Structural features of the diatom photosystem II-light-harvesting antenna complex.

In photosynthesis, light energy is captured by pigments bound to light-harvesting antenna proteins (LHC) that then transfer the energy to the photosystem (PS) cores to initiate photochemical reactions. The LHC proteins surround the PS cores to form PS-LHC supercomplexes. In order to adapt to a wide range of light environments, photosynthetic organisms have developed a large variety of pigments and antenna proteins to utilize the light energy efficiently under different environments. Diatoms are a group of important eukaryotic algae and possess fucoxanthin (Fx) chlorophyll a/c proteins (FCP) as antenna which have exceptional capabilities of harvesting blue-green light under water and dissipate excess energy under strong light conditions. We have solved the structure of a PSII-FCPII supercomplex from a centric diatom Chaetoceros gracilis by cryo-electron microscopy, and also the structure of an isolated FCP dimer from a pennate diatom Phaeodactylum tricornutum by X-ray crystallography at a high resolution. These results revealed the oligomerization states of FCPs distinctly different from those of LHCII found in the green lineage organisms, the detailed binding patterns of Chl c and Fxs, a huge pigment network, and extensive protein-protein, pigment-protein, and pigment-pigment interactions within the PSII-FCPII supercomplex. These results therefore provide a solid structural basis for examining the detailed mechanisms of the highly efficient energy transfer and quenching processes in diatoms.

Chlorophyll-carotenoid excitation energy transfer and charge transfer in Nannochloropsis oceanica for the regulation of photosynthesis.

Nonphotochemical quenching (NPQ) is a proxy for photoprotective thermal dissipation processes that regulate photosynthetic light harvesting. The identification of NPQ mechanisms and their molecular or physiological triggering factors under in vivo conditions is a matter of controversy. Here, to investigate chlorophyll (Chl)-zeaxanthin (Zea) excitation energy transfer (EET) and charge transfer (CT) as possible NPQ mechanisms, we performed transient absorption (TA) spectroscopy on live cells of the microalga Nannochloropsis oceanica We obtained evidence for the operation of both EET and CT quenching by observing spectral features associated with the Zea S1 and Zea●+ excited-state absorption (ESA) signals, respectively, after Chl excitation. Knockout mutants for genes encoding either violaxanthin de-epoxidase or LHCX1 proteins exhibited strongly inhibited NPQ capabilities and lacked detectable Zea S1 and Zea●+ ESA signals in vivo, which strongly suggests that the accumulation of Zea and active LHCX1 is essential for both EET and CT quenching in N. oceanica.

Using nanoBRET and CRISPR/Cas9 to monitor proximity to a genome-edited protein in real-time.

Bioluminescence resonance energy transfer (BRET) has been a vital tool for understanding G protein-coupled receptor (GPCR) function. It has been used to investigate GPCR-protein and/or -ligand interactions as well as GPCR oligomerisation. However the utility of BRET is limited by the requirement that the fusion proteins, and in particular the donor, need to be exogenously expressed. To address this, we have used CRISPR/Cas9-mediated homology-directed repair to generate protein-Nanoluciferase (Nluc) fusions under endogenous promotion, thus allowing investigation of proximity between the genome-edited protein and an exogenously expressed protein by BRET. Here we report BRET monitoring of GPCR-mediated ß-arrestin2 recruitment and internalisation where the donor luciferase was under endogenous promotion, in live cells and in real time. We have investigated the utility of CRISPR/Cas9 genome editing to create genome-edited fusion proteins that can be used as BRET donors and propose that this strategy can be used to overcome the need for exogenous donor expression.

Carotenoids in Microalgae.

Carotenoids are a class of isoprenoids synthesized by all photosynthetic organisms as well as by some non-photosynthetic bacteria and fungi with broad applications in food, feed and cosmetics, and also in the nutraceutical and pharmaceutical industries. Microalgae represent an important source of high-value products, which include carotenoids, among others. Carotenoids play key roles in light harvesting and energy transfer during photosynthesis and in the protection of the photosynthetic apparatus against photooxidative damage. Carotenoids are generally divided into carotenes and xanthophyls, but accumulation in microalgae can also be classified as primary (essential for survival) and secondary (by exposure to specific stimuli).In this chapter, we outline the high value carotenoids produced by commercially important microalgae, their production pathways, the improved production rates that can be achieved by genetic engineering as well as their biotechnological applications.

