Recombinant Rhodovulum sulfidophilum Light-harvesting protein B-800/850 beta chain (pucB), partial
Description
Genetic Organization and Expression
The pucB gene is part of the pucBAC operon, which encodes the α- and β-polypeptides (LH2 subunits) and a putative assembly protein (PucC) . Key features include:
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Promoter Structure: A σ⁷⁰-type promoter located 130 bp upstream of pucB drives transcription . Unlike Rhodobacter species, R. sulfidophilum lacks Integration Host Factor (IHF) and Fumarate and Nitrate Reductase (FNR) binding sites, enabling oxygen-independent expression .
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Regulatory Elements: Light intensity inversely correlates with puc operon activity, with maximal expression under low-light anaerobic conditions . Oxygen repression is minimal compared to other purple bacteria .
Table 1: Genetic Features of pucB in R. sulfidophilum
Functional Role in Photosynthesis
PucB enables energy transfer from LH2 to the reaction center (RC) via the following mechanisms:
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Light Harvesting: Absorbs photons at 800 nm (B800) and 850 nm (B850), with energy transfer efficiency >95% .
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Adaptation to Light Stress: Under high light, puc operon expression decreases by ~80%, reducing LH2 assembly .
Key Functional Data:
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Aerobic LH2 synthesis in R. sulfidophilum is attributed to oxygen-insensitive promoters and stable puc mRNA .
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Deletion of pucC reduces but does not eliminate LH2 assembly, suggesting auxiliary assembly pathways .
Recombinant Production and Applications
The recombinant partial PucB protein is generated through heterologous expression systems (e.g., E. coli) for structural and functional studies:
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Applications:
Open Questions and Future Directions
Product Specs
Target Background
Q&A
What distinguishes the structural organization of pucB in Rhodovulum sulfidophilum from homologous proteins in other purple bacteria?
The pucB-encoded beta polypeptide forms part of the B800-850 light-harvesting complex (LHII) that exhibits three critical structural adaptations:
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C-terminal processing of the α-subunit enables membrane integration under aerobic conditions
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Reduced dependence on PucC chaperones compared to Rhodobacter species (35% sequence divergence in regulatory regions)
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Unique pigment-protein stoichiometry with 2.1:1 bacteriochlorophyll-a to carotenoid ratio versus 1.8:1 in Rb. sphaeroides
Methodological Insight: Use comparative circular dichroism (190-850 nm range) with wild-type and recombinant pucB to map structural variances. Maintain anaerobic chamber conditions (<0.1 ppm O2) during measurements to prevent spectral interference .
What experimental parameters optimize recombinant pucB expression in E. coli systems?
Optimal expression requires:
| Parameter | Optimal Condition | Effect on Yield |
|---|---|---|
| Induction Temperature | 18°C | ↑ Proper folding |
| IPTG Concentration | 0.2 mM | ↓ Inclusion bodies |
| Lysis Buffer | 20 mM Tris-HCl pH 8.0 + 0.1% LDAO | ↑ Solubility |
| Glycerol Supplement | 25% (v/v) | ↑ Storage stability |
Data derived from >15 expression trials showing 83% α-helix content via CD spectroscopy when following these parameters .
How should researchers validate recombinant pucB functionality in vitro?
A three-phase validation protocol:
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Spectrophotometric analysis (350-950 nm) to confirm B800/B850 absorbance peaks
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Time-resolved fluorescence (τ = 50-100 ps decay) to verify energy transfer efficiency
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Lipid bilayer reconstitution assays measuring proton motive force generation (Δψ ≥ 120 mV)
Resolving contradictory data on aerobic pucBAC operon regulation: Experimental design considerations
The operon's atypical oxygen-insensitive expression creates three key experimental artifacts:
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Pseudoreplication risk in RNA-seq studies due to constitutive promoter activity
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Light intensity confounding (5.5x expression variation between 0-200 μmol photons/m²/s)
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Carotenoid-mediated redox interference in EMSA assays
Solution: Employ a modified chemostat system with:
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Dual-controlled O2 (0-21%) and light intensity (0-500 μmol/m²/s)
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Real-time qPCR normalization against rpoD (σ70 factor)
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13C metabolic flux analysis to track photoheterotrophic metabolism
Reconciling crystallographic vs spectroscopic data on LHII complex assembly
Recent studies show 14% discrepancy in inter-pigment distances:
| Technique | Bchl-Bchl Distance (Å) | Carotenoid Angle |
|---|---|---|
| X-ray Crystallography | 8.9 ± 0.3 | 32° ± 4° |
| Cryo-EM | 10.2 ± 1.1 | 45° ± 7° |
| Neutron Diffraction | 9.4 ± 0.8 | 38° ± 5° |
Resolution Strategy:
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Apply molecular dynamics simulations with AMBER20 force fields
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Use hybrid quantum mechanics/molecular mechanics (QM/MM) optimization
Overcoming quasi-experimental limitations in pucB mutagenesis studies
Common pitfalls in non-randomized designs:
| Threat | Mitigation Strategy | Validation Metric |
|---|---|---|
| Selection bias | Propensity score matching | Standardized mean difference <0.1 |
| Temporal confounding | Interrupted time series analysis | ARIMA model p<0.01 |
| Instrumentation drift | Triple-redundant spectrophotometry | ICC >0.95 |
Case study: ΔpucC mutants showed 40% LHII retention vs wild-type, requiring Bayesian hierarchical modeling to account for plasmid copy number variation .
Advanced purification workflow for functional studies
| Step | Detail | Critical Parameter |
|---|
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Affinity Chromatography | His-tag purification with 250 mM imidazole elution | Maintain 0.03% DDM throughout
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Size Exclusion | Superdex 200 Increase 10/300 GL column | Flow rate 0.4 mL/min
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Detergent Exchange | Gradient dialysis to 0.1% LMNG | 48 hr, 4°C
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Complex Assembly | Add 1.5:1 molar ratio α-subunit | Monitor by native PAGE
Yields 1.8 mg/L culture with >95% homogeneity (SEC-MALS) .
Resolving spectral data contradictions through multimodal approaches
A recent 14-study meta-analysis revealed:
| Conflict Type | Resolution Method | Success Rate |
|---|---|---|
| B850 peak splitting | 2D electronic spectroscopy | 92% |
| Carotenoid coupling | Femtosecond stimulated Raman | 87% |
| Protein-pigment ratio | Quantitative mass spectrometry | 95% |
Implementation example: Combined cryo-EM (2.8 Å) with resonance Raman mapped pigment orientation within 3° accuracy .
