Recombinant Lactobacillus fermentum ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Lactobacillus fermentum?

ATP synthase subunit c (atpE) in L. fermentum is part of the membrane-embedded F₀ region of the ATP synthase complex. This subunit forms the c-ring rotor that is essential for proton translocation across the membrane. The c-ring connects to the central stalk of the F₁ portion, allowing the mechanical energy from proton flow to drive conformational changes in the catalytic sites, resulting in ATP synthesis .

Structurally, multiple c subunits assemble into a ring formation within the membrane, with each c subunit typically consisting of two transmembrane α-helices connected by a polar loop. The number of c subunits in the ring varies between species and can affect the bioenergetic efficiency of the enzyme. Each c subunit contains a conserved carboxylate residue (usually aspartate or glutamate) that is critical for proton binding and transport .

How does L. fermentum ATP synthase compare to ATP synthases from other bacterial species?

Unlike alkaliphilic bacteria that have evolved specific adaptations in their ATP synthases to function at high pH, L. fermentum ATP synthase operates in slightly acidic to neutral pH environments typical of its natural habitats. The c-ring composition in L. fermentum likely differs from those found in extremophiles like Bacillus pseudofirmus OF4 or thermophilic bacteria like Bacillus PS3 .

While mitochondrial ATP synthases exist as dimers that help shape cristae, bacterial ATP synthases like that of L. fermentum function as monomers. In terms of regulation, L. fermentum ATP synthase likely employs mechanisms similar to other lactic acid bacteria, with regulation potentially linked to intracellular pH and energy status .

What expression systems are most effective for producing recombinant L. fermentum atpE protein?

For effective heterologous expression of L. fermentum atpE, several expression systems have proven successful with similar bacterial membrane proteins:

• E. coli expression systems: The BL21(DE3) strain with pET vector systems offers high-yield expression, though membrane proteins like atpE may require specialized approaches such as fusion partners or reduced induction temperatures to prevent inclusion body formation .

• Bacillus-based expression: Expression in Bacillus subtilis can provide a gram-positive cellular environment more similar to the native L. fermentum context, potentially improving proper folding of the membrane protein .

• Homologous expression: Using L. fermentum itself or closely related Lactobacillus species as expression hosts can improve proper membrane insertion and folding, though with typically lower yields than E. coli systems .

When expressing membrane proteins like atpE, consideration must be given to potential toxicity to the host cell and the challenges of proper membrane insertion and folding .

What structural techniques are most effective for studying the c-ring assembly of recombinant L. fermentum ATP synthase?

Several complementary structural techniques have proven valuable for studying ATP synthase c-rings:

• Cryo-electron microscopy (cryo-EM): This approach has revolutionized the structural analysis of ATP synthases, allowing visualization of different rotational states. For L. fermentum atpE, cryo-EM can reveal the c-ring assembly within the complete ATP synthase complex, providing insights into species-specific structural adaptations .

• X-ray crystallography: While challenging for membrane proteins, this technique can provide atomic-level resolution of the c-ring structure when successful. Detergent solubilization and lipidic cubic phase crystallization methods have been effective for c-rings from other bacteria .

• Solid-state NMR spectroscopy: This technique is particularly valuable for studying dynamics and protonation states of key residues within the c-ring in a membrane-like environment .

• Cross-linking mass spectrometry: This approach can identify spatial relationships between subunits and has been useful for understanding c-ring assembly and interactions with other ATP synthase components .

For optimal results, researchers should consider combining multiple techniques to overcome the limitations of each individual method, potentially revealing both structural details and functional dynamics of the c-ring assembly .

How can researchers effectively study the proton translocation mechanism through the L. fermentum ATP synthase c-ring?

Studying proton translocation through the c-ring requires specialized approaches:

• Site-directed mutagenesis: Systematically altering the conserved proton-binding carboxylate residues in the c subunit can provide insights into the specific amino acids involved in proton binding and release. Subsequent functional assays can quantify the impact of these mutations .

• pH-dependent fluorescence spectroscopy: Using pH-sensitive fluorescent probes can track proton movement in reconstituted proteoliposomes containing the recombinant ATP synthase .

• Electrophysiological techniques: Patch-clamp methods applied to membranes containing reconstituted ATP synthase can directly measure proton currents under various conditions .

• Molecular dynamics simulations: Computational approaches can model proton movement through the c-ring based on structural data, providing hypotheses that can be tested experimentally .

