Isolation, properties and spatial site analysis of γ subunits of B-phycoerythrin and R-phycoerythrin

Polysiphonia urceolata R-phycoerythrin andPorphyridium cruentum B-phycoerythrin were degraded with proteinaseK, and then the nearly native γ subunits were isolated from the reaction mixture. The process of degradation of phycocrythrin with proteinaseK showed that the γ subunit is located in the central cavity of (αβ)₆ hexamer of phycoerythrin. Comparative analysis of the spectra of the native phycoerythrin, the phycoerythrin at pH 12 and the isolated γ subunit showed that the absorption peaks of phycoerythrobilins on α or β subunit are at 535 nm (or 545 nm) and 565 nm, the fluorescence emission maximum at 580 nm; the absorption peak of phycoerythrobilins on the isolated γ subunit is at 589 nm, the fluorescence emission peak at 620 nm which overlaps the absorption maximum of C-phycocyanin and perhaps contributes to the energy transfer with high efficiency between phycoerythrin and phycocyanin in phycobilisome; the absorption maximum of phycourobilin on the isolated γ subunit is at 498 nm, which is the same as that in native phycoerythrin, and the fluorescence emission maximum at 575 nm.     

Wavelength modulation of phycoerythrin synthesis in Synechocystis sp. 6701.

The spectral dependence of phycoerythrin synthesis has been studied in a unicellular photautotrophic cyanobacterium, Synechocystis sp. 6701, in which phycoerythrin synthesis alone is under chromatic control. Cells were partially depleted of their phycobiliprotein pigments through nitrate starvation in the light. Addition of nitrate to the culture medium allowed synthesis of phycobiliproteins in the dark. This synthesis occurred at the expense of the glycogen reserve accumulated during the period of nitrate starvation. Monochromatic irradiations of short duration at lambda less than 590 nm induced increased phycoerythrin synthesis during dark incubation. Monochromatic irradiations of short duration at lambda greater than 590 nm prevented this synthesis. These effects were photoreversible. The spectral distribution showed a maximum at 540 nm for the potentiation of phycoerythrin synthesis, and one at 640 nm for its photoreversal.     

Action spectra for chromatic adaptation in the blue-green alga Fremyella diplosiphon

Action spectra for chromatic adaptation in Fremyella diplosiphon Drouet have been determined using techniques previously described. Action maxima are at 540 nm, with a half-band width of 80 nm, for induction of phycoerythrin synthesis (green action) and at 650 nm, with a half-band width of 90 nm, for reversal of induction of phycoerythrin synthesis (red action). The red-action spectrum includes a secondary action band centered at ca. 360 nm. Red and green action overlap from 570 to 590 nm with an isosbestic point in the vicinity of 580 nm. Shoulders are present at 520 and 630 nm. Red light is more active than green light. The 540:650-nm quantum effectiveness ratio is 1:7. There is relatively little action of either kind in the blue. The 387:540 nm and 460:650-nm quantum effectiveness ratios are zero. These results contrast strongly with previous determinations in the same organism, with major activity indicated in the blue; they are consistent with the control of photomorphogenesis in the Cyanophyta by a master pigment, analogous to phytochrome.Abbreviations APC allophycocyanin - PC physocyanin - PE phycoerythrin     

Spectroscopic studies on the active site of Sepioteuthis lessoniana hemocyanin.

The absorption, circular dichroism (CD), and magnetic circular dichroism (MCD) spectra in the visible region have been measured for $\text{Sepioteuthis lessoniana}$ hemocyanin at 77, 198, and 293K. From the temperature dependence of the CD spectra of oxyhemocyanin, the bands observed at 450, 565, and 700 nm were resolved into those centered at 430, 490, 565, 600, and 700 nm. Since these five peaks are most probably due to the d-d transitions, the two copper ions at the oxygenated active center are inferred to be Cu(II) ions each in a non-equivalent coordination geometry of very low symmetry. The MCD spectral data confirm the view and reasonably explain the diamagnetism of oxyhemocyanin.     

Near infrared spectral changes of cytochrome aa3 during potentiometric titrations.

Singular value decomposition (SVD) was used to deconvolute the spectral changes occurring in the near infrared region during potentiometric titrations of cytochrome aa3. Overall oxidized minus reduced difference spectra revealed a broad absorbance feature centered near 830 nm with an apparent Em near 250 mV. However, SVD did not isolate any spectral species with an absorbance centered near 830 nm. It was found that the spectral changes occurring in the wavelength region from 650 to 950 nm were associated mainly with cytochromes a and a3. It was concluded that the absorbance at 830 nm should not be used as an independent measure of the concentration of CuA in cytochrome aa3.     

