Inherent chirality dominates the visible/near-ultraviolet CD spectrum of rhodopsin

The visible (alpha) and near-UV (beta) CD bands of rhodopsin have been studied extensively, but their source(s) have never been definitively established. Do they result from the intrinsic chirality of the polyene chromophore of the protonated Schiff base of retinal (retPSB) or from the coupling of the transitions of this chromophore with those of protein groups? We have calculated the contributions of these two mechanisms to the CD of rhodopsin. The intrinsic CD of the retPSB chromophore was calculated using time-dependent density functional theory (TDDFT) and, for comparison, the semiempirical ZINDO method. First-order perturbation theory was used to calculate the effects of coupling of the retPSB transitions with the pi pi* transitions of the aromatic chromophores and the pi pi* and n pi* transitions of the peptide groups in rhodopsin. Calculations were performed for eight structures based upon the two molecules in the asymmetric unit of four crystal structures. The most reliable results were obtained from TDDFT calculations on the structure of Okada et al. (J. Mol. Biol. 2004, 342, 571), PDB 1U19. Averaging over the two molecules in the asymmetric unit, the intrinsic rotational strengths are 0.62 +/- 0.00 DBM (Debye-Bohr magneton) and 0.90 +/- 0.03 DBM for the alpha- and beta-bands, respectively. The contributions from coupling with protein groups are, respectively, -0.32 +/- 0.05 and -0.01 +/- 0.03 DBM. Our results show that the visible/near-UV CD bands of rhodopsin are determined by the intrinsic chirality of the retPSB chromophore and that the contributions of coupling with the protein are significantly smaller for the alpha-band and negligible for the beta-band.     

FLAVIN TYPE ACTION SPECTRUM OF NITRATE UTILIZATION BY Monoraphidium braunii

The light-dependent utilization of nitrate by the green alga Monoraphidium braunii, coming from nocturnal dark periods, shows an action spectrum of flavin type with two main bands: one in the blue, peaking at 450 and 480 nm, and the other in the near-UV region with a maximum at 365 nm. Other results indicate that cells growing on nitrate as the only nitrogen source resynthesize nitrate reductase daily, which implies the nocturnal loss of this enzyme. The biosynthesis of nitrate reductase at the beginning of the light periods can proceed under red light. In addition, blue or near-UV light is required for the activation of the previously formed nitrate reductase.     

SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—II. PHOTODYNAMIC ACTION OF RIBOFLAVIN ON BOVINE α-CRYSTALLIN

Abstract— Photolysis of α-crystallin in the presence of riboflavin under both aerobic and anaerobic conditions causes a rapid decrease in Trp emission; photooxidation most likely occurs via non-covalent complex formation between the sensitizer molecule and the substrate. However, the change in the tertiary structure of the protein, as manifested in the near-UV CD, is very different between aerobic and anaerobic photolysis. Riboflavin-sensitized reaction under aerobic condition causes a major change in the microenvironments of thiol groups as well as in the near-UV CD, whereas under anaerobic condition the change in the near-UV CD is much less and SH-group environments remain unaltered. The sensitizer in this photoinduced change in conformation of the protein is very selective and specific.     

MUTATIONAL INTERACTIONS BETWEEN NEAR-UV RADIATION AND DNA DAMAGING AGENTS IN Escherichia coli: THE ROLE OF NEAR-UV-INDUCED MODIFICATIONS IN GROWTH AND MACROMOLECULAR SYNTHESIS

Abstract— The mutational interactions between near-ultraviolet (near-UV, 334 nm, 365 nm) radiation and DNA damaging agents (far-UV (254 nm) and ethyl-methanesulphonate (EMS)) were studied in strains of Escherichia coli B/r trp thy with different susceptibilities to near-UV-induced growth delay (wild-type, rel and srd ). Far-UV induced reversion to tryptophan independence is reduced while forward mutation to streptomycin is enhanced by prior exposure of the rel+ srd+ strains to near-UV radiation. The observed interactions are reduced ( rel ) or absent ( srd ) in the two mutant strains as are the corresponding growth and macromolecular synthesis delays normally observed after near-UV treatment. Quantitatively, the degree of interaction induced by near-UV pre-treatment correlates closely with the degree of protein synthesis inhibition. We propose a mechanism for the contrasting interactions at the two genetic loci based on the different pathways by which pre-mutagenic lesions may be processed. The primary chromophore for the mutational interactions would appear to be 4-thiouracil-containing transfer RNA.     

