Zeng X.-L.,Huazhong Agricultural University |
Tang K.,Huazhong Agricultural University |
Zhou N.,Huazhong Agricultural University |
Zhou M.,Huazhong Agricultural University |
And 5 more authors.
Journal of the American Chemical Society | Year: 2013
The phycobilisomes of cyanobacteria and red-algae are highly efficient peripheral light-harvesting complexes that capture and transfer light energy in a cascade of excitation energy transfer steps through multiple phycobilin chromophores to the chlorophylls of core photosystems. In this work, we focus on the last step of this process by constructing simple functional analogs of natural phycobilisome-photosystem complexes that are based on bichromophoric protein complexes comprising a phycobilin- and a chlorophyll- or porphyrin-binding domain. The former is based on ApcE(1-240), the N-terminal chromophore-binding domain of the phycobilisome's LCM core-membrane linker, and the latter on HP7, a de novo designed four-helix bundle protein that was originally planned as a high-affinity heme-binding protein, analogous to b-type cytochromes. We fused a modified HP7 protein sequence to ApcEΔ, a water-soluble fragment of ApcE(1-240) obtained by excising a putative hydrophobic loop sequence of residues 77-153. HP7 was fused either to the N- or the C-terminus of ApcEΔ or inserted between residues 76 and 78, thereby replacing the native hydrophobic loop domain. We describe the assembly, spectral characteristics, and intramolecular excitation energy transfer of two unique systems: in the first, the short-wavelength absorbing zinc-mesoporphyrin is bound to the HP7 domain and serves as an excitation-energy donor to the long-wavelength absorbing phycocyanobilin bound to the ApcE domain; in the second, the short-wavelength absorbing phycoerythrobilin is bound to the ApcE domain and serves as an excitation energy donor to the long-wavelength absorbing zinc-bacteriochlorophyllide bound to the HP7 domain. All the systems that were constructed and tested exhibited significant intramolecular fluorescence resonance energy transfer with yields ranging from 21% to 50%. This confirms that our modular, covalent approach for studying EET between the cyclic and open chain tetrapyrroles is reasonable, and may be extended to larger structures mimicking light-harvesting in cyanobacteria. The design, construction, and characterization process demonstrated many of the advances in constructing such model systems, particularly in our ability to control the fold and aggregation state of protein-based systems. At the same time, it underlines the potential of exploiting the versatility and flexibility of protein-based systems in assembling multiple pigments into effective light-harvesting arrays and tuning the spectral properties of multichromophore systems. © 2013 American Chemical Society.
Bednarczyk D.,Weizmann Institute of Science |
Takahashi S.,Toho University |
Satoh H.,Toho University |
Noy D.,Migal Galilee Research Institute
Biochimica et Biophysica Acta - Bioenergetics | Year: 2015
The challenges involved in studying cofactor binding and assembly, as well as energy- and electron transfer mechanisms in the large and elaborate transmembrane protein complexes of photosynthesis and respiration have prompted considerable interest in constructing simplified model systems based on their water-soluble protein analogs. Such analogs are also promising templates and building blocks for artificial bioinspired energy conversion systems. Yet, development is limited by the challenge of introducing the essential cofactors of natural proteins that are highly water-insoluble into the water-soluble protein analogs. Here we introduce a new efficient method based on water-in-oil emulsions for overcoming this challenge. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four recombinant variants of the water-soluble chlorophyll-binding protein of Brassicaceae plants. We use the method to gain new insights into the protein-chlorophyll assembly process, and demonstrate its potential as a fast screening system for developing novel chlorophyll-protein complexes. © 2014 Elsevier B.V. All rights reserved.
Galili G.,Weizmann Institute of Science |
Amir R.,MIGAL Galilee Research Institute |
Fernie A.R.,Max Planck Institute of Molecular Plant Physiology
Annual Review of Plant Biology | Year: 2016
Although amino acids are critical for all forms of life, only proteogenic amino acids that humans and animals cannot synthesize de novo and therefore must acquire in their diets are classified as essential. Nine amino acids-lysine, methionine, threonine, phenylalanine, tryptophan, valine, isoleucine, leucine, and histidine-fit this definition. Despite their nutritional importance, several of these amino acids are present in limiting quantities in many of the world's major crops. In recent years, a combination of reverse genetic and biochemical approaches has been used to define the genes encoding the enzymes responsible for synthesizing, degrading, and regulating these amino acids. In this review, we describe recent advances in our understanding of the metabolism of the essential amino acids, discuss approaches for enhancing their levels in plants, and appraise efforts toward their biofortification in crop plants. Copyright © 2016 by Annual Reviews. All rights reserved.