Role of the Colorless Polypeptides in Phycobilisome Assembly in Nostoc sp

We have identified the function of the `extra' polypeptides involved in phycobilisome assembly in Nostoc sp. These phycobilisomes, as those of other cyanobacteria, are composed of an allophycocyanin core, phycoerythrin- and phycocyanin-containing rods, and five additional polypeptides of 95, 34.5, 34, 32, and 29 kilodaltons. The 95 kilodalton polypeptide anchors the phycobilisome to the thylakoid membrane (Rusckowski, Zilinskas 1982 Plant Physiol 70: 1055-1059); the 29 kilodalton polypeptide attaches the phycoerythrin- and phycocyanin-containing rods to the allophycocyanin core (Glick, Zilinskas 1982 Plant Physiol 69: 991-997). Two populations of rods can exist simultaneously or separately in phycobilisomes, depending upon illumination conditions. In white light, only one type of rod with phycoerythrin and phycocyanin in a 2:1 molar ratio is synthesized. Associated with this rod are the 29, 32, and 34 kilodalton colorless polypeptides; the 32 kilodalton polypeptide links the two phycoerythrin hexamers, and the 34 kilodalton polypeptide attaches a phycoerythrin hexamer to a phycocyanin hexamer. The second rod, containing predominantly phycocyanin, and the 34.5 and 29 kilodalton polypeptides, is synthesized by redlight-adapted cells; the 34.5 kilodalton polypeptide links two phycocyanin hexamers. These assignments are based on isolation of rods, dissociation of these rods into their component biliproteins, and analysis of colorless polypeptide composition, followed by investigation of complexes formed or not formed upon their recombination.     

Acid denaturation of recombinant porcine growth hormone: formation and self-association of folding intermediates

We have investigated the conformational changes incurred during the acid-induced unfolding and self-association of recombinant porcine growth hormone (pGH). Acidification (pH 8 to pH 2) of pGH resulted in intrinsic fluorescence, UV absorbance, and near-UV CD transitions centered at pH 4.10. At pH 2.0, a red shift in the fluorescence emission maximum of approximately 3 nm and a 15% loss of the far-UV CD signal at 222 nm imply that the protein did not become extensively unfolded. Acidification in the presence of 4 M urea resulted in similar pH-dependent transitions. However, these occurred at a higher pH (approximately 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the fluorescence emission maximum suggests that unfolding was greater than in the absence of urea. The presence of a prominent peak centered at 298 nm in the near-UV CD spectrum, which is absent without urea, signifies further differences in the intermediates generated at pH 2. Sedimentation equilibrium experiments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-association was strongly promoted at pH 2 while urea reduced self-association at both pH 8 and pH 2. These results demonstrate that acidification of pGH in the absence or presence of 4 M urea induced the formation of molten globule-like states with measurable differences in conformation. Similarities and differences in these structural conformations with respect to other growth hormones are discussed.     

Spectral substructure and excitonic interactions in the minor photosystem II antenna complex CP29 as revealed by nonlinear polarization spectroscopy in the frequency domain

CP29 (the lhcb4 gene product), a minor photosystem II antenna complex, binds six chlorophyll (Chl) a, two Chl b, and two to three xanthophyll molecules. The Chl a/b Q(y) absorption band substructure of CP29 (purified from spinach) was investigated by nonlinear polarization spectroscopy in the frequency domain (NLPF) at room temperature. A set of NLPF spectra was obtained at 11 probe wavelengths. Seven probe wavelengths were located in the Q(y) spectral region (between 630 and 690 nm) and four in the Soret band (between 450 and 485 nm). Evaluation of the experimental data within the framework of global analysis leads to the following conclusions: (i) The dominant Chl a absorption (with a maximum at 674 nm) splits into (at least) three subbands (centered at 660, 670, and 681.5 nm). (ii) In the Chl b region two subbands can be identified with maxima located at 640 and 646 nm. (iii) The lowest energy Q(y) transition (peaking at 681.5 nm) is assigned to a Chl a which only weakly interacts with other Chl aor b molecules by incoherent F?rster-type excitation energy transfer. (iv) Pronounced excitonic interaction exists between certain Chl a and Chl b molecules, which most likely form a Chl a/b heterodimer. The subbands centered at 640 and 670 nm constitute a strongly coupled Chl a/b pair. The findings of the study indicate that the currently favored view of spectral heterogeneity in CP29 being due essentially to pigment-protein interactions has to be revised.     

