FLUORESCENCE AND CIRCULAR DICHROISM STUDIES ON THE PHYCOERYTHROCYANINS FROM THE CYANOBACTERIUM

Two phycoerythrocyanin (PEC) fractions have been obtained from the phycobilisomes of the cyanobac-terium Westiellopsis prolifica ARM 365. They have been characterized by absorption, fluorescence and circular dichroism spectroscopy. One of them is spectroscopically similar to a PEC trimer known from other organisms. Whereas efficient energy transfer from its violin (α-84) to the cyanin (β-84, 155) chromophores is efficient in the trimer (αβ it is impeded after dissociation to the monomer (α,β). A second fraction of PEC which we earlier termed PEC(X) (Maruthi Sai et al., Photochem. Photobiol. 55,119–124, 1992), exhibited the spectral properties similar to that of the α-subunit of PEC from Mastigocladus laminosus. With this highly photoactive fraction, the circular dichroism spectra of the violobilin chromophore in both photoreversible states were obtained

TWO DIFFERENT TYPES OF PHOTOCHEMISTRY IN PHYCOERYTHROCYANIN α-SUBUNIT

The photochemical activities of phycoerythrocyanin α-subunits from Mastigocladus laminosus separated by isoelectric focusing were tested by irradiating at 500, 550, 577 and 600 nm. Two types of photoreversible photochromic responses have been characterized by absorption and absorption difference spectroscopy. Type I is the well-known absorption shift from 571 to 506 nm. Type II is a new response characterized by a line-broadening of the 570 nm absorption

THEORETICAL STUDIES OF BILIPROTEIN CHROMOPHORES AND RELATED BILE PIGMENTS BY MOLECULAR ORBITAL AND RAMACHANDRAN TYPE CALCULATIONS

Ramachandran calculations have been used to gain insight into steric hindrance in bile pigments related to biliprotein chromophores. The high optical activity of denatured phycocyanin, as compared to phycoerythrin, has been related to the asymmetric substitution at ring A, which shifts the equilibrium towards the P-helical form of the chromophore. Geometric effects on the electronic structures and transitions have then been studied by molecular orbital calculations for several conjugation systems including the chromophores of phycocyanin. phytochrome P,, cations, cation radicals and tautomeric forms. For these different chromophores some general trends can be deduced. For instance, for a given change in the gross shape (e.g. either unfolding of the molecule from a cyclic-helical to a fully extended geometry, or upon out-of-plane twists of the pyrrole ring A) of the molecules under study, the predicted absorption spectra all change in a simikar way. Nonetheless, there are characteristic distinctions between the different n-systems, both in the transition energies and the charge distribution, which can be related to their known differences in spectroscopic properties and their reactivity

Influence of chromophores on quarternary structure of phycobiliproteins from the cyanobacterium, Mastigocladus laminosus

Chromophores of C-phycocyanin and phycoerythrο-cyanin have been chemically modified by reduction to rubins , bleaching , photoisomerization , or perturbation with bulky substituents. Pigments containing modified chromophores, or hybrids containing modified and unmodified chromophores in individual protomers have been prepared. All modifications inhibit the association of the (aß)-protomers of these pigments to higher aggregates. The results demonstrate a pronounced effect of the state of the chromophores on biliprotein quaternary structure. It may be important in phycobi1isome assembly , and also in the dual function of biliproteins as (i) antenna pigments for photosynthesis and (ii) reaction centers for photomor-phogenesis

PHOTOCHEMISTRY OF PHYCOBILIPROTEINS

Native PEC from the cyanobacterium, Mastigocladus laminosus, and its isolated α-subunit show photoreversibly photochromic reactions with difference-maxima around 502 and 570 nm in the spectral region of the α-84 phycoviolobilin chromophore. (b) Native PEC and its β-subunit show little if any reversible photochemistry in the 600–620 nm region, where the phycocyanobilin chromophores on the β-subunit absorb maximally, (c) Reversible photochemistry is retained in ureadenatured PEC at pH = 7.0 or pH ≤ 3. The difference maxima are shifted to 510 and 600 nm, and the amplitudes are decreased. An irreversible absorbance increase occurs around 670 nm (pH ≤ 3). (d) The amplitude of the reversible photoreaction difference spectrum is maximum in the presence of 4–5 M urea or 1 M KSCN, conditions known to dissociate phycobiliprotein aggregates into monomers. At the same time, the phycocyanobilin chromophore(s) are bleached irreversibly, (e) The amplitude becomes very small in high aggregates, e.g. in phycobilisomes. (f) In a reciprocal manner, the phototransformation of native PEC leads to a reversible shift of its aggregation equilibrium between trimer and monomer. The latter is favored by orange, the former by green light, (g) It is concluded that the phycoviolobilin chromophore of PEC is responsible for reversible photochemistry in PEC, and that there is not only an influence of aggregation state on photochemistry, but also vice versa an effect of the status of the chromophore on aggregation state. This could constitute a primary signal in the putative function as sensory pigment, either directly, or indirectly via the release of other polypeptides, via photodynamic effects, or the like

Photophysics of phycoerythrocyanins from the cyanobacterium Westiellopsis prolifica studied by time-resolved fluorescence and coherent anti-Stokes Raman scattering spectroscopy