Origin of bimodal fluorescence enhancement factors of Chlorobaculum tepidum reaction centers on silver island films.

We focus on the spectral dependence of plasmon-induced enhancement of fluorescence of Chlorobaculum tepidum reaction centers. When deposited on silver island film, they exhibit up to a 60-fold increase in fluorescence. The dependence of enhancement factors on the excitation wavelength is not correlated with the absorption spectrum of the plasmonic structure. In particular, the presence of one (or multiple) trimers of the Fenna-Matthews-Olson (FMO) protein reveals itself in bimodal distribution of enhancement factors for the excitation at 589 nm, the wavelength corresponding to bacteriochlorophyll absorption of FMO and the core of the RC. We conclude that the structure of multichromophoric complexes can substantially affect the impact of plasmonic excitations, which is important in the context of assembling functional biohybrid systems.

Excitation energy transfer in Chlamydomonas reinhardtii deficient in the PSI core or the PSII core under conditions mimicking state transitions.

The efficient use of excitation energy in photosynthetic membranes is achieved by a dense network of pigment-protein complexes. These complexes fulfill specific functions and interact dynamically with each other in response to rapidly changing environmental conditions. Here, we studied how in the intact cells of Chlamydomonas reinhardtii (C.r.) the lack of the photosystem I (PSI) core or the photosystem II (PSII) core affects these interactions. To that end the mutants F15 and M18 (both PSI-deficient) and FUD7 (PSII-deficient) were incubated under conditions known to promote state transitions in wild-type. The intact cells were then instantly frozen to 77K and the full-spectrum time-resolved fluorescence emission of the cells was measured by means of streak camera. In the PSI-deficient mutants excitation energy transfer (EET) towards light-harvesting complexes of PSI (Lhca) occurs in less than 0.5 ns, and fluorescence from Lhca decays in 3.1 ns. Decreased trapping by PSII and increased fluorescence of Lhca upon state 1 (S1)→state 2 (S2) transition appears in the F15 and less in the M18 mutant. In the PSII-deficient mutant FUD7, quenched (0.5 ns) and unquenched (2 ns) light-harvesting complexes of PSII (LHCII) are present in both states, with the quenched form more abundant in S2 than in S1. Moreover, EET of 0.4 ns from the remaining LHCII to PSI increases upon S1→S2 transition. We relate the excitation energy kinetics observed in F15, M18 and FUD7 to the remodeling of the photosynthetic apparatus in these mutants under S1 and S2 conditions.

Equilibrium distributions of simple biochemical reaction systems for time-scale separation in stochastic reaction networks.

Many biochemical reaction networks are inherently multiscale in time and in the counts of participating molecular species. A standard technique to treat different time scales in the stochastic kinetics framework is averaging or quasi-steady-state analysis: it is assumed that the fast dynamics reaches its equilibrium (stationary) distribution on a time scale where the slowly varying molecular counts are unlikely to have changed. We derive analytic equilibrium distributions for various simple biochemical systems, such as enzymatic reactions and gene regulation models. These can be directly inserted into simulations of the slow time-scale dynamics. They also provide insight into the stimulus-response of these systems. An important model for which we derive the analytic equilibrium distribution is the binding of dimer transcription factors (TFs) that first have to form from monomers. This gene regulation mechanism is compared to the cases of the binding of simple monomer TFs to one gene or to multiple copies of a gene, and to the cases of the cooperative binding of two or multiple TFs to a gene. The results apply equally to ligands binding to enzyme molecules.

The architecture of Rhodobacter sphaeroides chromatophores.

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.

Internalization dissociates ß2-adrenergic receptors.