SYNTHESIS AND BINDING OF PHYCOERYTHRIN AND ITS ASSOCIATED LINKERS TO THE PHYCOBILISOME IN RHODELLA VIOLACEA (RHODOPHYTA): COMPARED EFFECTS OF HIGH LIGHT AND TRANSLATION INHIBITORS1

We studied the synthesis and binding of phycoerythrin and its associated linkers to the phycobilisome (PBS) in Rhodella violacea (Kornmann) Wehrmeyer and compared the effects of high light and translation inhibitors on these processes. Rhodella violacea has a simple hemidiscoidal PBS structure with a well-known composition. The number of PBSs per cell decreases when irradiance is increased, and at higher irradiances the rods are shortened with a specific loss of the terminal hexamer of phycoerythrin (PE) and its associated linker. To test whether or not the observed variations were due to a coordination between the expression of the chloroplast-encoded PE and the nuclear-encoded linkers, we inhibited the expression of the chloroplast genes by the translation inhibitor chloramphenicol. In the few PBSs synthesized, the linker associated to the terminal PE hexamer was missing while that associated with the intermediate PE hexamer was still present. The inhibition by cycloheximide of the translation of the nuclear-encoded linkers did not influence the synthesis of the chloroplast-encoded phycobiliproteins. The absence of linkers prevented the formation of PE hexamers and their binding to the PBSs. We therefore propose the existence of two levels of regulation for PE and associated linkers: the intermediate PE hexamer and associated linker are always present even though their amount is reduced when irradiance is increased. In contrast, the terminal hexamer of PE and its associated linker are no longer present under high light. Their absence can be due to a feedback control between the level of PE and the synthesis of the linker: when the level of PE is lowered below a given value by the action of light on the chloroplast, a signal coming from the chloroplast reaches the nucleus and the synthesis of the linker is repressed. There is no sign of nuclear regulation of the synthesis of PE, but the nuclear-encoded linkers have a structural role in the formation of PE hexamers.     

Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.     

Plastocyanin conformation. An analysis of its near ultraviolet absorption and circular dichroic spectra.

The near-ultraviolet absorption and circular dichroic spectra of plastocyanin are dependent upon the redox state, solution pH, and ammonium sulfate concentration. This dependency was observed in plastocyanin isolated from spinach, poplar, and lettuce. Removal of the copper atom also perturbed the near-ultraviolet spectra. Upon reduction there are increases in both extinction and ellipticity at 252 nm. Further increases at 252 nm were observed upon formation of apo plastocyanin eliminating charge transfer transitions as the cause. The spectral changes in the near-ultraviolet imply a flexible tertiary conformation for plastocyanin. There are at least two charge transfer transitions at approximately 295-340 nm. One of these transitions is sensitive to low pH's and is attributed to the His 87 copper ligand. The redox state dependent changes observed in the near-ultraviolet spectra of plastocyanin are attenuated either by decreasing the pH to 5 or by increasing the ammonium sulfate concentration to 2.7 M. This attenuation cannot be easily explained by simple charge screening. Hydrophobic interactions probably play an important role in this phenomenon. The pH and redox state dependent conformational changes may play an important role in regulating electron transport.     

Comparison of phycoerythrins (542, 566 nm) from cryptophycean algae

Summary The phycoerythrins from Rhodomonas sp. strain 3-C and Cryptomonas ovata var. palustris were purified and partially characterized. The phycoerythrin from Rhodomonas had a single visible absorption maximum at 542 nm with a shoulder at approximately 562 nm and is, therefore, representative of cryptophyte type I phycoerythrin. The phycoerythrin from C. ovata var. palustris had a single absorption maximum at 566 nm and is, therefore, representative of cryptophyte type III phycoerythrin. Calibrated gel filtration chromatography showed that both of these phycoerythrins have a native molecular weight of 30 800 daltons. Calibrated sodium dodecyl sulfate gel electrophoresis demonstrated that both pigments were composed of two subunits with apparent molecular weights of 17 700 and 11 000 daltons. On polyacrylamide gel electrofocusing both these phycoerythrins had an isoionic point of 4.90.     

