Characterization of chlorophyll binding to LIL3.
Ontology highlight
ABSTRACT:
SUBMITTER: Mork-Jansson AE
PROVIDER: S-EPMC5794176 | biostudies-literature | 2018
REPOSITORIES: biostudies-literature
Publications
Characterization of chlorophyll binding to LIL3.
Mork-Jansson Astrid Elisabeth AE Eichacker Lutz Andreas LA
PloS one 20180201 2
The light harvesting like protein 3 (LIL 3) from higher plants, has been linked to functions in chlorophyll and tocopherol biosynthesis, photo-protection and chlorophyll transfer. However, the binding of chlorophyll to LIL3 is unclear. We present a reconstitution protocol for chlorophyll binding to LIL3 in DDM micelles. It is shown in the absence of lipids and carotenoids that reconstitution of chlorophyll binding to in vitro expressed LIL3 requires pre-incubation of reaction partners at room te ...[more]
Similar Datasets
Project description:Background:The function of proteins is at large determined by cofactors selectively bound to protein structure. Without chlorophyll specifically bound to protein, light harvesting and photosynthesis would not be possible. The binding of chlorophyll to light harvesting proteins has been extensively studied in reconstitution assays using proteins expressed in vitro; however, the mechanism of the reconstitution reaction remained unclear. We have shown that membrane integral light-harvesting-like protein, LIL3, binds chlorophyll a with a Kd of 146 nM in vitro by thermophoresis. Here, reconstitution of chlorophyll binding to LIL3 has been characterized by four different methods. Results:Structural changes in the reconstitution process have been investigated by light-scattering and differential Trp-fluorescence. For characterization of the chlorophyll binding site at LIL3, the analysis of LIL3 mutants has been conducted using native PAGE and thermophoresis. We find that the oxidized state of dithiothreitol is the essential component for reconstitution of chlorophyll binding to LIL3 in n-Dodecyl ?-d-maltoside micelles at RT. Chlorophyll increased the polydispersity of the micellar states while dithiothreitol maintained LIL3 in a partially unfolded state at RT. Dimerization of LIL3 was abolished if amino acids N174, R176, and E171 were mutated to Ala; while, chlorophyll binding to LIL3 was abolished in mutant N174A, but retained in E171A, and R176A albeit at an about six- and five-fold decreased dissociation constant. Results show that N174 of LIL3 is essential for binding chlorophyll a. Conclusions:Chlorophyll binding to LIL3 can be shown by thermophoresis, and native gel electrophoresis, while analysis of reconstitution conditions by dynamic light scattering and differential scanning fluorometry are of critical importance for method optimization.
| S-EPMC6320596 | biostudies-literature
Project description:The light-harvesting-like (LIL) proteins are a family of membrane proteins that share a chlorophyll a/b-binding motif with the major light-harvesting antenna proteins of oxygenic photoautotrophs. LIL proteins have been associated with the regulation of tetrapyrrol biosynthesis, and plant responses to light-stress. Here, it was found in a native PAGE approach that chlorophyllide, and chlorophyllide plus geranylgeraniolpyrophosphate trigger assembly of Lil3 in three chlorine binding fluorescent protein bands, termed F1, F2, and F3. It is shown that light and chlorophyllide trigger accumulation of protochlorophyllide-oxidoreductase, and chlorophyll synthase in band F3. Chlorophyllide and chlorophyll esterified to geranylgeraniol were identified as basis of fluorescence recorded from band F3. A direct interaction between Lil3, CHS and POR was confirmed in a split ubiquitin assay. In the presence of light or chlorophyllide, geranylgeraniolpyrophosphate was shown to trigger a loss of the F3 band and accumulation of Lil3 and geranylgeranyl reductase in F1 and F2. No direct interaction between Lil3 and geranylgeraniolreductase was identified in a split ubiquitin assay; however, accumulation of chlorophyll esterified to phytol in F1 and F2 corroborated the enzymes assembly. Chlorophyll esterified to phytol and the reaction center protein psbD of photosystem II were identified to accumulate together with psb29, and APX in the fluorescent band F2. Data show that Lil3 assembles with proteins regulating chlorophyll synthesis in etioplasts from barley (Hordeum vulgare L.).
