Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

Reconstitution of the Peridinin–chlorophyll a Protein (PCP): Evidence for Functional Flexibility in Chlorophyll Binding

The coding regions for the N-domain, and full length peridinin–chlorophyll a apoprotein (full length PCP), were expressed in Escherichia coli. The apoproteins formed inclusion bodies from which the peptides could be released by hot buffer. Both the above constructs were reconstituted by addition of a total pigment extract from native PCP. After purification by ion exchange chromatography, the absorbance, fluorescence excitation and CD spectra resembled those of the native PCP. Energy transfer from peridinin to Chl a was restored and a specific fluorescence activity calculated which was ~86% of that of native PCP. Size exclusion analysis and CD spectra showed that the N-domain PCP dimerized on reconstitution. Chl a could be replaced by Chl b, 3-acetyl Chl a, Chl d and Bchl using the N-domain apo protein. The specific fluorescence activity was the same for constructs with Chl a, 3-acetyl Chl a, and Chl d but significantly reduced for those made with Chl b. Reconstitutions with mixtures of chlorophylls were also made with eg Chl b and Chl d and energy transfer from the higher energy Qy band to the lower was demonstrated.     

Effect of the Vibrational/Rotational Energy Partitioning on the Energy Transfer in Atom–Triatomic Molecule Collisions

The effect of the initial partitioning of the molecular energy between vibrational and rotational modes of a triatomic molecule on the collisional energy transfer is studied for a model atom–triatomic molecule system. We considered the collisions of thermal bath Ar atoms with SO2 molecules, and used the trajectory calculations for determining the energy transfer for three different samplings of initial conditions of the molecule. The first sampling method generated the microcanonical distribution over all states, entering into the vibrational and rotational manifolds, while two others produced distributions with relatively lower values of the rotational energies. It is shown that both the average energy transfer per collision and the mechanism of the energy exchange are significantly affected by the vibrational/rotational energy partitioning before the collisions. Relative decrease in the rotational energy results in the decrease of the averaged energy transfer and progressively emphasizes the role of active rotation as the gateway for translation-vibration energy exchange.     

Spectroscopic Characterization of the Excitation Energy Transfer in the Fucoxanthin–Chlorophyll Protein of Diatoms

We characterized the energy transfer pathways in the fucoxanthin–chlorophyll protein (FCP) complex of the diatom Cyclotella meneghiniana by conducting ultrafast transient absorption measurements. This light harvesting antenna has a distinct pigment composition and binds chlorophyll a (Chl-a), fucoxanthin and chlorophyll c (Chl-c) molecules in a 4:4:1 ratio. We find that upon excitation of fucoxanthin to its S₂ state, a significant amount of excitation energy is transferred rapidly to Chl-a. The ensuing dynamics illustrate the presence of a complex energy transfer network that also involves energy transfer from the unrelaxed or ‘hot’ intermediates. Chl-c to Chl-a energy transfer occurs on a timescale of a 100 fs. We observe no significant spectral evolution in the Chl-a region of the spectrum. We have applied global and target analysis to model the measured excited state dynamics and estimate the spectra of the states involved; the energy transfer network is discussed in relation to the pigment organization of the FCP complex.     

Analysis of Nucleosome Structure in Polyacrylamide Gel by the Förster Resonance Energy Transfer Method

A technique for analyzing the structure of (Cy3, Cy5)-labeled nucleosomes in polyacrylamide gel after electrophoresis under native conditions was developed based on the Förster resonance energy transfer (FRET) effect. It has been shown that the correct application of this technique requires monitoring of nonspecific intermolecular FRET and fluorescence reabsorption. A comparative analysis of the results of the FRET measurements of two types of nucleosomes and their complexes with yeast protein FACT was performed, which confirmed the similarity of the structural features of nucleosomes detected in the gel and in aqueous solution. Application of FRET analysis in combination with electrophoresis makes it possible not only to separate, visualize components of a complex mixture, and to evaluate their relative content but also to characterize the structural differences between these complexes in situ.     

A Four-Level Theory on Energy Transfer of the Light-Harvesting Complex ￠ò in PS ￠ò in Higher Plants (in English)

With reference to the recent achivements about the structure, spectra and kinetics of light-harvesting complex (LHCⅡ) in PSⅡ of higher plants, a four-level model was provided to simulate the energy transfer process from LHCⅡ to the reaction center. On the basis of this model, a set of rate equation was established. Analysis of its algebra solution led to a general picture of energy transfer process in LHCⅡ of higher plants and the strong interaction among pigment molecules in this process. Based on the spectra, kinetics and biological structural data providing some information of energy transfer path and energy dissipation mechanism, it has been found that energy transfer mainly happened between the pigments whose energy level was most closely adjacent, the loss of energy had a close relation to the process of energy transfer and tended to increase with the decrease of energy level. The protective mechanism of antenna system was also discussed.     