G protein-coupled receptors (GPCRs) self-associate as dimers or higher-order oligomers in living cells. The stability of associated GPCRs has not been extensively studied, but it is generally thought that these receptors move between the plasma membrane and intracellular compartments as intact dimers or oligomers. Here we show that ß(2)-adrenergic receptors (ß(2)ARs) that self-associate at the plasma membrane can dissociate during agonist-induced internalization. We use bioluminescence-resonance energy transfer (BRET) to monitor movement of ß(2)ARs between subcellular compartments. BRET between ß(2)ARs and plasma membrane markers decreases in response to agonist activation, while at the same time BRET between ß(2)ARs and endosome markers increases. Energy transfer between ß(2)ARs is decreased in a similar manner if either the donor- or acceptor-labeled receptor is mutated to impair agonist binding and internalization. These changes take place over the course of 30 minutes, persist after agonist is removed, and are sensitive to several inhibitors of arrestin- and clathrin-mediated endocytosis. The magnitude of the decrease in BRET between donor- and acceptor-labeled ß(2)ARs suggests that at least half of the receptors that contribute to the BRET signal are physically segregated by internalization. These results are consistent with the possibility that ß(2)ARs associate transiently with each other in the plasma membrane, or that ß(2)AR dimers or oligomers are actively disrupted during internalization.

Band shape heterogeneity of the low-energy chlorophylls of CP29: absence of mixed binding sites and excitonic interactions.

A number of spectroscopic characteristics of three almost isoenergetic, red-shifted chlorophylls (chls) in the PS II antenna complex CP29 are investigated with the aim of (i) determining whether their band shapes are substantially identical or not, (ii) addressing the topical problem of whether they are involved in excitonic interactions with other chls, and (iii) establishing whether their binding sites may be defined as "mixed" with respect to their capacity to bind chls a and b. The three chls A2-CHL612, A3-CHL613, and B3-CHL614 were analyzed after in vitro apoprotein-pigment reconstitution using the CP29 coding sequence from Arabidopsis thaliana for both the wild-type and mutant complexes. Difference spectra thermal broadening analyses indicated that the half-bandwidths varied between 12 and 15 nm (at room temperature), due mainly to differences in the optical reorganization energy (25-40 cm(-1)). Moreover, only the A2 chl displayed an intense vibrational band in the 300-600 cm(-1) interval from the 0-0 transition. We conclude that within the red absorbing (approximately 680 nm) antenna chls of a single chl-protein complex a marked spectral band shape heterogeneity exists. By analysis of the absorption and circular dichroism spectra no evidence was found of significantly strong excitonic interactions. The single gene mutation of the A3 and B3 binding sites causes absorption changes in both the long wavelength chl a absorbing region and in the chl b spectral region. This has previously been observed and was attributed to "mixed" chl a/b binding sites [Bassi, R., Croce, R., Cugini, D., and Sandona, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96,10056-10061]. This interpretation, while in principle not being unreasonable, is shown to be incorrect for these two chls.

A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

ApcD is necessary for efficient energy transfer from phycobilisomes to photosystem I and helps to prevent photoinhibition in the cyanobacterium Synechococcus sp. PCC 7002.

Phycobilisomes (PBS) are the major light-harvesting, protein-pigment complexes in cyanobacteria and red algae. PBS absorb and transfer light energy to photosystem (PS) II as well as PS I, and the distribution of light energy from PBS to the two photosystems is regulated by light conditions through a mechanism known as state transitions. In this study the quantum efficiency of excitation energy transfer from PBS to PS I in the cyanobacterium Synechococcus sp. PCC 7002 was determined, and the results showed that energy transfer from PBS to PS I is extremely efficient. The results further demonstrated that energy transfer from PBS to PS I occurred directly and that efficient energy transfer was dependent upon the allophycocyanin-B alpha subunit, ApcD. In the absence of ApcD, cells were unable to perform state transitions and were trapped in state 1. Action spectra showed that light energy transfer from PBS to PS I was severely impaired in the absence of ApcD. An apcD mutant grew more slowly than the wild type in light preferentially absorbed by phycobiliproteins and was more sensitive to high light intensity. On the other hand, a mutant lacking ApcF, which is required for efficient energy transfer from PBS to PS II, showed greater resistance to high light treatment. Therefore, state transitions in cyanobacteria have two roles: (1) they regulate light energy distribution between the two photosystems; and (2) they help to protect cells from the effects of light energy excess at high light intensities.