SENSITIZER-INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N-FORMYLKYNURENINE ON BOVINE α-CRYSTALLIN

Abstract— Fluorescence and circular dichroic properties of bovine a-crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N-formylkynurenine. Methylene blue-sensitized photooxidation causes a change in the tertiary structure as manifested in the near-UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non-polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near-UV CD (tertiary structure) suffers a drastic alteration whereas the far-UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron-ments, orientation, and intermolecular interactions of different amino acids. N-formylkynurenine also shows some changes in the near-UV CD, presumably, caused by H₂O₂ generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near-UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N-formylkynurenine and air.     

RECOVERY FROM DAMAGE INDUCED BY ACRIDINE PLUS NEAR-ULTRAVIOLET LIGHT IN ESCHERICHIA COLI

Abstract— Escherichia coli cells treated with sublethal doses of acridine plus near-UV light exhibit an effective split-dose recovery response that requires an incubation period of about 30–45 min. Studies of the metabolic requirements for split-dose recovery revealed the following: (a) DNA synthesis is not required for split-dose recovery; (b) inhibition of electron transport or protein synthesis reduces the efficiency of split-dose recovery by about one-half; (c) inhibition of phospholipid synthesis or cell wall synthesis completely eliminates the split-dose recovery response. These results suggest an involvement of membrane repair mechanisms in response to damage by acridine plus near-UV light. Additional evidence for such a process was provided by more direct assays for membrane recovery. It was found that cells treated with sublethal doses of acridine plus near-UV light are sensitive to low concentrations of detergents, and lose that sensitivity upon incubation. Likewise, treated cells are susceptible to lethal osmotic shock, but can recover from this susceptibility if incubated after treatment but prior to exposure to low osmotic conditions. Based on accumulating evidence, we propose that E. coli cells are capable of repairing membrane damage resulting from exposure to acridine plus near-UV light.     

WAVELENGTH-DEPENDENT ACTION DICHROISM: A THEORETICAL CONSIDERATION

Abstract— For a model structure the wavelength dependence of action dichroism is computed as a function of the angle(△) between the transition moments at two different wavelengths. With as small an angle as △ α = 20. inversion of action dichroism for parallel vs perpendicularly vibrating radiation is expected for different absorption bands if the sensory pigment molecules are in proper orientations. Thus published data on opposite action dichroism in the blue vs near-UV range are compatible with flavins as sensory pigments, although the calculations must not be considered as proof for the flavin hypothesis.     

CHLORPROMAZINE PHOTOSENSITIZATION—I. EFFECT OF NEAR-UV IRRADIATION ON BACTERIOPHAGES SENSITIZED WITH CHLORPROMAZINE

Abstract— Both DNA bacteriophage and RNA bacteriophage were inactivated when they were irradiated with near-UV light (black light) in the presence of chlorpromazine. The far-UV sensitive mutants of T4D, i.e. T4D v , T4D px and T4D y , were no more sensitive to near-UV light plus chlorpromazine than the wild type. Electron microscopic observations showed that adsorption of T4D was greatly influenced by the treatment. The present results may indicate that the inactivation of T4D is due to the loss of adsorption caused by impairment in the tail or the tail fiber protein rather than the inactivation of DNA.     

