respectively, suggesting C50 derivatives are suitable for tunable optical applications.     

Absorption band shape and emission in rhodopsin

Abstract— –The Planck law relationship between absorption and emission spectra is applied to the spectral sensitivity curve of human rod vision, assumed to be equivalent to the absorption spectrum of the visual pigment rhodopsin, to compute a hypothetical emission spectrum for rhodopsin. When 17, 100 cm^-1 is used as the reflection axis, the mirror image of this hypothetical emission substantially matches the activation spectrum over 2500 cm^-1. The predicted emission increases exponentially at long wavelengths, in contrast to published observations of fluorescence at 580 nm; this discrepancy, and an estimate of excited state lifetime based on the Planck law theory, suggest that emission occurs from a meta-stable vibrationally-excited state. The exceptionally slow falloff in absorption at long wavelengths is explained as being due to the smaller dependence of potential energy on angle of twist about bonds in the polyene chain in the lowest π, π* singlet state of rhodopsin than in its ground state; a model assuming six or seven essentially flat vibronic modes in the excited state accurately fits the observed action spectrum.     

Photosensory Functions of Channelrhodopsins in Native Algal Cells†

Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light‐gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin‐2) is biochemical activation of secondary Ca²⁺‐channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin‐1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low‐efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells.     

MINIMUM DOSES OF ULTRAVIOLET RADIATION REQUIRED TO INDUCE MURINE SKIN EDEMA and IMMUNOSUPPRESSION ARE DIFFERENT and DEPEND ON THE ULTRAVIOLET EMISSION SPECTRUM OF THE SOURCE*

Many photoimmunological studies have used UV radiation sources that emit nonsolar UV spectral energy and UV doses based on nonimmunological endpoints, e.g. erythema and skin edema. Interpretation of these data has led to misunderstanding when extrapolated to hypothetical effects in humans exposed to solar UV. The purpose of this study was to: (1) establish UV dose response relationships for murine skin edema and immunosuppression, and (2) determine how different UV spectra affect these relationships. Back skin and ear minimum edema doses (MEdD) for Kodacel-filtered FS20 sunlamp UV (290–400nm) were greater than two-fold higher than those for unfiltered FS20 sunlamp UV (250–400nm). Xenon arc solar simulator UV (295–400nm) MEdD were > 10-fold higher than those for unfiltered sunlamp UV. Back skin and ear MEdD differed two- to five-fold between C3H/ HeN, SWR/J and HRA/Skh-1 mice. The minimum immunosuppression doses (MISD) in C3H mice showed similar UV source spectrum dependence. The solar simulator UV MISD was 5.4- and 1.5-fold higher than for unfiltered and Kodacel-filtered sunlamp UV MISD, respectively. Furthermore, MISD were from 3- to 50-fold higher than the MEdD for the three UV sources. The UV bioeffectiveness spectra indicated that UVC energy (250–290nm) contributed 12% and 18%, respectively, of the total skin edema and immunosuppression UV energy. These data demonstrate the variability in UV sensitivity among mouse strains, the significant differences between murine MEdD and MISD and how these differences are influenced by nonsolar regions (below 295 nm) of the UV spectrum.     

RESEARCH NOTE

Two mutants of Escherichia coli unable to synthesize riboflavin were grown with limiting (2 micrograms ml-1) and non-limiting (10 micrograms ml-1) concentrations of riboflavin. These riboflavin auxotrophs when grown to exponential phase with non-limiting riboflavin are more sensitive to broad spectrum near-ultraviolet light (NUV, 320-400 nm) inactivation than when they are grown with limiting riboflavin. Exponential phase cells of the riboflavin auxotrophs grown with limiting riboflavin are sensitized when irradiated in saline supplemented with riboflavin. This suggests that extracellular riboflavin is important as a NUV sensitizer when intracellular levels of riboflavin are reduced. The concentration of riboflavin in crude extracts from exponentially growing cells correlates well with the sensitivity of these mutants to NUV inactivation. The level of riboflavin supplementation has little effect on the NUV sensitivity of the parental strain.     

Photochemical Properties of the Red‐shifted Channelrhodopsin Chrimson

Color‐tuned variants of channelrhodopsins allow for selective optogenetic manipulation of different host cell populations. Chrimson is the channelrhodopsin with the longest wavelength absorbance maximum. We characterize its photochemical properties at different pH values corresponding to two protonation states of the counterion for the protonated Schiff base. Both states will lead to a functional channel opening, but the route is different as reflected in the photochemical states observed spectroscopically. The light‐induced isomerization kinetics change with the local electrostatic environment, becoming faster with the presence of an anionic counterion. The spectral effect is stronger on the ground‐state energy surface. From the excited state, a bifurcated pathway leads to the electronic ground state resulting in a pronounced excitation wavelength dependence. The subsequent steps in the photocycles at pH 6 and pH 9.5 differ in the accumulation of states with a protonated and deprotonated Schiff base, respectively, that can be correlated with the open channel. Therefore, different protonation states are preserved in the open and the initial states. Chrimson's photocycle at alkaline pH shows features observed in other rhodopsins without an internal proton donor to the Schiff base, but it accumulates an intermediate with an even longer lifetime reflecting slow recovery of the initial state.