Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

Thermal denaturation of a recombinant mouse amelogenin: circular dichroism and differential scanning calorimetric studies

Conformational analyses of a recombinant mouse tooth enamel amelogenin (rM179) were performed using circular dichroism (CD), fluorescence, differential scanning calorimetry, and sedimentation equilibrium studies. The results show that the far-UV CD spectra of rM179 at acidic pH and 10 degrees C are different from the spectra of random coil in 6 M GdnHCl. A near-UV CD spectrum of rM179 at 10 degrees C is similar to that of rM179 in 6 M GdnHCl, which indicates that aromatic residues of native structure are exposed to solvent and rotate freely. Far-UV CD values of rM179 at 80 degrees C are different from that of random-coil structure in 6 M GdnHCl, which suggests that rM179 at 80 degrees C has specific secondary structures. A gradual thermal transition was observed by far-UV CD, which is interpreted as a weak cooperative transition from specific secondary structures to other specific secondary structures. The fluorescence emission maximum for the spectrum due to Trp residues in rM179 at 10 degrees C shows the same fluorescence emission maximum as rM179 in 6 M GdnHCl and amino acid Trp, which indicates that the three Trp in rM179 are exposed to solvent. Deconvolution of differential scanning calorimetry curve gives the population of three states (A, I, and C states). These results indicate that three states (A, I, and C) have specific secondary structures, in which hydrophobic and Trp residues are exposed to the solvent. The thermodynamic characteristics of rM179 are unique and different from a typical globular protein, proline-rich peptides, and a molten globule state.     

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.     

Fluorescence ratio intrinsic basis states analysis: a novel approach to monitor and analyze protein unfolding by fluorescence

Fluorescence ratio intrinsic basis states analysis (FRIBSTA) is a novel method allowing quantitative estimation of the stability of proteins in aqueous solution as a function of temperature. In FRIBSTA emission fluorescence spectra are repeatedly recorded while ramping temperature from < or =-15 to > or =100 degrees C. Subsets of these are identified as reference spectra of the protein in either its folded or in its heat denatured configuration. Each reference spectrum of both sets is normalized by its own integrated fluorescence intensity to give a fractional area spectrum. Linear extrapolations of these normalized reference spectral shapes over the entire temperature range of measurement are then used to deconvolute each experimental emission spectrum to give a fraction of emission from native state and a fraction from denatured state. Additionally, the integrated emission fluorescence intensity for the native configuration is fitted and extrapolated over the temperature range of measurement. Division of the deconvoluted native integrated fluorescence intensity by the fitted-extrapolated integrated emission fluorescence intensity yields the fraction folded. The free energy functions derived from fraction unfolded are presented for beta-lactoglobulin and phosphoglycerate kinase. According to these results both proteins are considerably less stable than heretofore assumed at ambient temperatures and partially denatured at temperatures < or =0 degrees C. The method is employed to study the effect of denaturants on these proteins as well. The major usefulness of FRIBSTA is that one can directly measure the protein stability at ambient and subambient temperatures in the absence of denaturants rather than predicting it by extrapolation from heat denaturation data.     

Pulse fluorometry of cyclo-(glycyl-L-tryptophyl).

The fluorescence of cyclo-(glycyl-L-tryptophyl) in trimethyl phosphate has been studied in a temperature range varying from room temperature to -85 degrees C. At room temperature, the fluorescence decay is the sum of two exponentials, the relative amplitude of which depends on the emission wavelength. This can be explained by the presence of the two following emitting molecular states: on one hand the unfolded state, the fluorescence decay time and the emission spectrum of which are close to these of skatole; on the other hand the folded state which has a shorter decay time and a blue-shifted spectrum. By lowering the temperature, the fluorescence spectrum shifts to the blue, while the skatole spectrum shifts to the red. This behavior corresponds to an increase of the folded conformation concentration in agreement with the NMR results. Furthermore the rate of exchange between the folded and the unfolded conformations decreases. Accordingly the wavelength dependence of the fluorescence decay lessens. There are two possible values of the conformational angle x2 differing by 180 degrees, which correspond to the folded state; due to the indole asymmetry, the interactions between the indole and diketopiperazine rings differ in these conformers. Consequently the fluorescence decay remains biexponential even at -85 degrees C.     