[Molecular responses of photosynthetic apparatus of plants to long term irradiance changes]. / Molekularne odpowiedzi aparatu fotosyntetycznego roslin na dlugoterminowe zmiany natezenia swiatla.

In response to long term (at least 1-3 h) irradiance changes the responses are elicited at the level of structure and function of photosynthetic apparatus of plants which are thought to be aimed to keep the balance between the level of excitation energy funneled to the reaction centers of the photosystems by energetic antennae and the utilization of this energy in the form of photosynthetic electron transfer and dark reactions. At high vs medium irradiances the rate of excitation energy transfer via LHCII is reduced while the rate of electron flow and photosynthetic dark reactions is increased. The reaction at LHCII level stems from the reduction of its pool per PSII reaction center and the regulatory events comprise changes in the expression of LHCII apoproteins and/or chi b biosynthesis. The basis for higher electron flow capabilities lies in significant increases in the content of some electron carriers and the catalytic activity of ATP synthase. The upregulation of photosynthetic dark reaction in turn is due to the activation of signaling pathways leading to the increase in the pool and catalytic activities of rubisco and other Calvin cycle enzymes.

Exploring the energy landscape of the genetic code.

New insights into the arrangement of the genetic code table, based on the analysis of the physico-chemical properties of its molecular constituents, are reported in this paper. It will be demonstrated that the code has a twofold symmetry that is not apparent from the conventional code table, but becomes apparent when the codon-anticodon energies are listed for each triplet. The evolutionary development of the current code based on single base replacement mutations (transitions) from an 'iso-energetic' degenerated subset of 16 of the 64 codons is discussed. The energy landscape of all 64 codons is presented. A detailed analysis of the energy changes due to mutations in the 3rd, 1st or 2nd position of a codon reveals that the modern genetic code is highly robust. Changes come in small discrete steps that can be quantified in relation to the thermal noise of the system. The relation of the individual codon to its neighbours in the rearranged codon table can be completely understood based on thermodynamic considerations.

Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round?

It is doubtful that diffusion can deliver sufficient ATP from the mitochondria to sustain activity at the distal end of the sperm flagellum. Glycolytic enzymes bound to the fibrous sheath could provide energy along the flagellum at the point it is required. An obligatory role for glycolysis is supported by the lack of progressive motility in sperm from mice where the gene for sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDHs) had been 'knocked out'. Here, I review some evidence against this idea. First, pure diffusion from the mitochondrion is likely to be adequate in species with smaller sperm, and it is possible that rapid ATP delivery required in larger sperm could be achieved by an adenylate kinase shuttle. Second, experience with alpha-chlorohydrin demonstrates that sperm can remain motile with normal ATP concentrations despite inhibition of GAPDHs; adverse effects only occur if glucose is added and high levels of glycolytic intermediates accumulate. These observations undermine the GAPDHs knockout mouse as evidence for an essential role of local glycolysis. Third, sperm from many species can remain motile for long periods in sugar-free media and excepting dog sperm, evidence that gluconeogenesis is a possible explanation, is weak. In most species, it is unlikely that local glycolysis is the only way that ATP can be supplied to the distal flagellum.

A combined approach for locating box H/ACA snoRNAs in the human genome.

A novel combined method for locating box H/ACA small nucleolar RNAs (snoRNAs) is described, together with a software tool. The method adopts both a probabilistic hidden Markov model (HMM) and a minimum free energy (MFE) rule, and filters possible candidate box H/ACA snoRNAs obtained from genomic DNA sequences. With our novel method 12 known box H/ACA snoRNAs, and one strong candidate were identified in 30 nucleolar protein genomic sequences.

Thermosynthesis as energy source for the RNA World: a model for the bioenergetics of the origin of life.