Bednarczyk D.,Weizmann Institute of Science |
Noy D.,Migal Galilee Research Institute
Journal of Visualized Experiments | Year: 2016
Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the watersoluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes. © 2016 Journal of Visualized Experiments.
Stern R.A.,MIGAL Galilee Research Institute
Acta Horticulturae | Year: 2016
Small fruit size is a limiting factor in marketing apples (Malus × domestica). Several techniques have been used to improve this trait, among them blossom and fruit thinning with plant growth regulators such as auxins and cytokinins to reduce crop load. In the present study, carried out in 3 consecutive years (2011-2013), we evaluated the photosynthesis inhibitor metamitron (MM) as a thinner for 'Gala' apples. MM was applied twice at 150 g ha-1 (in the form of 0.085% Brevis®) at the 6- and 10-mm diameter stages, or once at 180 g ha-1 (0.1% Brevis® at 1,200 L ha-1) at the 6-mm stage, resulted in significant thinning. Consequently, the average time spent on hand thinning was reduced from 40-80 day ha-1 to 10-20 day ha-1. However, crop load was only slightly affected, depending on the year. In "ON" years, there was a considerable and significant shift to larger fruit size. The relatively low effective doses of MM, compared to those used in Europe and the USA, were likely to be due to the higher night temperatures for 3 weeks post application, which increased respiration and caused assimilation deficiencies during that critical period of fruit development. No negative effects were observed on the return bloom in the following year.
Smock R.G.,Weizmann Institute of Science |
Yadid I.,Migal Galilee Research Institute |
Dym O.,Weizmann Institute of Science |
Clarke J.,University of Cambridge |
Tawfik D.S.,Weizmann Institute of Science
Cell | Year: 2016
Summary Molecular evolution has focused on the divergence of molecular functions, yet we know little about how structurally distinct protein folds emerge de novo. We characterized the evolutionary trajectories and selection forces underlying emergence of β-propeller proteins, a globular and symmetric fold group with diverse functions. The identification of short propeller-like motifs (<50 amino acids) in natural genomes indicated that they expanded via tandem duplications to form extant propellers. We phylogenetically reconstructed 47-residue ancestral motifs that form five-bladed lectin propellers via oligomeric assembly. We demonstrate a functional trajectory of tandem duplications of these motifs leading to monomeric lectins. Foldability, i.e., higher efficiency of folding, was the main parameter leading to improved functionality along the entire evolutionary trajectory. However, folding constraints changed along the trajectory: initially, conflicts between monomer folding and oligomer assembly dominated, whereas subsequently, upon tandem duplication, tradeoffs between monomer stability and foldability took precedence. © 2016 The Authors.
Hacham Y.,Migal Galilee Research Institute |
Matityahu I.,Migal Galilee Research Institute |
Amir R.,Migal Galilee Research Institute |
Amir R.,Galilée College
Amino Acids | Year: 2013
The sulfur-containing essential amino acid methionine controls the level of important metabolites and processes in plants. In addition, methionine levels limit the nutritional quality of many crop plants. The level of methionine is regulated mainly by cystathionine γ-synthase (CGS), the first enzyme committed to its biosynthesis. Within our efforts to reveal factors that regulate CGS and methionine content in plants, we have analyzed how light regulates the transcript and protein level of Arabidopsis CGS (AtCGS). The expression of AtCGS is up-regulated in the light and reduced in the dark, independent of the diurnal cycle. Using tobacco plants overexpressing AtCGS, we have found that the light sensitive motives of the AtCGS gene are found within the coding sequence of AtCGS and not in its promoter, terminator or the untranslated regions of the gene. Sucrose can partially mimic the effect of light in dark grown plants while the addition of nitrogen and sulfur sources does not have any effect. The kinetics of the changes in the expression level of AtCGS suggest that its level can be maintained during extended darkness, or even when the sucrose content is reduced, such as during abiotic stresses. The up-regulation of AtCGS by light is in agreement with previous studies showing that other enzymes regulating the level of the carbon/amino skeleton and the sulfur group of Met, are up-regulated by light. The results indicate that light and dark participate in the regulation of the carbon/amino skeleton flux in the synthesis of amino acids of the aspartate family. © 2013 Springer-Verlag Wien.