Effect of probucol on the physical properties of low-density lipoproteins oxidized by copper

Human plasma low-density lipoproteins (LDL) were incubated with 10 microM probucol for 1 h at 37 degrees C. Probucol incorporation into the LDL was complete as judged by filtration through a 0.2-micron filter, ultracentrifugation, and gel filtration. LDL with and without probucol were incubated for up to 24 h with 5 microM Cu2+ at 37 degrees C. Copper oxidation increased the content of random structure in the LDL protein from 30% to 36% at the expense of beta-structure (which decreased from 22% to 16%) without a change in alpha-helical content as measured by circular dichroism spectroscopy. This loss of beta-structure was prevented by the presence of probucol in the LDL during the copper incubation. Probucol reduced the rate of increase of fluorescence during copper oxidation at 37 degrees C. After 6 h, the fluorescence intensity at 360-nm excitation and 430-nm emission was 30% less in probucol-containing samples. Probucol had no effect on the circular dichroic spectrum of LDL and only minimal effects (less than 5%) on the fluorescence emission spectrum at wavelengths below 500 nm. Two fluorescence peaks, with emission at 420 nm and excitation at 340 and 360 nm, are resolved in three-dimensional fluorescence spectra of oxidized LDL. Probucol reduces the intensity of both peaks equally. The binding of a highly reactive heparin (HRH) fraction to LDL was measured by titration of LDL with HRH in the presence of fluoresceinamine-labeled HRH. The decrease in fluorescence anisotropy of the labeled HRH is proportional to the concentration of bound HRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.     

Excitation spectra for photosystem I and photosystem II in chloroplasts and the spectral characteristics of the distributions of quanta between the two photosystems.

The parameters listed in the title were determined within the context of a model for the photochemical apparatus of photosynthesis. The fluorescence of variable yield at 750 nm at -196 degrees C is due to energy transfer from Photosystem II to Photosystem I. Fluorescence excitation spectra were measured at -196 degrees C at the minimum, FO, level and the maximum, FM, level of the emission at 750 nm. The difference spectrum, FM-FO, which represents the excitation spectrum for FV is presented as a pure Photosystem II excitation spectrum. This spectrum shows a maximum at 677 nm, attributable to the antenna chlorophyll a of Photosystem II units, with a shoulder at 670 nm and a smaller maximum at 650 nm, presumably due to chlorophyll a and chlorophyll b of the light-harvesting chlorophyll complex. Fluoresence at the FO level at 750 nm can be considered in two parts; one part due to the fraction of absorbed quanta, alpha, which excites Photosystem I more-or-less directly and another part due to energy transfer from Photosystem II to Photosystem I. The latter contribution can be estimated from the ratio of FO/FV measured at 692 nm and the extent of FV at 750 nm. According to this procedure the excitation spectrum of Photosystem I at -196 degrees C was determined by subtracting 1/3 of the excitation spectrum of FV at 750 nm from the excitation spectrum of FO at 750 nm. The spectrum shows a relatively sharp maximum at 681 nm due to the antenna chlorophyll a of Photosystem I units with probably some energy transfer from the light-harvesting chlorophyll complex. The wavelength dependence of alpha was determined from fluorescence measurements at 692 and 750 nm at -196 degrees C. Alpha is constant to within a few percent from 400 to 680 nm, the maximum deviation being at 515 nm where alpha shows a broad maximum increasing from 0.30 to 0.34. At wavelengths between 680 and 700 nm, alpha increases to unity as Photosystem I becomes the dominant absorber in the photochemical apparatus.     