Characterization of the peridinin–chlorophyll a-protein complex in the dinoflagellate Symbiodinium

The water-soluble peridinin–chlorophyll a-proteins (PCPs) are one of the major light harvesting complexes in photosynthetic dinoflagellates. PCP contains the carotenoid peridinin as its primary pigment. In this study, we identified and characterized the PCP protein and the PCP gene organization in Symbiodinium sp. CS-156. The protein molecular mass is 32.7 kDa, revealing that the PCP is of the monomeric form. The intronless PCP genes are organized in tandem arrays. The PCP gene cassette is composed of 1095-bp coding regions and spacers in between. Despite the heterogeneity of PCP gene tandem repeats, we identified a single form of PCP, the sequence of which exactly matches the deduced sequence of PCP gene clone 7 (JQ395030) by LC–MS/MS analysis of tryptic digested PCP, revealing the mature PCP apoprotein is 312 amino acids in length. Pigment analysis showed a peridinin-to-Chl a ratio of 4. The peridinin-to-Chl a Q_y energy transfer efficiency is 95% in this complex.     

Changes with age in the absorption spectrum of chlorophyll α in a diatom

Summary Absorption spectra of a young and an old culture of the diatom Pheodactylum tricornutum were measured in thin layers between two opal glass sheets. The spectra at 24° and at -196°C were replotted to give equal areas from 730–625 m to allow direct comparison. At 24°C the spectrum for the difference between the two cultures had a negative component of 18 m half width centered at 675 m and a positive region of W_0.5=26 m near 700 m.The spectra at -196°C may be somewhat distorted by clumping of the cells during freezing but nevertheless the 16 day culture clearly showed a smaller proportion of Ca 670 to Ca 680. This older culture has a shoulder due to a 707 m component. The difference curve at -196°C shows the decrease of an unsymmetrical band peaking at 669 m and an increase at 695 m in addition to the 707 m component. Due to the possibility of distortion, the presence of an actual component at 695 is doubtful in these particular cultures.The room temperature spectrum in the chloropyhll a region for the 5 day culture can be closely fitted by a single probability curve at 675 m having a half-width of 31 m. The sum of two components, with widths more reasonable for chlorophylls, also matched the data well enough. These two probability curves, of 22 m half width, centered on 669 and 683.2 m and had a height ratio, h₆₆₉/h₆₈₃ of 1.18. In the 16 day culture the ratio for these bands changed to 1.11 and there was extra absorption around 700 m.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday     

Relative binding affinities of chlorophylls in peridinin–chlorophyll–protein reconstituted with heterochlorophyllous mixtures

Peridinin–chlorophyll–protein (PCP), containing differently absorbing chlorophyll derivatives, are good models with which to study energy transfer among monomeric chlorophylls (Chls) by both bulk and single-molecule spectroscopy. They can be obtained by reconstituting the N-terminal domain of the protein (N-PCP) with peridinin and chlorophyll mixtures. Upon dimerization of these “half-mers”, homo- and heterochlorophyllous complexes are generated, that correspond structurally to monomeric protomers of native PCP from Amphidinium carterae. Heterochlorophyllous complexes contain two different Chls in the two halves of the complete structure. Here, we report reconstitution of N-PCP with binary mixtures of Chl a, Chl b, and [3-acetyl]-Chl a. The ratios of the pigments were varied in the reconstitution mixture, and relative binding constants were determined from quantification of these pigments in the reconstituted PCPs. We find higher affinities for both Chl b and [3-acetyl]-Chl a than for the native pigment, Chl a.     

Förster Resonance Energy Transfer Between Molecules in the Vicinity of Graphene-Coated Nanoparticles

The recent demonstration of the plasmonic-enhanced Förster resonance energy transfer (FRET) between two molecules in the vicinity of planar graphene monolayers is further investigated using graphene-coated nanoparticles (GNP). Due to the flexibility of these nanostructures in terms of their geometric (size) and dielectric (e.g., core material) properties, greater tunability of the FRET enhancement can be achieved employing the localized surface plasmons. It is found that while the typical characteristic graphene plasmonic enhancements are manifested from using these GNPs, even higher enhancements can be possible via doping and manipulating the core materials. In addition, the broadband characteristics are further expanded by the closely spaced multipolar plasmon resonances of the GNPs.     

Improve the Hole Size–Dependent Refractive Index Sensitivity of Au–Ag Nanocages by Tuning the Alloy Composition

Plasmonics - In this study, Au–Ag nanoboxes are converted into Au–Ag alloy nanocages by increasing the hole size. The extinction spectrum and the refractive index sensing...     