| S-EPMC4501709 | biostudies-literature
Project description:The light-harvesting complex (LHC) constitutes the major light-harvesting antenna of photosynthetic eukaryotes. LHC contains a characteristic sequence motif, termed LHC motif, consisting of 25-30 mostly hydrophobic amino acids. This motif is shared by a number of transmembrane proteins from oxygenic photoautotrophs that are termed light-harvesting-like (LIL) proteins. To gain insights into the functions of LIL proteins and their LHC motifs, we functionally characterized a plant LIL protein, LIL3. This protein has been shown previously to stabilize geranylgeranyl reductase (GGR), a key enzyme in phytol biosynthesis. It is hypothesized that LIL3 functions to anchor GGR to membranes. First, we conjugated the transmembrane domain of LIL3 or that of ascorbate peroxidase to GGR and expressed these chimeric proteins in an Arabidopsis mutant lacking LIL3 protein. As a result, the transgenic plants restored phytol-synthesizing activity. These results indicate that GGR is active as long as it is anchored to membranes, even in the absence of LIL3. Subsequently, we addressed the question why the LHC motif is conserved in the LIL3 sequences. We modified the transmembrane domain of LIL3, which contains the LHC motif, by substituting its conserved amino acids (Glu-171, Asn-174, and Asp-189) with alanine. As a result, the Arabidopsis transgenic plants partly recovered the phytol-biosynthesizing activity. However, in these transgenic plants, the LIL3-GGR complexes were partially dissociated. Collectively, these results indicate that the LHC motif of LIL3 is involved in the complex formation of LIL3 and GGR, which might contribute to the GGR reaction.
| S-EPMC3887221 | biostudies-literature
Project description:The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in ?-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.
| S-EPMC2944722 | biostudies-literature
Project description:BACKGROUND:Phytyl residues are the common side chains of chlorophyll (Chl) and tocopherols. Geranylgeranyl reductase (GGR), which is encoded by CHLP gene, is responsible for phytyl biosynthesis. The light-harvesting like protein LIL3 was suggested to be required for stability of GGR and protochlorophyllide oxidoreductase in Arabidopsis. RESULTS:In this study, we isolated a yellow-green leaf mutant, 637ys, in rice (Oryza sativa). The mutant accumulated majority of Chls with unsaturated geranylgeraniol side chains and displayed a yellow-green leaf phenotype through the whole growth period. The development of chloroplasts was suppressed, and the major agronomic traits, especially No. of productive panicles per plant and of spikelets per panicle, dramatically decreased in 637ys. Besides, the mutant exhibited to be sensitive to light intensity and deficiency of tocopherols without obvious alteration in tocotrienols in leaves and grains. Map-based cloning and complementation experiment demonstrated that a point mutation on the OsLIL3 gene accounted for the mutant phenotype of 637ys. OsLIL3 is mainly expressed in green tissues, and its encoded protein is targeted to the chloroplast. Furthermore, the 637ys 502ys (lil3 chlp) double mutant exclusively accumulated geranylgeranyl Chl and exhibited lethality at the three-leaf stage. CONCLUSIONS:We identified the OsLIL3 gene through a map-based cloning approach. Meanwhile, we demonstrated that OsLIL3 is of extreme importance to the function of OsGGR, and that the complete replacement of phytyl side chain of chlorophyll by geranylgeranyl chain could be fatal to plant survival in rice.
| S-EPMC6819399 | biostudies-literature
Project description:The cyclic tetrapyrroles, viz. chlorophylls (Chl), their bacterial analogs bacteriochlorophylls, and hemes are ubiquitous cofactors of biological catalysis that are involved in a multitude of reactions. One systematic approach for understanding how Nature achieves functional diversity with only this handful of cofactors is by designing de novo simple and robust protein scaffolds with heme and/or (bacterio)chlorophyll [(B)Chls]-binding sites. This strategy is currently mostly implemented for heme-binding proteins. To gain more insight into the factors that determine heme-/(B)Chl-binding selectivity, we explored the geometric parameters of (B)Chl-binding sites in a nonredundant subset of natural (B)Chl protein structures. Comparing our analysis to the study of a nonredundant database of heme-binding helical histidines by Negron et al. (Proteins 2009;74:400-416), we found a preference for the m-rotamer in (B)Chl-binding helical histidines, in contrast to the preferred t-rotamer in heme-binding helical histidines. This may be used for the design of specific heme- or (B)Chl-binding sites in water-soluble helical bundles, because the rotamer type defines the positioning of the bound cofactor with respect to the helix interface and thus the protein-binding site. Consensus sequences for (B)Chl binding were identified by combining a computational and database-derived approach and shown to be significantly different from the consensus sequences recommended by Negron et al. (Proteins 2009;74:400-416) for heme-binding helical proteins. The insights gained in this work on helix- (B)Chls-binding pockets provide useful guidelines for the construction of reasonable (B)Chl-binding protein templates that can be optimized by computational tools.