GENETIC CONTROL OF NEAR-UV (300–400nm) SENSITIVITY INDEPENDENT OF THE recA GENE IN STRAINS OF ESCHERICHIA COLI K12

Abstract— Stationary cells of isogenic pairs of Escherichia coli K12 strains presumably differing only in the recA function have been inactivated with near-UV (300–400 nm) radiation. Based on near-UV inactivation kinetics, the strains can be divided into two discrete categories in which near-UV sensitivity does not necessarily correlate with far-UV sensitivity conferred by two different recA alleles. Lack of overlap between near-UV and far-UV ( recA ) sensitivity can be explained hy assuming that a different chromosomal gene ( nur ) controls near-UV sensitivity. Support for this hypothesis comes from a mating experiment in which four selected recombinants, isogenic with respect to auxotrophic markers, were identified exhibiting all four possible combinations of far-UV ( recA 1 vs recA ⁺ ) and near-UV sensitivity ( nur vs nur⁺ ). Transduction with phase P1 has shown that introduction of the recA 1 allele into a recA⁺ recipient does not affect the near-UV sensitivity of the recipient. Additional matings together with transduction experiments suggest that the nur gene is located at a position on the E. coli linkage map clearly separable from recA (minute 58).     

IN SITU HYDROGEN PEROXIDE PRODUCTION MAY ACCOUNT FOR A PORTION OF NUV (300–400 nm) INACTIVATION OF STATIONARY PHASE Escherichia coli

Abstract— Pre-irradiation of stationary phase cells of Escherichia coli K-12 with broadband near-UV radiation potentiates the lethal effects of subsequent exposure to near-UV radiation plus hydrogen peroxide. Identical fluences failed to modulate killing due to far-UV radiation. These data indicate that biologically revelant levels of hydrogen peroxide may be generated in situ upon the near-UV irradiation of cells.     

SIMILARITIES IN THE INDUCTION OF SYNTHESIS OF A CELL-SURFACE POLYPEPTIDE IN Arthrobacter sp. BY NEAR-UV IRRADIATION and PHOTODYNAMIC CONDITIONS

Irradiation of aerobic suspensions of Arthrobacter sp. with near-UV light (310-400 nm) induced synthesis of a 21 000 dalton, cell-surface polypeptide. Synthesis of this polypeptide also was induced by visible light in the presence of photodynamic dyes, as shown previously (Hoober, 1978). Induction of the polypeptide in near-UV light and with visible light plus dyes was inhibited by histidine. Hemin inhibited induction in near-UV light and in visible light with methylene blue, neutral red and acrifiavin, which are cationic dyes, but failed to inhibit induction in visible light with rose bengal, an anionic dye. These results suggested that inhibition by hemin required electrostatically favored interaction between the anionic porphyrin and the sensitizer, and that the near-UV light effect was mediated by a cationic or neutral endogenous sensitizer. The similarities in the responses of the cells to near-UV irradiation and visible light plus dyes suggested that the mechanism of induction under the two conditions was the same.     

Enhancement of the non-tryptophan fluorescence of human lens proteins after near-UV light exposure

Abstract. In this work, the non-tryptophan fluorescence (360 nm excited; 440 nm emitted) of human lens proteins was found to be intensified by exposing whole lens homogenates to near-UV light in the presence of tryptophan photoproducts. The induced fluorescence accumulates mainly in the soluble phase proteins, whereas in aging and brown cataractous lenses, the major fluorescence is found in the insoluble proteins. Using SDS-polyacrylamide gel electrophoresis with densitometric and fluorescence scanning techniques, the polypeptide chains of the three major protein fractions were analyzed for their specific non-tryptophan fluorescences. The same chains were found in all fractions. Two chains (11,000 and 45,000 daltons) were found to accumulate most of the induced fluorescence. These also contained the greatest intrinsic fluorescence initially. The data indicates that specific polypeptide chains in the lens proteins are most sensitive to modifications due to their exposure to near-UV light in the presence of tryptophan photoproducts.     

DEPENDENCE ON STAGE OF GROWTH OF MAMMALIAN CELL SENSITIVITY TO NEAR-UV IRRADIATION*

Abstract The variable sensitivity of exponentially growing mouse myeloma cells (66.2 subclone of MPC11) to monochromatic near-UV (365 nm) radiation was studied by determination of the relative cloning efficiency (i.e. survival) of cells in soft agar. Maximum sensitivity of the cells to near-UV light was found during the middle and late stages of exponential growth when the survival was only one-tenth of that during the early stage of growth. In addition, the shapes of near-UV survival curves changed with the stage of the cells irradiated. These data indicate that stages of growth can substantially alter the response of mammalian cells to near-UV radiation. These data further suggest the importance of testing the response of the cells throughout the growth employed in the experimental procedure.     