Evolution of New Disease Specificity at a Simple Resistance Locus in a Crop–Weed Complex: Reconstitution of the Lr21 Gene in Wheat

The wheat leaf-rust resistance gene Lr21 was first identified in an Iranian accession of goatgrass, Aegilops tauschii Coss., the D-genome donor of hexaploid bread wheat, and was introgressed into modern wheat cultivars by breeding. To elucidate the origin of the gene, we analyzed sequences of Lr21 and lr21 alleles from 24 wheat cultivars and 25 accessions of Ae. tauschii collected along the Caspian Sea in Iran and Azerbaijan. Three basic nonfunctional lr21 haplotypes, H1, H2, and H3, were identified. Lr21 was found to be a chimera of H1 and H2, which were found only in wheat. We attempted to reconstitute a functional Lr21 allele by crossing the cultivars Fielder (H1) and Wichita (H2). Rust inoculation of 5876 F₂ progeny revealed a single resistant plant that proved to carry the H1H2 haplotype, a result attributed to intragenic recombination. These findings reflect how plants balance the penalty and the necessity of a resistance gene and suggest that plants can reuse “dead” alleles to generate new disease-resistance specificity, leading to a “death–recycle” model of plant-resistance gene evolution at simple loci. We suggest that selection pressure in crop–weed complexes contributes to this process.PLANTS possess large numbers of resistance genes (R gene) as a part of an elaborate plant defense system. In different plants, an R-gene locus may consist of a single-copy (simple) or of multiple copies of R genes (complex) in clusters as a result of gene duplication events. This duplication is considered as the birth of an R gene. R genes are necessary for plants to respond to pathogen attacks and to survive when pathogens are in the environment. Mutations, gene conversion, and recombination were found to be the means to create new specificities for various pathogens (for review, Leister 2004). However, an R gene could bring a penalty when the pathogen is absent (Stahl et al. 1999). In such a case, plants have better fitness when they get rid of the R-gene function. In nature, the presence of different pathogens maintains the diversity of R-gene specificities. So far, there is no report on the fates of nonfunctional R genes.In native agricultural ecosystems, wild plants often grow as weeds intermixed with or adjacent to their crop relatives. Extensive gene flow occurs between wild and domesticated forms, spawning numerous crop landraces adapted to diverse environments and occasionally new species. Common (hexaploid or bread) wheat (Triticum aestivum L., 2n = 6x = 42, genome formula AABBDD) arose from such a process by hybridization of domesticated tetraploid wheat (T. turgidum L., 2n = 4x = 28, AABB) with goatgrass (Aegilops tauschii Coss., 2n = 2x = 14, DD) growing as a weed in farmers'' fields along the Caspian Sea in Iran ca. 8000 years ago (Kihara 1944; McFadden and Sears 1946; Nesbitt and Samuel 1998).Because of the pivotal importance of Ae. tauschii in wheat evolution and crop improvement, Kihara et al. (1965) gathered extensive collections from Iran, Afghanistan, and adjacent regions. They suggested that Caspian Iran was the center of the genetic diversity of Ae. tauschii, a proposition later confirmed by molecular-marker analysis (Lubbers et al. 1991), as well as of resistance to leaf rust. Nine named and 12 new leaf-rust resistance genes have been documented in Ae. tauschii, and many more remain to be identified (Gill et al. 2008). Leaf rust, a scourge of wheat since before Roman times, is caused by the fungus Puccinia triticina (Eriks). It attacks mainly the leaf blade, producing small, elliptical, orange-red pustules on the upper surface, causing premature defoliation that results in as much as a 40% yield loss (McIntosh et al. 1995).One Ae. tauschii accession, TA1599, collected in Caspian Iran, carries a gene named Lr21 that confers resistance to all known P. triticina races. Lr21, transferred to wheat in the 1970s (Rowland and Kerber 1974; McIntosh et al. 1995), was recently cloned (Huang et al. 2003) and shown to be a simple (single-copy) locus encoding a nucleotide-binding site–leucine-rich repeats (NBS–LRR) protein of 1080 amino acids. Here we report how a simple locus such as Lr21 evolved novel resistance specificities in a unique crop–weed system and how fragments of nonfunctional alleles could be reused in this process.     