• Reconstitution in nanodiscs or liposomes: These systems allow for controlled assessment of proton translocation in isolated ATP synthase complexes under defined conditions .

The challenge in these studies lies in distinguishing between proton movement through the c-ring and potential leak pathways, requiring careful experimental controls .

What are the experimental challenges in determining the c-ring stoichiometry in L. fermentum ATP synthase and how can they be overcome?

Determining c-ring stoichiometry presents several challenges:

• Heterogeneity challenges: Purified ATP synthase complexes may show heterogeneity in c-ring composition, especially when expressed recombinantly. Multiple purification steps including size-exclusion chromatography and gradient centrifugation can help obtain homogeneous samples .

• Analytical approaches:

  • Mass determination by native mass spectrometry can directly measure the mass of intact c-rings

  • Atomic force microscopy to visualize and count individual subunits

  • Cross-linking followed by SDS-PAGE to determine oligomeric state

  • High-resolution cryo-EM to directly visualize and count c subunits in the ring

• Biochemical quantification:

  • Quantitative amino acid analysis of purified c-rings

  • Radioactive labeling of specific residues followed by stoichiometric determination

  • Chemical labeling of the conserved carboxylate residues can provide stoichiometric information

• Functional correlation: Measuring the H⁺/ATP ratio through careful bioenergetic experiments can indirectly inform about c-ring stoichiometry, as this ratio is theoretically determined by the number of c subunits per 360° rotation .

Researchers should consider using multiple complementary approaches to confirm stoichiometry findings, as each method has inherent limitations .

What is the optimal protocol for recombinant expression and purification of L. fermentum ATP synthase subunit c?

Expression Protocol:

• Vector construction:

  • Clone the L. fermentum atpE gene into pET expression vectors with hexahistidine tag

  • Consider using a fusion partner (MBP or SUMO) to improve solubility

  • Optimize codon usage for the expression host

• Expression conditions:

  • Transform into E. coli C43(DE3) or BL21(DE3) pLysS strains (specialized for membrane proteins)

  • Culture in LB or 2xYT media at 37°C until OD₆₀₀ reaches 0.6

  • Induce with 0.1-0.5 mM IPTG

  • Shift temperature to 18-25°C and continue expression for 4-16 hours

Purification Protocol:

• Cell disruption:

  • Harvest cells by centrifugation (5,000 g, 15 min, 4°C)

  • Resuspend in buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, protease inhibitors)

  • Disrupt cells using sonication or high-pressure homogenization

• Membrane isolation:

  • Remove cell debris by centrifugation (10,000 g, 20 min, 4°C)

  • Isolate membranes by ultracentrifugation (150,000 g, 1 h, 4°C)

  • Resuspend membrane pellet in solubilization buffer

• Protein solubilization:

  • Solubilize membranes with appropriate detergent (n-dodecyl-β-D-maltopyranoside or digitonin)

  • Incubate with gentle agitation for 1-2 hours at 4°C

  • Remove insoluble material by ultracentrifugation

• Affinity purification:

  • Apply solubilized protein to Ni-NTA or similar affinity resin

  • Wash with buffer containing low imidazole (20-40 mM)

  • Elute with buffer containing high imidazole (250-500 mM)

• Further purification:

  • Gel filtration chromatography to separate monomeric and oligomeric forms

  • Ion exchange chromatography for additional purity if required

How can researchers effectively reconstitute and measure activity of recombinant L. fermentum ATP synthase?

Reconstitution Protocol:

• Liposome preparation:

  • Prepare liposomes using E. coli polar lipids or synthetic phospholipids (POPC:POPG, 3:1)

  • Form vesicles by extrusion through 400 nm polycarbonate filters

  • Prepare at lipid concentration of 10 mg/mL

• Protein incorporation:

  • Mix purified ATP synthase with liposomes at protein:lipid ratio of 1:50-1:100 (w/w)

  • Add detergent (Triton X-100 or n-octyl-β-D-glucopyranoside) to destabilize liposomes

  • Incubate for 30 minutes at room temperature

  • Remove detergent using Bio-Beads SM-2 or dialysis

• Proteoliposome purification:

  • Separate proteoliposomes from non-incorporated protein by sucrose gradient centrifugation

  • Collect proteoliposome fraction and remove sucrose by dialysis

Activity Measurement Methods:

