Recombinant Rhodocyclus tenuis Light-harvesting polypeptide B-800/860 alpha chain, partial
Description
Introduction to the B-800/860 Alpha Chain
The B-800/860 complex (LH2) in Rhodocyclus tenuis consists of α- and β-polypeptides that non-covalently bind bacteriochlorophyll (BChl) a and carotenoids. The α-chain plays a critical role in stabilizing the arrangement of BChl molecules, particularly those absorbing at 800 nm and 860 nm wavelengths . Recombinant versions of this polypeptide enable detailed biochemical and biophysical studies without requiring native membrane extraction.
Primary Sequence
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The native α-subunit from Rhodocyclus tenuis has been purified using reverse-phase HPLC and sequenced via micro-sequencing and mass spectrometry .
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Partial recombinant sequences likely retain conserved regions critical for pigment binding, such as histidine residues coordinating BChl’s Mg²⁺ .
Tertiary Structure and Pigment Binding
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The α-chain forms a transmembrane helix, creating a scaffold for BChl a and carotenoids.
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Each αβ-dimer binds 3 BChl a molecules (2 B860, 1 B800) and 1.5 lycopenes in Rhodocyclus tenuis . Resonance Raman spectroscopy confirms a 5-coordinated Mg²⁺ in BChl, critical for spectral tuning .
Energy Transfer Mechanism
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The B-800/860 complex absorbs light at 800 nm (B800) and transfers energy to B860, which funnels it to the core LH1-reaction center complex .
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Sequence similarities between Rhodocyclus tenuis α-chains and LH1 complexes (e.g., B870) suggest evolutionary conservation of energy transfer efficiency .
Genetic and Metabolic Context
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Genomic analyses of Rhodocyclus tenuis strains (DSM 109, DSM 110) reveal conserved operons encoding LH2 proteins, alongside cytochrome genes for electron transport .
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Unlike Rhodocyclus purpureus, R. tenuis retains genes for high-potential iron-sulfur proteins (HiPIP), which facilitate cyclic electron transfer .
Expression and Purification
Applications in Biotechnology
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Engineered LH2 complexes are studied for solar energy harvesting and nanotechnology. For example, Rhodococcus spp. (related to Rhodocyclus) are leveraged for enzymatic catalysis, suggesting potential hybrid systems .
Table 2: Functional Differentiation of LH2 Complexes
Open Research Questions
Product Specs
Target Background
Q&A
Basic Research Questions
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What is the structural organization and function of the Light-harvesting polypeptide B-800/860 alpha chain in Rhodocyclus tenuis?
The B-800/860 alpha chain is a critical component of the peripheral light-harvesting complex in Rhodocyclus tenuis, functioning primarily to bind bacteriochlorophyll a (BChl a) and carotenoid pigments. This polypeptide, similar to those studied in other purple bacteria, forms part of a transmembrane protein complex that captures light energy and transfers it to photosynthetic reaction centers with remarkable efficiency .
The structural organization likely resembles that of similar light-harvesting complexes in other purple bacteria, containing a single transmembrane alpha-helix with specific amino acid residues that coordinate the binding of bacteriochlorophyll molecules. Based on studies of similar systems, the B-800/860 designation refers to the absorption maxima of the bacteriochlorophyll molecules when properly bound to the protein complex, with peaks at approximately 800 and 860 nm .
In comparable light-harvesting complexes, such as the B800-850 complex in Rhodopseudomonas capsulata, chemical cross-linking studies have revealed that these polypeptides are organized in clusters containing at least four of each polypeptide species, forming both homo-oligomers and hetero-oligomers . This specific spatial arrangement is crucial for efficient energy transfer between the pigment molecules.
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How do light-harvesting complexes from Rhodocyclus tenuis compare with those of other photosynthetic bacteria?
Light-harvesting complexes across different purple photosynthetic bacteria share fundamental structural and functional similarities while exhibiting species-specific variations. The table below compares key characteristics of light-harvesting complexes from several purple bacteria:
| Characteristic | Rhodocyclus tenuis | Rhodopseudomonas capsulata | Rhodobacter sphaeroides |
|---|---|---|---|
| Complex designation | B-800/860 | B800-850 | LH2 (B800-850) |
| Polypeptide components | α and β chains | Three polypeptides (Mr 8000, 10000, 14000) | α and β polypeptides |
| Pigment association | BChl a and carotenoids | BChl a and carotenoids with two smaller polypeptides | BChl a and carotenoids |
| Oligomeric structure | Likely ring-like arrangement | Clusters with ≥4 of each polypeptide | Ring of 8-9 αβ-heterodimers |
| Membrane association | Integral membrane proteins | Integral membrane proteins | Integral membrane proteins |
Recent comparative genomic analysis reveals significant heterogeneity among strains previously all assigned to Rhodocyclus tenuis, with some strains now reclassified as a new species, Rhodocyclus gracilis . This genomic diversity likely extends to variations in their light-harvesting complex components and may explain functional differences observed among strains.
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What expression systems and methodologies are recommended for recombinant production of the B-800/860 alpha chain?
Successful expression of membrane proteins like the B-800/860 alpha chain requires careful consideration of expression systems and methodologies. Although the search results don't specifically address recombinant production of this particular protein, insights can be drawn from similar studies:
Recommended Expression Systems:
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Escherichia coli: The successful overproduction of other Rhodocyclus tenuis proteins (such as HiPIP) in E. coli demonstrates compatibility . For membrane proteins, specialized strains like C41(DE3), C43(DE3), or BL21-AI are recommended to minimize toxicity issues.
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Photosynthetic bacterial hosts: Expression in a related purple bacterium lacking endogenous light-harvesting complexes may provide the native-like membrane environment and accessory factors needed for proper assembly.
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Cell-free expression systems: These provide better control over the reaction environment and can be coupled with direct incorporation into nanodiscs or liposomes.
Methodological Considerations:
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Construct design: Include appropriate affinity tags for purification while ensuring they don't interfere with protein folding or pigment binding. A cleavable tag is often preferable.
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Co-expression strategies: Consider co-expressing the alpha and beta chains together, as they form a functional heterodimer in the native complex. This may improve stability and folding.
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Induction conditions: Low temperature (16-20°C) and reduced inducer concentrations often improve the yield of correctly folded membrane proteins.
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Pigment supplementation: For functional reconstitution, bacteriochlorophyll and carotenoids must be available either through co-expression in a photosynthetic host or addition during purification/reconstitution.
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Membrane extraction: Careful selection of detergents is critical; in studies of similar complexes, dodecyl dimethylamine-N-oxide has been shown to maintain the native arrangement of polypeptides .