Vaya J.,MIGAL Galilee Research Institute |
Vaya J.,Tel-Hai Academic College
Biochemical Pharmacology | Year: 2013
Human atherosclerotic plaque is composed of a large mixture of elements, predominantly lipids and oxidized lipids, lipid-loaded macrophages and smooth muscle cells, forming foam cells. Plaque contents undergo dynamic changes during the plaque's progression, being in a constant interaction with the circulating blood. During the mutual interaction between blood and plaque and the specific biochemical processes occurring in both, specific molecules can be generated in the serum which might provide information on plaque status. This information, mostly on plaque vulnerability, is highly important for making appropriate treatment decisions before neurological symptoms appear. The present review summarizes plaque contents, mostly lipids, oxidized lipids, oxidized products of cholesterol (oxysterols), and covers the recent literature on their association with biomarkers in the blood and on the possibility of using them for providing information on plaque status. © 2013 Elsevier Inc. All rights reserved.
Chen C.,Bar - Ilan University |
Letnik I.,Bar - Ilan University |
Hacham Y.,Migal Galilee Research Institute |
Hacham Y.,Galilée College |
And 6 more authors.
Plant Physiology | Year: 2014
A seed’s ability to properly germinate largely depends on its oxidative poise. The level of reactive oxygen species (ROS) in Arabidopsis (Arabidopsis thaliana) is controlled by a large gene network, which includes the gene coding for the hydrogen peroxide-scavenging enzyme, cytosolic ASCORBATE PEROXIDASE6 (APX6), yet its specific function has remained unknown. In this study, we show that seeds lacking APX6 accumulate higher levels of ROS, exhibit increased oxidative damage, and display reduced germination on soil under control conditions and that these effects are further exacerbated under osmotic, salt, or heat stress. In addition, ripening APX6-deficient seeds exposed to heat stress displayed reduced germination vigor. This, together with the increased abundance of APX6 during late stages of maturation, indicates that APX6 activity is critical for the maturation-drying phase. Metabolic profiling revealed an altered activity of the tricarboxylic acid cycle, changes in amino acid levels, and elevated metabolism of abscisic acid (ABA) and auxin in drying apx6 mutant seeds. Further germination assays showed an impaired response of the apx6 mutants to ABA and to indole-3-acetic acid. Relative suppression of abscisic acid insensitive3 (ABI3) and ABI5 expression, two of the major ABA signaling downstream components controlling dormancy, suggested that an alternative signaling route inhibiting germination was activated. Thus, our study uncovered a new role for APX6, in protecting mature desiccating and germinating seeds from excessive oxidative damage, and suggested that APX6 modulate the ROS signal cross talk with hormone signals to properly execute the germination program in Arabidopsis. © 2014 American Society of Plant Biologists. All rights reserved.
Wahadoszamen M.,VU University Amsterdam |
Wahadoszamen M.,University of Dhaka |
Margalit I.,Weizmann Institute of Science |
Margalit I.,Migal Galilee Research Institute |
And 4 more authors.
Nature Communications | Year: 2014
Understanding how specific protein environments affect the mechanisms of non-radiative energy dissipation within densely assembled chlorophylls in photosynthetic protein complexes is of great interest to the construction of bioinspired solar energy conversion devices. Mixing of charge-transfer and excitonic states in excitonically interacting chlorophylls was implicated in shortening excited states lifetimes, but its relevance to active control of energy dissipation in natural systems is under considerable debate. Here we show that the degree of fluorescence quenching in two similar pairs of excitonically interacting bacteriochlorophyll derivatives is directly associated with increasing charge-transfer character in the excited state, and that the protein environment may control non-radiative dissipation by affecting the mixing of charge-transfer and excitonic states. The capability of local protein environments to determine the fate of excited states, and thereby to confer different functionalities to excitonically coupled dimers substantiates the dimer as the basic functional element of photosynthetic enzymes. © 2015 Macmillan Publishers Limited. All rights reserved.