Three building blocks of the antenna complexes of the cyanobacterium Westiellopsis prolifica were studied: PEC(X), which is similar to the α-subunit of phycoerythrocyanin (PEC), trimers of PEC and monomers derived from these by deaggregation with KSCN. The fit of the fluorescence decay curve of PEC(X) requires at least four exponentials, although it supposedly contains only one chromophore. The coherent anti-Stokes Raman scattering (CARS) spectra indicate that the heterogeneity observed is due to geometrical isomers, which are in part generated by photoinduced processes. A similar heterogeneity in chromophore structure and properties is also found in the monomers, where four exponentials are needed to fit the fluorescence decay curve. As in trimers, there is a long-lived, low-amplitude component, which can be assigned to impurities and/or oxidation products. The energy transfer time between the two phyocyanobilin chromophores in the β-subunit is about 500 ps; the lifetime of the fluorescing β-chromophore is 1.5 ns. The phycoviolobilin chromophore in the α-subunit adopts different geometries characterized by fluorescence lifetimes of about 240 and 800 ps. No evidence was found for energy transfer between the α-chromophore and the β-chromophores. This energy transfer occurs in trimers on a time scale of less than 20 ps; the energy transfer time between the two different types of β-chromophore is about 250 ps and the lifetime of the terminal emitter is about 1.5 ns. The excited state kinetics are therefore similar to those of PEC trimers from Mastigocladus laminosus, as are the CARS spectra, indicating a similar chromophore—protein arrangement. In comparison with phycocyanin, the ordering of the excited states of chromophores β84 and β155 may be changed. Although PEC trimers of Westiellopsis prolifica show almost as good a photostability as trimers of Mastigocladus laminosus, monomers are so photolabile that no CARS spectra could be recorded

Picosecond time-resolved fluorescence of phycobiliproteins

The α- and β-subunits of C-phycocyanin from Mastigocladus laminosus were prepared according to revised procedures. Both subunits are isolated as dimers, which can be dissociated into monomers with detergent mixtures. The fluorescence decay kinetics are similar for the respective monomers and dimers. In no case could they be fitted by only one (α-subunit) or two exponentials (β-subunit) which are predicted by theory for samples with a unique chromophore—protein arrangement containing one and two chromophores, respectively. It is suggested that there exists a heterogeneity among the chromophores of the subunits, which may persist in the highly aggregated complexes present in cyanobacterial antennas

Dendritic Cells Release HLA-B-Associated Transcript-3 Positive Exosomes to Regulate Natural Killer Function

NKp30, a natural cytotoxicity receptor expressed on NK cells is critically involved in direct cytotoxicity against various tumor cells and directs both maturation and selective killing of dendritic cells. Recently the intracellular protein BAT3, which is involved in DNA damage induced apoptosis, was identified as a ligand for NKp30. However, the mechanisms underlying the exposure of the intracellular ligand BAT3 to surface NKp30 and its role in NK-DC cross talk remained elusive. Electron microscopy and flow cytometry demonstrate that exosomes released from 293T cells and iDCs express BAT3 on the surface and are recognized by NKp30-Ig. Overexpression and depletion of BAT3 in 293T cells directly correlates with the exosomal expression level and the activation of NK cell-mediated cytokine release. Furthermore, the NKp30-mediated NK/DC cross talk resulting either in iDC killing or maturation was BAT3-dependent. Taken together this puts forward a new model for the activation of NK cells through intracellular signals that are released via exosomes from accessory cells. The manipulation of the exosomal regulation may offer a novel strategy to induce tumor immunity or inhibit autoimmune diseases caused by NK cell-activation

THERMAL DENATURATION OF MONOMERIC AND TRIMERIC PHYCOCYANINS STUDIED BY STATIC AND SPECTROSCOPY POLARIZED TIME-RESOLVED FLUORESCENCE

C-Phycocyanin (PC) and allophycocyanin (APC). as well as the a-subunit of PC. have been isolated from the blue-green alga (cyanobacterium). Spirulina platensis. The effects of partial thermal denaturation of PC and of its state of aggregation have been studied by ps time-resolved, polarized fluorescence spectroscopy. All measurements have been performed under low photon fluxes (< 10’ ’ photonsipulse x cm’) to minimize singlet-singlet annihilation processes. A complex decay is obtained under most conditions, which can be fitted satisfactorily with a bi-exponential (7’ = 70400 ps. T? = 1000-3000 ps) for both the isotropic and the polarized part, but with different intensities and time constants for the two decay curves. The data are interpreted in the frameworkof the model first developed by Teak and Dale (Biochern. J. 116, 161 (1970)], which divides the spectroscopically different chromophores in (predominantly) sensitizing (s) and fluorescing U, ones. If one assumes temperature dependent losses in the energy transfer from the s to the f and between f chromophores. both the biexponential nature of the isotropic fluorescence decay and the polarization data can be rationalized. In the isotropic emission (corresponding to the population of excited states) the short lifetime is related to the s-,f transfer. the longer one to the “free“ decay of the final acceptor(s) (= f). The polarized part is dominated by an extremely short decay time. which is related to s+f transfer, as well as to resonance transfer between the f-chromophores