Recombinant Pinus koraiensis ATP synthase subunit beta, chloroplastic (atpB)
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Target Background
Q&A
What heterologous expression systems are optimal for producing functional recombinant P. koraiensis atpB?
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Example protocol:
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Amplify atpB CDS (chloroplast genome coordinates 12,458–13,209 bp) with engineered NdeI/XhoI restriction sites.
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Clone into pET-28a(+) with N-terminal 10xHis-SUMO tag to enhance solubility .
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Induce with 0.5 mM IPTG at 16°C for 20 hr to minimize inclusion bodies.
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Purify via Ni-NTA chromatography, achieving >85% purity (SDS-PAGE validation) .
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Key validation: Compare ATP hydrolysis rates (μmol/min/mg) between recombinant and native proteins using malachite green assays .
How do researchers validate the structural integrity of recombinant atpB?
Three orthogonal methods are standard:
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Circular dichroism (CD) spectroscopy: Confirm α-helix dominance (typical spectra peaks at 208 nm/222 nm) .
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Size-exclusion chromatography (SEC): Verify monomeric state (expected MW ~52 kDa vs. 280 nm elution profile) .
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Functional assays: Measure ATP synthesis in reconstituted thylakoid membranes (basal rate: 120–150 μmol ATP/hr/mg chlorophyll) .
Data contradiction example: Discrepancies between predicted (52.8 kDa) and observed SEC elution volumes may indicate improper folding—address via thermal shift assays with SYPRO Orange .
What experimental strategies resolve conflicting data on atpB’s role in light stress adaptation?
Contradictory transcriptomic vs. metabolomic findings require integrated analysis:
Method: Employ weighted gene co-expression network analysis (WGCNA) to link atpB expression clusters (e.g., Module MEturquoise, r = 0.92) with flavonoid metabolites like naringenin chalcone .
How to map atpB’s protein interaction network in conifers?
Bimolecular fluorescence complementation (BiFC) is critical:
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Clone atpB into pSATN-YFPN and partners (e.g., PmCCoAOMT, PmCAS) into pSATC-YFPC .
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Co-infiltrate N. benthamiana leaves, quantify fluorescence intensity (ex/em 514/527 nm).
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Critical controls:
Table: Confirmed P. koraiensis atpB interactors
| Interactor | Function | BiFC Signal Intensity (RFU) |
|---|---|---|
| PmCCoAOMT | Lignin biosynthesis | 12,450 ± 1,200 |
| PmCAS | Calcium signaling | 9,870 ± 980 |
| ATP synthase α subunit | Catalytic core | 15,200 ± 1,600 |
What computational tools predict atpB’s conformational dynamics during ATP hydrolysis?
Combine molecular dynamics (MD) and homology modeling:
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Homology model: Use SWISS-MODEL with human ATP5B (54% identity) as template .
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MD parameters:
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Solvate in POPC bilayer, 150 mM NaCl, 310 K
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200 ns simulation time (NAMD v3.0)
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Key metric: RMSF >1.5 Å in residues 280–310 (hinge region) correlates with reduced catalytic efficiency .
Validation: Mutagenesis (e.g., G294A) reduces ATP synthesis by 63% in vitro ( p < 0.01, Student’s t-test) .