| S-EPMC6298425 | biostudies-literature
Project description:Chlorophyll degradation by aerobic marine bacterium
| PRJEB15523 | ENA
Project description:Type-II water-soluble chlorophyll (Chl) proteins (WSCPs) of Brassicaceae are promising models for understanding how protein sequence and structure affect Chl binding and spectral tuning in photosynthetic Chl-protein complexes. However, to date, their use has been limited by the small number of known WSCPs, which also limited understanding their physiological roles. To overcome these limitations, we performed a phylogenetic analysis to compile a more comprehensive and complete set of natural type-II WSCP homologues. The identified homologues were heterologously expressed in Escherichia coli, purified, tested for assembly with chlorophylls, and spectroscopically characterized. The analyses led to the discovery of previously unrecognized type-IIa and IIb subclass WSCPs, as well as of a new subclass that did not bind chlorophylls. Further analysis by ancestral sequence reconstruction yielded sequences of putative ancestors of the three subclasses, which were subsequently recombinantly expressed in E. coli, purified and characterized. Combining the phylogenetic and spectroscopic data with molecular structural information revealed distinct Chl-binding motifs, and identified residues critically impacting spectral tuning. The distinct Chl-binding properties of the WSCP archetypes suggest that the non-Chl-binding subclass evolved from a Chl-binding ancestor that most likely lost its Chl-binding capacity upon localization in the plant tissues with low Chl content. This dual evolutionary trajectory is consistent with WSCPs association with the Kunitz-type protease inhibitors superfamily, and indications of their inhibitory activity in response to various forms of stress in plants. These findings suggest new directions for exploring the physiological roles of WSCPs and the correlation, if any, between Chl-binding and protease inhibition functionality.
| S-EPMC7054047 | biostudies-literature
Project description:In this study, two chlorophyll A/B binding protein (CAB) genes (CsCP1 and CsCP2) in tea plant were cloned. The proteins encoded by these genes belong to the external or internal antenna proteins of PS II, respectively. They may be the targets of physiological regulation for tea leaf cell PS II because they all contain multiple functional domains and modifiable sites. The CAB gene family in the tea genome consists of 25 homologous genes. We measured the expression patterns of ten genes in the CsCP1 and CsCP2 subfamily under six different stresses. CsCP1 expression was inhibited in response to 6 kinds of stress; CsCP2 expression was slightly upregulated only after cold stress and ABA treatment. However, the expression levels of CSA016997 and CSA030476 were upregulated significantly in the six stresses. The results suggested that the 10 CAB genes may have different functions in tea leaves. Moreover, changes in the expression of the 10 genes under stress appear to be related to ABA- and MeJA-dependent signalling pathways, and their responses to MeJA treatment is faster than those to ABA. In addition, we introduced our experiences for cloning the genes in the context of complex genomes.
| S-EPMC7067855 | biostudies-literature
Project description:Chlorophyll is an indispensable constituent of the photosynthetic machinery in green organisms. Bound by apoproteins of photosystems I and II, chlorophyll performs light-harvesting and charge separation. Due to the phototoxic nature of free chlorophyll and its precursors, chlorophyll synthesis is regulated to comply with the availability of nascent chlorophyll-binding apoproteins. Conversely, the synthesis and co-translational insertion of such proteins into the thylakoid membrane have been suggested to be influenced by chlorophyll availability. In this study, we addressed these hypotheses by using ribosome profiling to examine the synthesis and membrane targeting of chlorophyll-binding apoproteins in chlorophyll-deficient chlH maize mutants (Zm-chlH). ChlH encodes the H subunit of the magnesium chelatase (also known as GUN5), which catalyzes the first committed step in chlorophyll synthesis. Our results show that the number and distribution of ribosomes on plastid mRNAs encoding chlorophyll-binding apoproteins are not substantially altered in Zm-chlH mutants, suggesting that chlorophyll has no impact on ribosome dynamics. Additionally, a Zm-chlH mutation does not change the amino acid position at which nascent chlorophyll-binding apoproteins engage the thylakoid membrane, nor the efficiency with which membrane-engagement occurs. Together, these results provide evidence that chlorophyll availability does not selectively activate the translation of plastid mRNAs encoding chlorophyll apoproteins. Our results imply that co- or post-translational proteolysis of apoproteins is the primary mechanism that adjusts apoprotein abundance to chlorophyll availability in plants.
| S-EPMC5368244 | biostudies-literature