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.     

Hsp85 conformational change within the heat shock temperature range.

One of the major mammalian heat shock proteins, hsp85, aggregates extensively when heated in the presence of non-ionic detergents (J Cell. Physiol. 140: 601-607, 1989). The present study used intrinsic fluorescence and susceptibility to tryptic proteolysis to probe hsp85 conformation within the physiological and heat shock temperature ranges. Fluorescence intensity decreased and the emission spectrum was red-shifted (2.5 nm) as hsp85 was heated from 15 degrees to 50 degrees C. Upon heating in the absence of detergent, the red shift, monitored by the ratio of fluorescence emission at 330 nm to that at 350 nm, began at 38 degrees-45 degrees C with a transition midpoint at 45 degrees-50 degrees C, depending on the rate of temperature increase. This transition was masked by 1% n-octyl-O-glucoside - a detergent previously shown to promote aggregation. The spectral changes were not reversible upon cooling to 15 degrees C. Susceptibility to proteolysis in the absence of detergent, measured by the degradation of characteristic large fragments, increased sharply between 40 degrees C and 45 degrees C. These findings suggest that hsp85 undergoes a major conformational change within the range of temperatures known to induce hsp synthesis. This change is consistent with partial unfolding which exposes additional sites to the aqueous environment and influences detergent binding.     

Regulation of isoprene emission in Populus trichocarpa leaves subjected to changing growth temperature

The hydrocarbon isoprene is emitted in large quantities from numerous plant species, and has a substantial impact on atmospheric chemistry. Temperature affects isoprene emission at several levels: the temperature at which emission is measured, the temperature at which leaves develop, and the temperatures to which a mature leaf is exposed in the days prior to emission measurement. The molecular regulation of the response to the last of these factors was investigated in this study. When plants were grown at 20 degrees C and moved from 20 to 30 degrees C and back, or grown at 30 degrees C and moved from 30 to 20 degrees C and back, their isoprene emission peaked within 3 h of the move and stabilized over the following 3 d. Trees that developed at 20 degrees C and experienced 30 degrees C episodes had higher isoprene emission capacities than did leaves grown exclusively at 20 degrees C, even 2 weeks after the last 30 degrees C episode. The levels and extractable activities of isoprene synthase protein, which catalyses the synthesis of isoprene, and those of dimethylallyl diphosphate (DMADP), its substrate, alone could not explain observed variations in isoprene emission. Therefore, we conclude that control of isoprene emission in mature leaves is shared between isoprene synthase protein and DMADP supply.     

Fluorescence properties of allophycocyanin and a crosslinked allophycocyanin trimer

Data on the wavelength and temperature dependence of both time-resolved and steady state fluorescence emission are presented for allophycocyanin (AP) and for a crosslinked allophycocyanin trimer (XL-AP) (Ong LJ and Glazer AN: Physiol Veg 23:777-787, 1985). AP dissociates at high dilution and is not stable above 40 degrees C even at moderate protein concentration. In contrast, XL-AP does not dissociate even at very low protein concentrations and is completely stable up to 60 degrees C in the presence of 0.75 M NaK-phosphate, pH 7.0. The results show that XL-AP is superior to AP for use in conjugates that absorb and emit in the red region of the spectrum. The high stability of XL-AP at elevated temperatures at high phosphate concentrations suggests that this derivative may be useful in conjunction with nucleic acid probes.     

Three-photon excitation of N-acetyl-L-tyrosinamide.

We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.     

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.     