圆二色光谱分析蛋白质构象的方法及研究进展

介绍了远紫外圆二色光谱数据计算蛋白质二级结构,辨认蛋白质三级结构类型的原理、拟合方法、实验技术。对近紫外圆二色作为光谱探针,研究蛋白质中芳香氨基酸残基、二硫键微环境的变化作了简单介绍。     

USE OF THE DYE HOECHST 33258 TO INCREASE THE PHOTOSENSITIVITY OF BROMODEOXYURIDINE-CONTAINING CELLULAR SLIME MOLDS

The photosensitivity of amoebae of Dictyostelium discoideum to near-ultraviolet light (near-UV; 365 nm peak) was investigated following growth in 5-bromodeoxyuridine (BrdUrd) and treatment with the dye Hoeschst 33258. Cell killing was studied as a function of dye-treatment time and concentration, and BrdUrd incorporation time and concentration. For example, cells grown for 13 h in 50 μg BrdUrd/ml and then treated for 1 h in 10 μg dye/ml were reduced to 10% survival by a near-UV exposure of 0.8 min. Combinations of any two of the three factors (BrdUrd, dye, near-UV) were ineffective for killing. The sensitivity of the cells to near-UV decreased rapidly as the holding time between the end of the dye treatment and the start of the UV exposure was increased.     

A Unified Experimental/Theoretical Description of the Ultrafast Photophysics of Single and Double Thionated Uracils

Photoinduced processes in thiouracil derivatives have lately attracted considerable attention due to their suitability for innovative biological and pharmacological applications. Here, sub-20 fs broadband transient absorption spectroscopy in the near-UV are combined with CASPT2/MM decay path calculations to unravel the excited-state decay channels of water solvated 2-thio and 2,4-dithiouracil. These molecules feature linear absorption spectra with overlapping ππ* bands, leading to parallel decay routes which we systematically track for the first time. The results reveal that different processes lead to the triplet states population, both directly from the ππ* absorbing state and via the intermediate nπ* dark state. Moreover, the 2,4-dithiouracil decay pathways is shown to be strongly correlated either to those of 2- or 4-thiouracil, depending on the sulfur atom on which the electronic transition localizes.     

EFFECT OF FAR-UV AND NEAR-UV RADIATION ON THE CELL SURFACE CHARGE OF THE PROTOZOAN

Abstract— Cell electrophoresis was used to detect the effect of far-UV or near-UV radiation on the cell surface charge of the pathogenic protozoan Tritrichomonas foerus . Either far-UV or near-UV radiation interfered with the surface charge of T. foetus at fluences which inhibited cell growth by 50%. Both UV-radiations induced a significant decrease on surface charge of T. foetus , as evaluated by measurement of its electrophoretic mobility (EPM). Determinations of EPM of protozoa in solution of low ionic strength indicated that the decrease in the EPM induced by far-UV is much less pronounced than that observed for near-UV or control cells.     

Interaction of 7-hydroxyflavone with human serum albumin: a spectroscopic study

Numerous recent investigations have revealed that various synthetic as well as therapeutically active natural flavonoids possess novel luminescence properties that can serve as highly sensitive monitors for exploring their interactions with relevant physiological targets. Here we report a detailed study on the interactions of the model flavone, 7-hydroxyflavone (7HF) with the plasma protein human serum albumin (HSA), employing electronic absorption, fluorescence (steady state and time resolved) and induced circular dichroism (ICD) spectroscopy. The spectral data indicate that in the protein matrix, the neutral 7HF molecules are predominantly transformed to a conjugate anion (7HFA) by a proton abstraction in the ground state. The protein (HSA) environment induces dramatic enhancements in the fluorescence emission intensity, anisotropy (r) and lifetime (tau) values, as well as pronounced changes in the fluorescence excitation and emission profiles of the fluorophore. Moreover, evidence for efficient F?rster type resonance energy transfer (FRET, from tryptophan to 7HFA) is presented, from which we infer that the binding site of 7HF in HSA is proximal (estimated distance, R=23.6A) to the unique tryptophan - 214 residue present in the inter-domain (between IIA and IIIA domains) loop region of the protein. The binding constant (K=9.44x10(4)M(-1)) and the Gibbs free energy change (DeltaG=-28.33kJ/mol) for 7HFA-HSA interaction have been estimated from the emission data. Finally, the near-UV circular dichroism (CD) studies show that the electronic transitions of 7HF are strongly perturbed on binding to the chiral host (HSA), leading to the appearance of ICD bands. Implications of these results are discussed.     