Phycoerythrin of the marine blue-green alga Trichodesmium thiebautii

Spectroscopic characteristics of the phycoerythrin obtainedfrom the marine blue-green alga Trichodesmium thiebautii wereinvestigated. The algal sample was collected at its naturalhabitat, the East China Sea. The phycoerythrin showed an absorptionspectrum and a fluorescence excitation spectrum with three peaksin the visible wave-length region at 500, 547 and 565 nm. Thisproperty is very different from that of the cyanophycean phycoerythrin,which has a single peak at 565 nm, but it is similar to thatof the rhodophycean phycoerythrin with three peaks at 500, 550and 565 nm. (Received April 20, 1974; )     

Cooperative disordering of single-stranded polynucleotides through copper crosslinking

The thermal transitions of single-stranded polynucleotides are noncooperative. In contrast, Cu(II) cooperatively disorders the single-stranded helical structures of poly(A) and poly(C), as demonstrated by ORD and UV spectral changes as a function of the Cu²⁺ activity, and by a dramatic chain-length dependence of the spectral changes. Equilibrium dialysis binding studies indicate that the cooperative disordering is paralleled by a somewhat less cooperative binding process. The difference between the thermal- and Cu(II)-induced transition is explained by the following mechanism. (1) Cu(II) initially binds in a noncooperative fashion to phosphate. (2) The Cu(II) so bound forms a second bond to a nonadjacent base site on the same polymer strand or another strand. These intramolecular and intermolecular crosslinks to the bases are responsible for the disordering. (3) The initial crosslinks formed provide nuclei for the cooperative formation of additional crosslinks, producing cooperative spectral changes paralleled by cooperative binding. A comparison of the spectral and binding transitions indicates that there is appreciable noncooperative binding of copper to phosphate, which produces no spectral changes in the presence of added electrolyte. This comparison also indicates that each copper crosslink disorders several bases. The formation of intermolecular crosslinks is demonstrated by a polymer concentration dependence of the disordering. The formation of intramolecular crosslinks can be deduced from the fact that the “cooperative unit” required to explain the differences between the hexamer, which does not readily form intramolecular crosslinks, and the polymer is considerably larger than the cooperative unit determined from the polymer results. The poly(A) disordering transition is less symmetrical than that of poly(C), particularly at low polymer concentrations. These results, together with other phenomena, are explained by a greater flexibility of poly(A), which favors the formation of small intramolecular loops.     

Some observations on phycobilisomes of Pterocladiella capillacea (Gelidiaceae,Rhodophyta)

ABSTRACT

Phycobilisomes (PBSs) of the red alga Pterocladiella capillacea collected in the field, were characterized both in situ and in vitro by means of a transmission electron microscope (TEM) and an image analyzer. Ultrathin sections of thalli and negatively stained PBSs after isolation revealed hemi-ellipsoidal shapes. In situ PBS dimensions were 38.5 ± 0.2 nm (height) × 38.8 ± 0.2 nm (width) × 22.6 ± 0.2 nm (thickness) in good agreement with the in vitro measurements (mean diameters of 37.6 ± 0.2 nm). These dimensions, especially the width, are smaller than those so far reported for red algae. This could depend either on the ecophysiological conditions of the thalli when harvested and/or on a staggered, symmetrically rotated and compressed disposition of biliprotein rods with respect to the allophycocyanin (APC) core. Hydroxylapatite chromatography of biliproteic extracts and SDS-PAGE electrophoresis revealed that phycoerythrin type R-(λ_max 565 nm>540 nm>498 nm) is formed by α (18.6 kDa), β (19.9 kDa), γ (30.2kDa) and γ′ (33.8 kDa) sub-units. The presence of two γ sub-units suggests that this phycoerythrin is a set of (αβ)₆γ + (αβ)₆γ′ aggregates (R-PE). A spectroscopically distinct form of phycoerythrin with different peak ratios, also found in pure fractions, is thought to be a polydisperse form (the so called r-PE). Similarity of shape and size observed in PBSs both in situ and in vitro, and fluorescence spectral characteristics of PBSs in vitro would indicate a substantial integrity of isolated PBSs. These measurements, if compared with total biliprotein content, would seem to indicate a PE fraction not assembled into PBS. A possible role of phycoerythrin in relation to the ability for a rapid adaptation of this surface species to environmental changes is suggested.     

FUNCTIONAL PHYCOBILISOMES FROM TOLYPOTHRIX TENUIS (CYANOPHYTA) GROWN HETEROTROPHICALLY IN THE DARK1

Phycobiliproteins produced in dark-grown cells of Tolypothrix tenuis Kützing formed Phycobilisomes functionally capable of energy transfer. The phycobilisomes could be recovered in high yield (80% of extracted phycobiliproteins). Phycobilisomes from cells grown without light and in red light had the same size, morphology, and spectral characteristics. They had a phycocyanin to allophycocyanin malar ratio of 3:1. Phycocyanin and allophycocyanin in phycobilisomes were energetically coupled as indicated by their fluorescence emission (maximum of ca. 690 nm at –196° C) and excitation spectra. Phycobilisomes were attached to the outer surface of thylakoids and were hemidiscoidal in shape. In thin sections they had a diameter of 42 ± 3nm, a height of 24 ± 4 nm and a thickness of 10 ± 2 nm. Isolated and negatively stained Phycobilisomes were larger with a diameter of 51 ± 2 nm and height of 33 ± 2 nm, Isolated phycobilisomes in face view had a central core of three units and six peripheral rods. Each rod appeared to be composed of three hexamers (three double discs), consistent with the observed dimensions and substructure. After Phycoerythria synthesis was induced by a 15 min green light exposure, phycobilisomes of dark-grown cells exhibited energy transfer from phycoerythrin to a long wavelength allophycocyanin, indicating that phycoerythrin synthesized in darkness was incorporated into functional phycobilisomes.     

