Recombinant Lactobacillus brevis ATP synthase subunit c (atpE)

What expression systems are optimal for producing recombinant Lactobacillus brevis ATP synthase subunit c (atpE)?

The hydrophobic nature of atpE (70 residues, theoretical pI 5.2) necessitates fusion tags for solubility. E. coli BL21(DE3) strains with pET or pMAL vectors are commonly used, with maltose-binding protein (MBP) fusions outperforming His-tag-only constructs in solubility . Codon optimization for E. coli expression is critical—synonymous substitutions in rare codons (e.g., changing Lactobacillus-preferred AGA arginine codons to E. coli-optimized CGT) improve yields 3–5 fold . Induction at 18°C with 0.5 mM IPTG for 16 hours enhances proper folding, while co-expression with chaperones (DnaK/DnaJ/GrpE) reduces aggregation .

Table 1: Expression System Comparison

Data synthesized from Spinacia oleracea c-subunit trials , applicable to L. brevis homologs .

How can researchers verify the structural integrity of recombinant atpE post-purification?

Circular dichroism (CD) spectroscopy is mandatory to confirm α-helical content matching native structures (predicted 65–70% helicity for atpE). A characteristic minimum at 222 nm and maximum at 192 nm indicates proper folding . Compare against synthetic peptides (e.g., residues 20–55) to isolate transmembrane domain conformation. For L. brevis atpE, thermal denaturation experiments should show a cooperative transition with Tm ≈52°C in lipid bilayers . Mass spectrometry validates intact disulfide bonds (Cys28-Cys47 in L. brevis) and absence of methionine oxidation .

What purification strategies mitigate aggregation of recombinant atpE?

Reverse-phase HPLC with C4 columns (5 µm, 300 Å pore) using acetonitrile/TFA gradients effectively separates monomeric atpE from oligomers . Pre-purification steps:

• Solubilization: 20 mM n-dodecyl-β-D-maltoside (DDM) preserves α-helices better than Triton X-100 .

• Tag removal: His-MBP tags require 48-hour cleavage with thrombin (2 U/mg) at 4°C to prevent truncation .

• Lyophilization: Reconstitute in 20 mM Tris-HCl (pH 8.0), 6% trehalose to stabilize monomers during storage .

How do proton translocation kinetics of recombinant L. brevis atpE compare to mitochondrial homologs?

Single-channel electrophysiology in planar lipid bilayers reveals key differences:

Table 2: Proton Transport Properties

The higher conductance in L. brevis correlates with its acidophilic habitat, requiring efficient proton capture at lower pH . Mutagenesis studies show Glu56 (conserved in Lactobacilli) as critical for proton binding—E56Q abolishes H+ transport .

What experimental approaches resolve controversies about atpE’s role in membrane permeability transition?

Conflicting reports attribute membrane leakiness either to c-subunit oligomerization defects or oxidative modifications. A three-pronged methodology is recommended:

• Crosslinking: Treat purified atpE with 0.1% glutaraldehyde for 2 min, resolve oligomers via non-denaturing PAGE. L. brevis atpE primarily forms decamers (≈75 kDa), unlike mitochondrial 8-mers .

• Liposome assays: Incorporate atpE into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) vesicles. Monitor calcein release (ex/em 495/515 nm) under ΔpH conditions. Wild-type atpE increases permeability 4-fold vs. controls .

• Redox profiling: LC-MS/MS identifies oxidation at Met17 (unique to Lactobacilli) under H2O2 stress, which increases proton slippage by 30% .

How can CRISPR-interference (CRISPRi) optimize atpE expression in native L. brevis for functional studies?

Design sgRNAs targeting the atpE promoter region (5’-TACTATAAT-3’ for L. brevis DSM 20054). Use dCas9 under a nisin-inducible promoter for repression control . Key validations:

• qRT-PCR: Measure atpE mRNA levels (expected 70–80% knockdown)

• ATP synthesis assays: Compare membrane fractions’ ADP→ATP rates (↓60% in CRISPRi strains)

• Complementation: Express plasmid-borne atpE with silent mutations (C28S/C47S) to evade CRISPRi

Discrepancies in c-ring stoichiometry between cryo-EM and crosslinking data: How to reconcile?

Problem: Cryo-EM suggests 12-mer c-rings for L. brevis , while chemical crosslinking implies 10-mers .
Resolution protocol:

• Gradual crosslinking: Time-course (0–60 min) with DSG (disuccinimidyl glutarate) shows transient 10-mers progressing to 12-mers.

• Native MS: Intact complex analysis at 20 kV, 10 mM ammonium acetate confirms 12 subunits (calc. 84.4 kDa vs. observed 84.7 kDa).

• Molecular dynamics: Simulate c-ring flexibility—Lactobacillus rings exhibit 12-fold symmetry under proton gradient but collapse to 10-fold in detergent .

Why do ATP synthesis assays with recombinant atpE show variable coupling efficiency (H+/ATP)?

Four critical factors influence coupling ratios:

• Lipid composition: 20% cardiolipin increases efficiency to 3.8 H+/ATP vs. 4.7 in phosphatidylcholine-only membranes .

• Subunit a co-reconstitution: Co-express L. brevis subunit a (atpB) with atpE—coupling improves from 25% to 89% of theoretical maximum .

• Rotational assays: Single-molecule F0F1 labeling (Cy3B on c-ring, Cy5 on γ-subunit) detects stalling at 12 steps/rotation, confirming stoichiometry .

• Divalent cations: 5 mM Mg2+ reduces proton slippage by stabilizing Asp61 carboxylates .

Concluding Recommendations

• Prioritize MBP fusion systems over His-tag vectors for superior atpE solubility and yield.

• Combine CD spectroscopy with crosslinking/MS to resolve oligomeric state controversies.

• Use planar lipid bilayer electrophysiology to benchmark proton transport against evolutionary homologs.

• Address species-specific variations (e.g., L. brevis Met17 oxidation susceptibility) when extrapolating mechanisms from model organisms.