Reconstitution of TGF-β sensitivity in the VACO-411 human colon carcinoma line by somatic cell fusion with MCF-7

We characterized the mechanism of transforming growth factor beta (TGF-beta) resistance in the VACO-411 human colon carcinoma line. VACO-411 is unique for several reasons, including having a novel mutator phenotype and wild-type p53. Like many colon tumors, VACO-411 is not growth inhibited by TGF-beta. However, VACO-411 represents a subset of colon tumors that are resistant to TGF-beta-mediated growth inhibition, despite the expression of functional TGF-beta receptors. VACO-411 expresses cell surface TGF-beta receptor types I and II, and the coding regions of these receptors are wild type. To further characterize the nature of the VACO-411 defect, we fused VACO-411 with the human breast carcinoma line MCF-7. MCF-7 is also resistant to TGF-beta-mediated growth inhibition. However, unlike VACO-411, MCF-7 lacks cell surface expression of TGF-beta receptor type II, but does contain an intact postreceptor signaling pathway, as shown by regeneration of TGF-beta sensitivity following wild-type TGF-beta receptor type II transfection. In contrast to parental VACO-411 and MCF-7, the morphologically distinct cell hybrids were growth inhibited by TGF-beta. Therefore, the TGF-beta defect in VACO-411 is a postreceptor, loss-of-function mutation which can be genetically complemented. The data suggest that the VACO-411 defect in TGF-beta signaling will be able to be further complemented by microcell-mediated chromosome transfer.     

Predicting the Impact of Single-Nucleotide Polymorphisms in CDK2–Flavopiridol Complex by Molecular Dynamics Analysis

Cyclic-dependent kinase 2 (CDK2) is one of the primary protein kinases involved in the regulation of cell cycle progression. Flavopiridol is a flavonoid derived from an indigenous plant act as a potent antitumor drug showing increased inhibitory activity toward CDK2. The presence of deleterious variations in CDK2 may produce different effects in drug-binding adaptability. Studies on nsSNPs of CDK2 gene will provide information on the most likely variants associated with the disease. Furthermore, investigating the relationship between deleterious variants and its ripple effect in the inhibitory action with drug will provide fundamental information for the development of personalized therapies. In this study, we predicted four variants Y15S, V18L, P45L, and V69A of CDK2 as highly deleterious. Occurrence of these variations seriously affected the normal binding capacity of flavopiridol with CDK2. Analysis of 10-ns molecular dynamics (MD) simulation trajectories indicated that the predicted deleterious variants altered the CDK2 stability, flexibility, and surface area. Notably, we noticed the decrease in number of hydrogen bonds between CDK2 and flavopiridol mutant complexes in the whole dynamic period. Overall, this study explores the possible relationship between the CDK2 deleterious variants and the drug-binding ability with the help of molecular docking and MD approaches.     

Förster Energy Transfer in the Vicinity of Two Metallic Nanospheres (Dimer)

We present a detailed theoretical analysis of the Förster energy transfer process when a pair of molecules (donor and acceptor) is located nearby a cluster of two metallic nanospheres (dimer). We consider the case in which plasmonic resonances are within the overlap between the donor emission and acceptor absorption spectra, as well as the case that excludes such resonances from the aforementioned spectral overlap. Moreover, we explore the dependence of the Förster energy transfer rate on different dimer configurations (size and separation of nanospheres) and several dipole orientations of molecules. The dimer perturbs strongly the Förster energy transfer rate when plasmons are excited, donor dipole is oriented along the longitudinal axis of the dimer, and the radii of nanospheres and the sphere-gap distance are on the order of a few nanometers. In case of plasmonic excitation, the Förster energy transfer rate is degraded as the sphere-gap distance and size of the nanoparticles increase due to the dephasing of electronic motion arising from ohmic losses of metal. Also, we study the Förster efficiency influenced by the dimer, finding that the high efficiency region (delimited by the Förster radius curve) is reduced as a consequence of significant enhancement of the direct donor decay rate. Our study could impact applications that involve Förster energy transfer.     

Emi2-mediated Inhibition of E2-substrate Ubiquitin Transfer by the Anaphase-promoting Complex/Cyclosome through a D-Box–independent Mechanism

Vertebrate eggs are arrested at Metaphase II by Emi2, the meiotic anaphase-promoting complex/cyclosome (APC/C) inhibitor. Although the importance of Emi2 during oocyte maturation has been widely recognized and its regulation extensively studied, its mechanism of action remained elusive. Many APC/C inhibitors have been reported to act as pseudosubstrates, inhibiting the APC/C by preventing substrate binding. Here we show that a previously identified zinc-binding region is critical for the function of Emi2, whereas the D-box is largely dispensable. We further demonstrate that instead of acting through a “pseudosubstrate” mechanism as previously hypothesized, Emi2 can inhibit Cdc20-dependent activation of the APC/C substoichiometrically, blocking ubiquitin transfer from the ubiquitin-charged E2 to the substrate. These findings provide a novel mechanism of APC/C inhibition wherein the final step of ubiquitin transfer is targeted and raise the interesting possibility that APC/C is inhibited by Emi2 in a catalytic manner.