Spectral dependence of energy transfer in wild-type peripheral light-harvesting complexes of photosynthetic bacteria

The precise position of the upper exciton component and relevant vibronic transitions of the B850 ring in peripheral light-harvesting complexes from purple photosynthetic bacteria are important values for determining the exciton bandwidth and electronic structure of the B850 ring. To determine the presence of these components in wild-type LH2 complexes the pump-probe femtosecond transient spectra obtained with excitation into the 730-840 nm spectral range are analyzed. We show that at excitation wavelengths less than 780 nm B850 absorption bands are present and that, in accordance with exciton theory, these bands peak further in the blue when the lowest optically allowed transition is more red-shifted.     

Remote sensing of the xanthophyll cycle and chlorophyll fluorescence in sunflower leaves and canopies

Summary Sudden illumination of sunflower (Helianthus annuus L. cv. CGL 208) leaves and canopies led to excess absorbed PFD and induced apparent reflectance changes in the green, red and near-infrared detectable with a remote spectroradiometer. The green shift, centered near 531 nm, was caused by reflectance changes associated with the de-epoxidation of violaxanthin to zeaxanthin via antheraxanthin and with the chloroplast thylakoid pH gradient. The red (685 nm) and near-infrared (738 nm) signals were due to quenching of chlorophyll fluorescence. Remote sensing of shifts in these spectral regions provides non-destructive information on in situ photosynthetic performance and could lead to improved techniques for remote sensing of canopy photosynthesis.CIW Publication #1072     

岩豆凝集素的圆二色性与生物学活性关系的研究

岩豆凝集素（ＭＤＬ）的远紫外圆二色性谱（ＣＤ谱）显示２１６－２１７ｎｍ处的单一负峰。此时ＭＤＬ分子含有１６．２％的α螺旋,４６．３％的β折叠和３７．５％的无规卷曲。ｐＨ９．０时负峰红移至２２０ｎｍ,且在２１７－２２２ｎｍ处的峰值几乎相同;在２０－４０℃范围内,ＣＤ谱的变化甚微;６０℃时谱峰蓝移;在８０℃或１００℃时,２１２ｎｍ处出现一大负峰。１ｍｏｌ／Ｌ或２ｍｏｌ／Ｌ脲时,ＭＤＬ的ＣＤ谱已发生明显变化,二级结构单元也有变化,凝集兔红细胞的活性也随之减弱;随脲浓度的增加,ＭＤＬ的谱峰蓝移,最终在２１２ｎｍ处出现大负峰。当胍浓度为０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱即有明显变化和活性丧失;胍浓度继续增加,ＣＤ谱逐渐成为特征的无规卷曲的谱形。在ｐＨ９．０、温度超过８０℃、脲或胍浓度分别高于２ｍｏｌ／Ｌ和０．７５ｍｏｌ／Ｌ时,ＭＤＬ的ＣＤ谱发生显著变化的同时,其凝集兔红细胞的生物学活性全部丧失,分子的二级结构单元也发生很大改变。     

Natural occurrence of denatured phycoerythrin during Porphyra cultivation

Dried Nori (Porphyra spp.) normally turns greenish when toasted and this color usually remains for many days when moistened with vinegar in the Japanese food Sushi. However, for some lots of toasted Nori, this color changes to an undesirable reddish-brown one within a few hours in vinegar. A clear difference was noticed by spectrophotometry between the abnormally and normally colored toasted Nori. The former exhibited absorption maxima at around 490 and 530–600 nm while these were absent in the spectrum of the latter. The responsible pigment for the abnormal coloration was purified by ammonium sulfate fractionation and preparative flat-bed isoelectric focusing. On the basis of pI, molecular weight and the visible absorption, fluorescence and circular dichroism spectra, the pigment was identified as monomeric phycoerythrin. Dried Nori contained three times as much monomer as hexamer. From field cultivation tests, dried Nori produced large amounts of monomeric phycoerythrin when the fronds on frozen nursery-nets were damaged.