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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.

Pigment organization effects on energy transfer and Chl a emission imaged in the diatoms C. meneghiniana and P. tricornutum in vivo: a confocal laser scanning fluorescence (CLSF) microscopy and spectroscopy study.

The (auto)fluorescence from three diatom strains, Cyclotella meneghiniana (Cm), Phaeodactylum tricornutum 1a (Pt1a), and Phaeodactylum UTex (PtUTex), 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 (Fx) found within the fucoxanthin chlorophyll a/c 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 Chl a 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 Fx(red) light harvesters, and lesser amounts of Fx(green)s, 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 Fx--Fx(red), Fx(green) and Fx(blue)--as well as Chl c. The heterogeneous Chl a emission observed from the (C)LSF images indicates that the different Fx's serve different final emitters in P. tricornutum and suggest, at least in C. meneghiniana , a localization of FCPs with relatively greater Fx(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 Cm but not in Pt1a nor PtUTex.

DISCO synchrotron-radiation circular-dichroism endstation at SOLEIL.

The new synchrotron-radiation circular-dichroism (SRCD) endstation on the UV-visible synchrotron beamline DISCO has been commissioned at the SOLEIL synchrotron. The design has been focused on preservation of a high degree of linear polarization at high flux and moderate resolving power covering the vacuum ultraviolet to visible spectral range (125-600 nm). The beam dimensions have been set to 4 mm × 4 mm at 1 nm bandwidth for lower sample degradation. The nitrogen-purged sample chamber fits three types of sample holders accommodating conventional round cell mounting, automated rotation of the samples, as well as a microfluidic set-up. Automated temperature-controlled data collection on microvolumes is now available to the biology and chemistry communities. Macromolecules including membrane proteins, soluble proteins, bio-nanotubes, sugars, DNA and RNAs are now routinely investigated.

Pigment organization in fucoxanthin chlorophyll a/c(2) proteins (FCP) based on resonance Raman spectroscopy and sequence analysis.

Chlorophylls (Chls)-a and -c(2) are identified and characterized in fucoxanthin chlorophyll-a/c(2) protein (FCP) complexes in the trimeric (FCPa(trim)) and oligomeric (FCPb(olig)) forms of FCP from the diatom Cyclotella meneghiniana using resonance Raman (RR) spectroscopy. Importantly, two different Chl-c(2)s are identified in both FCPa(trim) and FCPb(olig) from their signature ring-breathing modes at approximately 1360 cm(-1). In addition, the C13(1)-keto carbonyl peaks indicate the presence of more than four Chl-a's in both FCP complexes and are broadly classified into three groups with strong, medium and weak external hydrogen bonds. Together, they provide the strongest spectroscopic evidence so far that there may be up to double the number of pigments previously estimated at 4Fx:4Chl-a:1Chl-c(2) per FCP monomer. Careful analysis of the protein sequences also strongly support the higher pigment content by showing that at least six Chl-a, and one Chl-b, binding sites found in LHCII are retained in the FCPs. The relative enhancement of the RR bands for 406.7 versus 413.1 nm further allows some distinction of blue- versus red-absorbing Chl-a's, respectively. Further differences between the Chls in FCPb(olig) and FCPa(trim) are present in the amino-acid sequences and the RR signals. Information about the Chl-binding sites, complemented by information about the structures and interactions of the Chls are used to characterize their local environments, and assign pigment locations (and functions) in FCPb(olig) and FCPa(trim), which along with the earlier characterization of the carotenoids (J. Phys. Chem. B. 112 (2009) 12565-12574) provide a first (global) framework for pigment organization in FCP.

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

The 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 approximately 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), >or=8 x 10(7) M(-1) for OGN 1a and >or=2 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) >or= 8 x 10(7) M(-1) for OGN 1a and >or=1 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.

Carotenoid structures and environments in trimeric and oligomeric fucoxanthin chlorophyll a/c2 proteins from resonance Raman spectroscopy.

Resonance Raman (RR) spectroscopy is used to characterize the structures and environments of the carotenoid fucoxanthin (Fx), the primary light harvester in the membrane-intrinsic fucoxanthin chlorophyll a/c2 proteins(FCP) from the diatom Cyclotella meneghiniana, thereby building on the findings from Stark spectroscopy and calculations (J. Phys. Chem. B 2008, 112 (37), 11838-11853). Solvent-dependent effects on the RR bands of isolated Fx and the xanthophyll-cycle carotenoid, diadinoxanthin (Ddx), are studied to better characterize the protein-specific environmental factors that affect their geometry and spectral signatures. In addition, excitation-wavelength-dependent (441.6-570 nm) changes in the RR bands of the nu1 and nu 3 modes,as well as the conjugated C8 carbonyl stretch, allow the identification of 5-6 in both the trimeric (FCPatrim)and oligomeric (FCPbolig) forms of FCP. These Fx's are broadly classified into two each of high (Fxblue) and low (Fxred) energy, and 1-2 of intermediate (Fxgreen) energy that are allied to their location and function in the protein. The CdC stretching frequencies (nu 1), which indicate conjugation over at least 7 double bonds, and the low intensity of the nu 4 C-H bending modes attest to their planar all-trans conformations both in the protein and in solution, with the protein-bound Fxred's exhibiting signs of nonlinearity. Additionally, rededge excitation of Fx in solution, and in the FCPs, exhibits the effect of mixing between the two lowest energy, 21Ag--like and 11Bu+-like, excited states, which underpins the high light-harvesting and energy transfer efficiency of the Fxred's. RR spectra also reveal differences between FCPatrim and FCPbolig complexes,such as the greater prevalence of Ddx in FCPbolig. Importantly, the identification of 5-6 Fx's per FCP monomer suggests that there may be more than the four Fx's previously assumed per FCP monomer, or else there is definite heterogeneity in Fx structures and/or binding sites.