Theoretical investigation of the photophysics of methyl salicylate isomers

The photophysics of methyl salicylate (MS) isomers has been studied using time-dependent density functional theory and large basis sets. First electronic singlet and triplet excited states energies, structure, and vibrational analysis were calculated for the ketoB, enol, and ketoA isomers. It is demonstrated that the photochemical pathway involving excited state intramolecular proton transfer (ESIPT) from the ketoB to the enol tautomer agrees well with the dual fluorescence in near-UV (from ketoB) and blue (from enol) wavelengths obtained from experiments. Our calculation confirms the existence of a double minimum in the excited state pathway along the O-H-O coordinate corresponding to two preferred energy regions: (1) the hydrogen belongs to the OH moiety and the structure of methyl salicylate is ketoB; (2) the hydrogen flips to the closest carboxyl entailing electronic rearrangement and tautomerization to the enol structure. This double well in the excited state is highly asymmetric. The Franck-Condon vibrational overlap is calculated and accounts for the broadening of the two bands. It is suggested that forward and backward ESIPT through the barrier separating the two minima is temperature-dependent and affects the intensity of the fluorescence as seen in experiments. When the enol fluoresces and returns to its ground state, a barrier-less back proton transfer repopulates the ground state of methyl salicylate ketoB. It is also demonstrated that the rotamer ketoA is not stable in an excited state close to the desired emission wavelength. This observation eliminates the conjecture that the near-UV emission of the dual fluorescence originates from the ketoA rotamer. New experimental results for pure MS in the liquid state are reported and theoretical results compared to them.     

LINEAR DICHROISM STUDY OF METALLOPORPHYRIN TRANSITION MOMENTS IN VIEW OF RADIATIONLESS INTERACTIONS WITH TRYPTOPHAN IN HEMOPROTEINS

We measured the linear dichroism of several metalloporphyrins embedded in stretched polyvinyl alcohol (PVA) films to estimate the orientation of the absorption transition moments, which in hemoproteins are relevant to the radiationless energy transfer between tryptophan and heme. The metalloporphyrins were derivatives of protoporphyrin IX (PPIX), namely Fe³⁺-PPIX (ferric-heme) and Fe²⁺CO-PPIX (CO-heme), Mg-PPIX (Mg-heme) and Zn-PPIX (Zn-heme). Measurements were conducted between 300 and 700 nm. In all cases the linear dichroism was wavelength dependent, indicating the presence of several transition moments with different orientations. We focused our attention on the near-UV (300–380 nm) and Soret (380450 nm) absorption bands. Deconvolution in terms of Gaussian components gave three components between 380 and 450 nm and only one in the 300–380 nm region. Deconvolution of the near-UV and Soret spectra of oxy-, deoxy- and carbonmonoxyhemoglobin gave very similar results, suggesting a very similar orientation of the various transition moments in the free and protein-embedded hemes. It should be stressed that the single 300–380 nm band is the only one responsible for the overlap integral that regulates the energy transfer from tryptophan to heme in hemoproteins (Gryczynski et al., Biophys. J . 63, 648–653, 1992). The dichroism of this single band indicated that its transition moment is oriented at about 60 from the α-γ meso-axis of the heme moiety. We conclude that the heme should be considered a linear oscillator when it acts as acceptor of energy transfer from tryptophans.