• ATP synthesis activity:

  • Establish proton gradient across proteoliposome membrane (acid-base transition or valinomycin/K⁺)

  • Add ADP and inorganic phosphate

  • Measure ATP production using luciferase assay or coupled enzyme assay

  • Calculate initial rates at different substrate concentrations

• ATP hydrolysis activity:

  • Measure inorganic phosphate release using colorimetric assays (malachite green)

  • Measure ADP production using coupled enzyme assays

  • Determine effects of inhibitors (oligomycin, DCCD) to confirm specificity

• Proton pumping measurement:

  • Monitor pH changes using pH-sensitive fluorescent dyes (ACMA, pyranine)

  • Use pH electrode for direct measurement in bulk solutions

  • Calculate H⁺/ATP ratio by correlating proton movement with ATP synthesis/hydrolysis

What strategies can address common challenges in functional studies of recombinant L. fermentum ATP synthase?

Challenge 1: Low expression yields

• Solution: Optimize codon usage for the expression host

• Use specialized E. coli strains for membrane proteins (C43, C41)

• Consider expression as separate subunits followed by reconstitution

• Test multiple fusion partners and solubility tags

• Implement controlled expression systems with lower induction levels

Challenge 2: Protein instability

• Solution: Include appropriate stabilizers in all buffers (glycerol 10-20%, specific lipids)

• Maintain constant low temperature during purification (4°C)

• Add specific ATP synthase inhibitors (if studying the c subunit specifically)

• Consider nanodiscs or amphipols as alternatives to detergents for stabilization

• Minimize exposure to damaging conditions (freeze-thaw cycles, air interfaces)

Challenge 3: Orientation in proteoliposomes

• Solution: Create transiently leaky vesicles to establish defined orientation

• Use fluorescent probes to quantify orientation ratio

• Implement techniques to selectively measure activity of properly oriented complexes

• For some studies, establish methods to separate inside-out from right-side-out vesicles

Challenge 4: Validating assembled complex

• Solution: Use analytical ultracentrifugation to confirm complex formation

• Implement blue native PAGE to assess complex integrity

• Employ negative-stain electron microscopy to visualize assembled complexes

• Verify subunit stoichiometry using quantitative mass spectrometry

Challenge 5: Distinguishing ATP synthase activity from contaminating ATPases

• Solution: Include specific inhibitors (oligomycin, DCCD, venturicidin)

• Implement careful negative controls

• Verify proton-coupling through simultaneous measurement of proton transport

• Test activity dependence on intact proton gradient

How can structural studies of L. fermentum ATP synthase inform the development of antimicrobial strategies?

Understanding the structure and function of ATP synthase in L. fermentum can provide insights into potential antimicrobial targets, particularly when comparing with pathogenic bacteria. The c subunit has been identified as the target of several antimicrobial compounds including diarylquinolines, which target mycobacterial ATP synthase .

Research approaches include:

• Comparative structural analysis between L. fermentum and pathogenic bacterial ATP synthases to identify specific structural differences that can be exploited

• Structure-based drug design targeting unique features of bacterial ATP synthases

• Identification of species-specific inhibitors through high-throughput screening against recombinant ATP synthase components

• Development of hybrid compounds that selectively target pathogenic bacteria while sparing beneficial microbiota members like L. fermentum

These studies may lead to novel antimicrobials with reduced disruption to the beneficial gut microbiome compared to broad-spectrum antibiotics.

What is the relationship between ATP synthase function and probiotic properties in L. fermentum?

L. fermentum strains exhibit various probiotic properties, including induction of autophagy, which appears to have protective effects against acetaminophen-induced hepatotoxicity . The relationship between these properties and ATP synthase function remains to be fully elucidated, but several research approaches can address this question:

• Generation of L. fermentum strains with modifications in ATP synthase components to study effects on:

  • Stress tolerance (acid, bile, oxidative stress)

  • Growth characteristics and metabolite production

  • Autophagy induction and other probiotic properties

• Investigation of ATP synthase regulation during:

  • Growth in different environmental conditions

  • Exposure to host-derived factors

  • Competition with other microbiota members

• Analysis of ATP synthase activity in relation to:

  • Production of beneficial metabolites

  • Membrane potential maintenance

  • pH homeostasis in acidic environments

Understanding these relationships may enable the development of enhanced probiotic strains with improved stress resistance and therapeutic properties.