The charge-transfer properties of the S2 state of fucoxanthin in solution and in fucoxanthin chlorophyll-a/c2 protein (FCP) based on stark spectroscopy and molecular-orbital theory.

Fucoxanthin chlorophyll-a/c 2 protein (FCP), the membrane-intrinsic light harvesting complex from the diatom Cyclotella meneghiniana, is characterized by Stark spectroscopy to obtain a quantitative measure of the excited-state dipolar properties of the constituent pigments. The electro-optical properties of the carotenoid fucoxanthin (Fx), the primary light harvester in FCP, were determined from the Stark spectrum measured in a MeTHF glass (77 K) and compared to the results from electronic-structure calculations. On photon absorption by Fx, a 17 D change in the static dipole moment (|Delta mu|exp), and a somewhat larger |Delta mu|exp at the red edge, are measured for the S 0 --> S 2 (1 (1)A g (-)-like -->1 (1)B u *+-like) transition. The large change in dipole moment indicates that Fx undergoes photoinduced charge transfer (CT), and underscores the influence of the S 2 state on the polarity-dependent excited-state dynamics of Fx that has so far been attributed to, and discussed in terms of, the S 0 and the S 1/ICT states. MNDO-PSDCI and SACCI-CISD calculations indicate that the 1 (1)B u (*+)-like state intrinsically possesses a dipole moment much smaller than the 2 (1)A g (*-)-like state, suggesting that solvent fields promote the mixing of these two states and could account for the large dipole moments measured here for the S 0 --> S 2 transition. These CT properties of the 1 (1)B u (*+)-like state of Fx are further enhanced in the protein and underpin its photosynthetic capabilities for light harvesting and energy transfer (ET). In FCP, the CT properties of the Fx's vary according to the energetic position: between 450 and 500 nm there appear to be two sets of Fx's that exhibit |Delta mu| exp values on the order of 5 and 15 D, whereas the red-most Fx's, that are very efficient in ET to chlorophyll-a (Chl-a), exhibit strikingly large |Delta mu| exp values on the order of 40 D. Such magnitudes of |Delta mu| exp suggest a mechanism that enhances Coulombic coupling to promote ET from the S 2 state of Fx to Chl-a. These three sets of Fx's, including a fourth red Fx, are identified by fitting the Stark spectrum of FCP with the Stark spectrum of Fx in MeTHF. In contrast to the Fx's in the protein, the electrostatic properties of the Chl's in FCP are comparatively much smaller. Notably, for the Q y band of Chl-a, a |Delta mu| exp of 0.92 D and a change in polarizability ( Delta alpha exp) of 20 A (3), indicate that the Chl-a's are monomeric in nature and decoupled from each other.

The charge-transfer character of the S0 --> S2 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 (|Deltamu|) was found between the ground state, S(0) (1(1)A(g)(-)), and the Franck-Condon region of the S(2) (1(1)B(u)(+)) excited state, in both MeTHF (22 D) and EG (approximately 27 D), thus revealing the previously unknown charge transfer (CT) character of this pi-pi transition in peridinin. Such a large |Deltamu| 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 S(2) 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 S(2) and S(1)/ICT states to the chlorophylls in PCP.

Stark spectroscopy of the light-harvesting complex II in different oligomerisation states.

The electric field-induced absorption changes (Stark effect) of light-harvesting complex II (LHCII) in different oligomerisation states-monomeric, trimeric and aggregated-have been probed at 77 K. All the chlorophyll (Chl) a molecules exhibit electro-optic properties in the Q(y) absorption region characterized by a change in dipole moment /Deltamu-->/ =0.6+/-0.06D/f and polarizability, Tr(Deltaalpha;) approximately 55+/-5 A(3)/f(2) upon electronic excitation, which are similar to those of unbound monomeric Chl a, indicating the absence of strong delocalization of the excitations which would be expected in the presence of strong excitonic interactions. The Stark effect in the Chl b absorption region is significantly bigger with /Deltamu-->/ values of the order of 2.0+/-0.2 D/f and it is attributed to strong interactions with neoxanthin molecules. Clear oligomerisation-dependent differences are observed in the carotenoid region, mainly due to the appearance of a new xanthophyll absorption band at 509 in the spectra of trimers and oligomers. It is ascribed to some lutein molecules, in agreement with previous experimental observations. The electro-optic properties of these lutein molecules are significantly different from those of the other xanthophylls in LHCII, which do not exhibit such a big change in dipole moment upon electronic excitation (/Deltamu-->/ =14.6+/-2.0 D/f). Upon aggregation of LHCII some extra absorption appears on the red side of the main Chl a Q(y) absorption band. In contrast to an earlier suggestion [J. Phys. Chem., A 103 (1999) 2422], no indications are found for the charge-transfer character of the corresponding band. The assignments of the S(2) electronic transitions of neoxanthin and lutein in LHCII and possible origins of the Stark effect are discussed.