Functional heterogeneity of the fucoxanthins and fucoxanthin-chlorophyll proteins in diatom cells revealed by their electrochromic response and fluorescence and linear dichroism spectra

In this work, by analyzing the electrochromic transient spectra, the 77 K fluorescence emission and excitation, as well as the linear dichroism (LD) and circular dichroism (CD) spectra of low-light (LL) and high-light (HL) grown Phaeodactylum tricornutum cells, we show that the fucoxanthins (Fx) and fucoxanthin-chlorophyll proteins (FCP) exhibit marked functional heterogeneity. Electrochromic transients reveal that LL and HL cells differ substantially in their relative contents of two Fx forms, which absorb at 501 and 550 nm; they exhibit distinct LD signals but are CD silent. Fluorescence emission and excitation spectra at 77 K reveal that although both forms efficiently transfer excitation energy to Chl a, the red form feeds somewhat more energy to photosystem II than to photosystem I. Similar data obtained in Cyclotella meneghiniana cells suggest that the heterogeneity of the FCP pool, with different Fx forms, plays a role in the regulation of energy utilization in FCP-containing organisms. © 2010 Elsevier B.V. All rights reserved

Acknowledging and citing core facilities Key contributions to data lifecycle should be recognised in the scientific literature

Core facilities play a central role in the life sciences by generating data and ensuring quality standards. Their contributions to research should be appropriately acknowledged or cited in research papers.Non peer reviewe

On the involvement of Single-Bond Rotation in the Primary Photochemistry of Photoactive Yellow Protein

AbstractPrior experimental observations, as well as theoretical considerations, have led to the proposal that C4-C7 single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C4-C7 single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C4-C7 single-bond rotation in PYP is not an alternative to C7=C8 double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore

Overview of EU-LIFE Core Facilities Benchmarking Survey 2021

<p>Overview of EU-LIFE Core Facilities Benchmarking Survey 2021</p&gt

Netropsin binding in five duplex-dimer DNA constructs as a function of size and distance between binding sites: circular dichroism and absorption spectroscopy

International audienceThe optical activity induced on binding the drug netrospin (NET) in the minor groove of DNA is studied in five oligonucleotides (OGNs) as a function of (1) the size of the binding site in (5'-(GC)(2)AATT(GC)(2)-3')(2) (OGN 1a) versus (5'-(GC)(2)AAATTT(GC)(2)-3')(2) (OGN 1b) and (2) the distance between two AATT binding sites in (5'-(GC)(2)AATT(GC) (x) AATT(GC)(2)-3')(2), with x = 1, 2, or 3 (OGNs 2a, b, c, respectively). NET binding is monitored via the induced circular dichroism (CD) at similar to 315 nm, where the nucleic acids are optically inactive. The CD titrations, fit to a tight binding model, yield lower limits for the binding constant, K-a, a parts per thousand yen8 x 10(7) M-1 for OGN 1a and a parts per thousand yen2 x 10(8) M-1 for OGNs 2a, b, c in 1 mM buffer. In 100 mM buffer, tight binding occurs in all five OGNs with K-a a parts per thousand yen 8 x 10(7) M-1 for OGN 1a and a parts per thousand yen1 x 10(8) M-1 for OGNs 1b and 2a, b, c. In contrast, the elongated AAATTT binding site of OGN 1b results in weak binding of NET in 1 mM buffer, where competing electrostatic interactions with the solvent environment are lower. In the constructs with two binding sites, the increase in flexibility introduced by intervening GC base pairs does not induce co-operative binding, although differences in the number of binding sites, n (2.05-2.65), indicate that there may be differences in the way NET is bound in OGNs 2a, b, c. In addition, the large shifts in the absorption spectra induced in bound versus free NET, and effects on the CD spectral bands at higher energy, are discussed in terms of electrostatic and excitonic interactions

How Light-Induced Charge Transfer Accelerates the Receptor-State Recovery of Photoactive Yellow Protein from its Signaling State

Stark (electroabsorption) spectra of the M100A mutant of photoactive yellow protein reveal that the neutral, cis cofactor of the pB intermediate undergoes a strikingly large change in the static dipole moment ([Formula: see text] = 19 Debye) on photon absorption. The formation of this charge-separated species, in the excited state, precedes the cis → trans isomerization of the pB cofactor and the regeneration of pG. The large [Formula: see text] , reminiscent of that produced on the excitation of pG, we propose, induces twisting of the cis cofactor as a result of translocation of negative charge, from the hydroxyl oxygen, O1, toward the C7-C8 double bond. The biological significance of this photoinduced charge transfer reaction underlies the significantly faster regeneration of pG from pB in vitro, on the absorption of blue light

Pigment organisation in the membrane-intrinsic major light-harvesting complex of Amphidinium carterae : structural characterisation of the peridinins and chlorophylls a and c₂ by resonance Raman spectroscopy and from sequence analysis

