Characterization of chlorophyll binding to LIL3.
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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]
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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.
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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.).
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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.
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