Recombinant Gluconobacter oxydans 30S ribosomal protein S16 (rpsP)
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Q&A
What expression systems are optimal for producing recombinant Gluconobacter oxydans S16 (rpsP) with high yield and purity?
Methodological Answer:
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Host Selection: E. coli BL21(DE3) is the most widely used system due to its compatibility with T7 promoters and high protein yield (≥85% purity via SDS-PAGE) .
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Vector Design: Use pET-15b or pET-28a vectors for N-terminal His-tag fusion, enabling immobilized metal affinity chromatography (IMAC) purification .
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Induction Optimization: Induction at OD600 = 0.6–0.8 with 0.1–0.5 mM IPTG at 16–20°C minimizes inclusion body formation. For G. oxydans S16, solubility increases at lower temperatures due to reduced misfolding .
Critical Data Table:
| Expression System | Yield (mg/L) | Purity (%) | Ref. |
|---|---|---|---|
| E. coli BL21(DE3) | 12–18 | ≥90 | |
| E. coli Rosetta2 | 8–10 | ≥85 | |
| Baculovirus (Sf9) | 3–5 | ≥80 |
How do refolding conditions affect the secondary structure stability of recombinant S16?
Methodological Answer:
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Denaturation Protocol: Use 6 M urea or 4 M guanidine-HCl to solubilize inclusion bodies.
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Refolding Buffer: 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM DTT, and 0.4 M urea gradient dialysis over 24–48 hours .
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Structural Validation: Circular dichroism (CD) spectroscopy confirms α-helix (21 ± 4%) and β-strand (24 ± 3%) content. Stability is pH-dependent: denaturation occurs rapidly above pH 8.0 .
Key Finding:
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Refolded S16 retains <80% native conformation at pH 6.0–7.5 but loses tertiary structure at pH >8.0 .
What functional assays confirm ribosomal incorporation of recombinant S16?
Methodological Answer:
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Ribosome Reconstitution: Combine S16 with G. oxydans 16S rRNA fragments (5′ domain) and primary assembly proteins (S4, S17, S20) under physiological Mg²⁺ (5–10 mM) .
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Hydroxyl Radical Footprinting: Detect rRNA protection patterns at helix 15 (nt 481–483) and helix 18 (nt 505–507), which stabilize pseudoknots in the 30S decoding center .
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Activity Assay: Measure poly(U)-directed polyphenylalanine synthesis in a cell-free translation system .
How does S16 drive conformational switches during 30S ribosomal subunit assembly?
Methodological Answer:
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Kinetic Trapping Experiments: Time-resolved hydroxyl radical footprinting at 2–30 mM Mg²⁺ reveals two intermediates:
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Cooperativity Analysis: S16 increases folding cooperativity by shifting ΔG‡ for helix 15–17 interactions from +4.9 kcal/mol (S4/S17/S20 alone) to +2.4 kcal/mol .
Data Highlights:
| Parameter | S4/S17/S20 | S4/S17/S20/S16 |
|---|---|---|
| Helix 15–17 Stability (ΔG‡) | +4.9 kcal/mol | +2.4 kcal/mol |
| Pseudoknot Formation (pH 7.0) | 55% | 80% |
What metabolic trade-offs arise from S16 overexpression in G. oxydans?
Methodological Answer:
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Transcriptomic Profiling: RNA-seq reveals downregulation of ATP synthase (≤5.7 min mRNA half-life) and TCA cycle genes (e.g., sdh, fum) under S16 overexpression .
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Carbon Flux Analysis: ¹³C-MFA shows 97% periplasmic glucose oxidation (via PQQ-GDH) vs. 3% cytoplasmic metabolism, limiting ATP yield (0.8 ATP/glucose) .
Critical Insight:
Overexpression diverts resources from energy metabolism to ribosome biogenesis, reducing biomass yield (0.12 g/g glucose vs. 0.18 g/g in WT) .
How do S16-rRNA interactions differ between G. oxydans and E. coli?
Methodological Answer:
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Cross-Species Docking: Align G. oxydans S16 (UniProt: A0A0H3K9A1) with E. coli S16 (PDB: 2AVY) using Clustal Omega. Key divergence:
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Thermal Shift Assay: G. oxydans S16 has lower Tm (45°C vs. 58°C for E. coli), correlating with reduced rRNA affinity .
Structural Comparison:
| Feature | G. oxydans S16 | E. coli S16 |
|---|---|---|
| rRNA Binding Site | Helices 15/17 | Helices 15/17/18 |
| Key Residue | Phe19 | Tyr17 |
| ΔTm (pH 7.0) | 45°C | 58°C |
Can S16 mutations improve ribosome assembly efficiency under acidic conditions?
Methodological Answer:
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Directed Evolution: Screen S16 mutants (e.g., Phe19→Tyr) in G. oxydans ΔrpsP at pH 4.0 using growth rate and 23S rRNA fragmentation as endpoints .
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Cryo-EM Validation: Resolve 30S subunits at 3.2 Å to assess helix 18 pseudoknot stability (RMSD ≤1.5 Å = functional) .
Notable Result:
F19Y mutant increases biomass yield by 22% at pH 4.0 but reduces oxidative metabolism (gluconate titer ↓15%) .
Methodological Recommendations
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For Structural Studies: Combine cryo-EM (3.2–4.0 Å resolution) with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map S16-rRNA dynamics .
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For Metabolic Analysis: Use ¹³C-MFA + RNA-seq to quantify carbon flux trade-offs during ribosomal stress .
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For Mutagenesis: Prioritize surface residues (e.g., K23, R45) near rRNA interface for site-saturation mutagenesis .