嗜碱细菌环状糊精葡糖基转移酶的纯化和性质

嗜碱细菌52—2除去菌体的培养液经硫酸铵沉淀和DEAE－纤维素离子交换柱层析,得到凝胶电泳均一的环状糊精葡糖基转移酶,纯化了11.5倍,酶活力回收为5.7％。用浓度梯度PAGE测分子量为151700。酶反应最适温度为65℃,50℃以下比较稳定。酶反应最适pH为7．0,在6．0～9.0范围内稳定。Zn²⁺、Hg²⁺、Pb²⁺、Al³⁺、Cu²⁺、Ag⁺和Fe²⁺强烈抑制酶活力。紫外光谱在270nm和244nm处分别有最大和最小吸收。荧光光谱的最大激发波长和发射波长分别为283nm和335nm。用NBS、NEM、NAI、DEP和EDC对酶进行了化学修饰,初步推测组氨酸和色氨酸残基可能为酶活力必需基因,羧基与酶活力有一定关系。     

Use of GFP tags to monitor localization of different luciferases in E. coli.

The utility of the green fluorescent protein (GFP) as a probe to monitor protein localization in living cells is gaining a great deal of attention. In this study, to understand the localization of luciferases in E. coli, we have attached GFP tags at both the N- and the C-terminus of firefly luciferase (FF-Luc)(from Pyrocoelia miyako) and of red (RE-Luc) and green (GR-Luc) bioluminescence-emitting luciferases (from Phrixothrix railroad-worms), respectively. There was no significant change in the bioluminescence emission spectrum for any of the three luciferases following the tagging with GFP at either the N- or C-terminus, confirming the absence of energy transfer between one another. Using confocal imaging microscopy, we observed that all three luciferases expressed in the E.coli cultured at 37 degrees C tend to aggregate and are seen to localize in the poles, thus confirming their poor folding properties. In contrast, in the E.coli cultured at 18 degrees C FF-Luc was found to be highly expressed in the soluble form when compared to RE-Luc and GR-Luc. These results support our previous finding that the folding properties of FF-Luc and RE/GR-Luc are totally different.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Fluorescence and bioluminescence of bacterial luciferase intermediates.

An intermediate in the luciferase-catalyzed bioluminescent oxidation of FMNH2, isolated and purified by chromatography at -20degrees, was postulated to be an oxygenated reduced flavin-luciferase. Maintained and studied at -20 to -30degrees, this material exhibits a relatively weak fluorescence emission peaking about 505 nm when excited at 370 nm. It may comprise more than one species. Upon continued exposure to light at 370 nm, the intensity of this fluorescence increases, often by a factor of 5 or more, and its emission spectrum is blue shifted to a maximum at about 485 nm. Upon warming its fluorescence is lost and the fluorescence of flaving mononucleotide appears. If warming is carried out in the presence of a long chain aldehyde, bioluminescence occurs, with the appearance of a similar amount of flavine fluorescence. The bioluminescence yield is about the same with irradiated and nonirradiated samples. The bioluminescence emission spectrum corresponds exactly to the fluorescence emission spectrum of the intermediate formed by irradiation, implicating the latter as being structurally close to the emitting species in bioluminescence.     

Effect of temperature on tryptophan fluorescence of beta-lactoglobulin B.

The effect of heat on the conformation of bovine beta-lactoglobulin has been studied using intrinsic fluorescence spectroscopy. Changes in the intensity, wave-length of maximum emission and emission peak width at half height of tryptophan fluorescence over the range 15-90 degrees C at pH 6.4-6.5 has allowed the environments of the two tryptophans in the molecule to be discriminated. At 20 degrees C both tryptophans are in hydrophobic environments. As the temperature is raised the conformation changes such that at about 50 degrees C one of the tryptophans is transferred to a more polar environment accessible to solvent. Conformational changes appear to be reversible if the protein is cooled to 20 degrees C after heat treatments up to 70 degrees C. Above 70 degrees C the second tryptophan residue becomes exposed to solvent. Complete exposure of one residue occurs at 80 degrees C while the other is still partially buried even at 90 degrees C. When the protein is then cooled to 20 degrees C the conformational changes appear to be irreversible with only one tryptophan residue returning to the hydrophobic interior of the molecule.