The structures and environments of the protein-bound peridinins (Pers) and chlorophylls (Chls) a/c₂ in the membrane-intrinsic major light-harvesting complex of the dinoflagellate Amphidinium carterae (LHCAmph) are characterised using resonance Raman (RR) spectroscopy with 11 excitation wavelengths, at 77K. The excitation-dependent variation in the CC stretching mode (ν₁) suggests the presence of three Pers with conjugation lengths over 8 double bonds (dBs), and one diadinoxanthin, between 413.7 and 528.7nm. Two Perred species are revealed on excitation at 550 and 560nm. These Perred species exhibit anomalously low ν₁ values, together with notable resonant enhancement of lactone ring-breathing and -deformation modes. To discern protein-specific effects, the RR spectra are compared to that of Per in polar (acetonitrile), polarisable (toluene) and polar-protic (ethanol) solvents. Resonantly enhanced lactone, ring-breathing (942cm⁻¹) and ring-deformation (~650cm⁻¹), modes are identified both in solution, and in the protein, and discussed in the context of the mixing of the S₁ and S₂ states, and formation of the intramolecular charge-transfer (ICT) state. In the Chl-absorbing region, two sets of Chl c₂'s, and (at least) six Chl a's can be differentiated. With a pigment ratio of 5-6 (Chl a):2 (Chl c₂):5-6 (Per):1 Ddx determined from the fit to the RT absorption and 77K RR spectra, sequence comparison of LHCAmp to LHCII, and the diatom LHC, fucoxanthin-chlorophyll-a/c-protein (FCP), a template for the conserved pigment binding sites is proposed, to fill the paucity of structural information in the absence of a crystal structure for LHCAmph.13 page(s

S-2 transition in the carotenoid peridinin is revealed by stark spectroscopy

Peridinin, the carotenoid in the peridinin chlorophyll a protein (PCP), was studied by Stark (electroabsorption) spectroscopy to determine the change in electrostatic properties produced on excitation within the absorption band, in methyl tetrahydrofuran (MeTHF) versus ethylene glycol (EG), at 77 K. Strikingly, a large change in the permanent dipole moment (|Δμ|) was found between the ground state, S0 (11Ag*-), and the Franck−Condon region of the S2 (11Bu*+) excited state, in both MeTHF (22 D) and EG (~27 D), thus revealing the previously unknown charge transfer (CT) character of this π−π* transition in peridinin. Such a large |Δμ| produced on excitation, we suggest, facilitates the bending of the lactone moiety, toward which charge transfer occurs, and the subsequent formation of the previously identified intramolecular CT (ICT) state at lower energy. This unexpectedly large S2 dipole moment, which has not been predicted even from high-level electronic structure calculations, is supported by calculating the shift of the peridinin absorption band as a function of solvent polarity, using the experimentally derived result. Overall, the photoinduced charge transfer uncovered here is expected to affect the excited-state reactivity of peridinin and, within the protein, be important for efficient energy transfer from the carotenoid S2 and S1/ICT states to the chlorophylls in PCP.9 page(s

Pigment Organization Effects on Energy Transfer and <i>Chl a</i> Emission Imaged in the Diatoms <i>C. meneghiniana</i> and <i>P. tricornutum</i> In Vivo: A Confocal Laser Scanning Fluorescence (CLSF) Microscopy and Spectroscopy Study

The (auto)­fluorescence from three diatom strains, Cyclotella meneghiniana (<i>Cm</i>), Phaeodactylum tricornutum 1a (<i>Pt1a</i>), and Phaeodactylum UTex (<i>PtUTex</i>), has been imaged in vivo to submicrometer resolution using confocal laser scanning fluorescence (CLSF) microscopy. The diatoms are excited at 473 and 532 nm, energy primarily absorbed by the carotenoid fucoxanthin (<i>Fx</i>) found within the fucoxanthin chlorophyll <i>a</i>/<i>c</i> proteins (FCPs). On the basis of the fluorescence spectra measured in each image voxel, we obtain information about the spatial and energetic distribution of the terminal <i>Chl a</i> emitters, localized in the FCPs and the reaction centers of the PSII protein complexes, and the nature and location of the primary absorbers that are linked to these emitters; 532 nm excites the highly efficient <i>Fx</i>_red light harvesters, and lesser amounts of <i>Fx</i>_greens, that are enriched in some FCPs and preferentially transfer energy to PSII, compared to 473 nm, which excites almost equal amounts of all three previously identified sets of <i>Fx</i> – <i>Fx</i>_red, <i>Fx</i>_green and <i>Fx</i>_blue – as well as <i>Chl c</i>. The heterogeneous <i>Chl a</i> emission observed from the (C)­LSF images indicates that the different <i>Fx</i>’s serve different final emitters in P. tricornutum and suggest, at least in C. meneghiniana, a localization of FCPs with relatively greater <i>Fx</i>_red content at the chloroplast edges, but with overall higher FCP concentration in the interior of the plastid. To better understand our results, the concentration-dependent ensemble-averaged diatom solution spectra are compared to the (auto)­fluorescence spectra of individual diatoms, which indicate that pigment packing effects at an intracellular level do affect the diatoms’ spectral properties, in particular, concerning a 710 nm emission band apparent under stress conditions. A species-specific response of the spectral signature to the incident light is also discussed in terms of the presence of a silica shell in <i>Cm</i> but not in <i>Pt1a</i> nor <i>PtUTex</i>