The thermosynthesis concept, biological free energy gain from thermal cycling, is combined with the concept of the RNA World. The resulting overall origin of life model suggests new explanations for the emergence of the genetic code and the ribosome. It is proposed that the first protein named pF(1) obtained the energy to support the RNA World by a thermal variation of F(1) ATP synthase's binding change mechanism. It is further proposed that this pF(1) was the single translation product during the emergence of the genetic machinery. During thermal cycling pF(1) condensed many substrates with broad specificity, yielding NTPs and randomly constituted protein and RNA libraries that contained self-replicating RNA. The smallness of pF(1) permitted the emergence of the genetic machinery by selection of RNA that increased the fraction of pF(1)s in the protein library: (1) an amino acids concatenating progenitor of rRNA bound to (2) a chain of 'positional tRNAs' linked by mutual recognition, and yielded a pF(1) (or its main motif); this positional tRNA set gradually evolved to a set of regular tRNAs functioning according to the genetic code, with concomitant emergence of (3) an mRNA coding for pF(1).

Quenching of chlorophyll triplet states by carotenoids in reconstituted Lhca4 subunit of peripheral light-harvesting complex of photosystem I.

In this study, triplet quenching, the major photoprotection mechanism in antenna proteins, has been studied in the light-harvesting complex of photosystem I (LHC-I). The ability of carotenoids bound to LHC-I subunit Lhca4, which is characterized by the presence of the red-most absorption components at wavelength >700 nm, to protect the system through quenching of the chlorophyll triplet states, has been probed, by analyzing the induction of carotenoid triplet formation. We have investigated this process at low temperature, when the funneling of the excitation toward the low-lying excited states of the Chls is stronger, by means of optically detected magnetic resonance (ODMR), which is well-suited for investigation of triplet states in photosynthetic systems. The high selectivity and sensitivity of the technique has made it possible to point out the presence of specific interactions between carotenoids forming the triplet states and specific chlorophylls characterized by red-shifted absorption, by detection of the microwave-induced Triplet minus Singlet (T-S) spectra. The effect of the red forms on the efficiency of triplet quenching was specifically probed by using the Asn47His mutant, in which the red forms have been selectively abolished (Morosinotto, T., Breton, J., Bassi, R., and Croce, R. (2003) J. Biol. Chem. 278, 49223-49229). Lack of the red forms yields into a reduced efficiency of the triplet quenching in LHC-I thus suggesting that the "red Chls" play a role in enhancing triplet quenching in LHC-I and, possibly, in the whole photosystem I.

Real-time measurement in living cells of insulin-like growth factor activity using bioluminescence resonance energy transfer.

Insulin-like growth factor (IGF)-I and -II function in normal physiology to control growth, development, and differentiation, but are also important in pathophysiological conditions, particularly in cancer. The biological effects of the IGFs are mediated by the IGF-I receptor (IGFR), a covalent homodimer composed of two alpha and two beta chains, similar in structure to the insulin receptor (IR). To allow measurement of the stimulation of IGFR in living cells, we developed an assay based on bioluminescence resonance energy transfer (BRET) between a donor molecule, Renilla luciferase, and an acceptor fluorophore, enhanced yellow fluorescent protein (EYFP). Initial attempts based on fusion of the luciferase to IGFR, and EYFP to IGFR, or to downstream signaling molecules, insulin receptor substrate-1 (IRS1) or protein tyrosine phosphatases-1B (PTP-1B), failed. However, similar experiments with IR, carried our in parallel, proved successful. We therefore, constructed assays based on chimeric IGFR/IR proteins, in which the ligand binding site was derived from IGFR. With the most efficient assay, in which the luciferase is fused to a chimeric receptor with the entire intracellular portion derived from IR, and EYFP fused to PTP-1B, IGF activity was measured specifically with sensitivity similar to the corresponding assay for insulin, based on IR. The established system allows efficient evaluation of candidate ligand- or receptor-directed molecules for the modulation of IGF activities. Furthermore, we demonstrate that a set of inhibitory IGF binding proteins (IGFBPs) or activating IGFBP-specific proteinases, unique to the IGF system, may serve as potential targets. In addition to screening, real-time measurement of IGFR stimulation may be important in efforts to understand the kinetics of receptor stimulation, in particular differences between IGFR and IR.

Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II.

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.