Recombinant Lactobacillus sakei subsp. sakei ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Lactobacillus sakei and what is its significance?

ATP synthase subunit c, encoded by the atpE gene in Lactobacillus sakei subsp. sakei, is a critical component of the F0 sector of F-type ATP synthase. This 70-amino acid protein (MNFLAAAIAAGLAAFAASYGNGKVISKTIESMARQPELSAQLRSTMFIGVGLIEAVPILSIVVSFLILFS) forms the membrane-embedded proton channel that facilitates proton translocation across the membrane, which is essential for ATP synthesis . The protein is particularly significant in L. sakei's energy metabolism, especially during fermentation processes. L. sakei is valuable in the fermentation of meat products and contributes to better preservation of meat and fish . Understanding the structure and function of atpE provides insights into the bacterium's bioenergetics and adaptation mechanisms in different growth environments.

How should recombinant L. sakei atpE protein be stored and handled for optimal stability?

For optimal stability of recombinant L. sakei atpE protein, follow these evidence-based handling protocols:

• Store the lyophilized protein powder at -20°C/-80°C upon receipt

• Perform proper aliquoting when reconstituting the protein to avoid repeated freeze-thaw cycles

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is recommended) for long-term storage at -20°C/-80°C

• For working solutions, store aliquots at 4°C for no longer than one week

• Avoid repeated freezing and thawing as this significantly degrades protein quality

• Briefly centrifuge the vial before opening to ensure material is at the bottom

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

Based on current research protocols, E. coli is the most commonly used and effective expression system for recombinant L. sakei atpE protein production . When designing expression experiments, researchers should consider:

• Codon optimization for E. coli expression, as L. sakei has different codon usage patterns

• Selection of appropriate fusion tags (His-tag is commonly used and positioned at the N-terminus)

• Optimization of induction conditions (temperature, inducer concentration, and duration)

• Use of specialized E. coli strains designed for membrane protein expression

• Implementation of appropriate extraction and purification protocols that maintain the native structure of this highly hydrophobic membrane protein

The choice of vector system should allow for tight regulation of expression, as membrane proteins can be toxic to host cells when overexpressed.

What purification methods yield the highest purity and activity for recombinant L. sakei atpE?

For high purity (>90%) recombinant L. sakei atpE protein, a multi-step purification strategy is recommended:

• Initial extraction using specialized detergents suitable for membrane proteins

• Affinity chromatography utilizing the His-tag (Immobilized Metal Affinity Chromatography, IMAC)

• Size-exclusion chromatography to remove aggregates and impurities

• Buffer optimization containing appropriate detergents to maintain protein solubility

• Quality assessment using SDS-PAGE to confirm purity (should exceed 90%)

• Activity validation using functional assays specific to ATP synthase function

Researchers should note that the choice of detergent is critical for maintaining the native structure and function of this membrane protein during purification.

How does ribose metabolism in L. sakei affect ATP synthase expression and function?

The relationship between ribose metabolism and ATP synthase function in L. sakei involves complex regulatory networks:

• Transcriptome analysis reveals that when L. sakei shifts from glucose to ribose as a carbon source, significant changes in energy metabolism gene expression occur, which may indirectly affect ATP synthase regulation .

• Ribose catabolism in L. sakei proceeds through the phosphoketolase pathway (PKP) rather than glycolysis, resulting in different ATP yields compared to glucose metabolism .

• The ribose uptake and catabolic machinery is highly regulated at the transcription level, with the deoxyribonucleoside synthesis operon transcriptional regulator being strongly upregulated during ribose metabolism .

• HPr kinase/phosphatase (encoded by hprK), which plays a major role in carbon metabolism regulation, shows increased expression during ribose metabolism and may indirectly modulate ATP synthase activity through global metabolic shifts .

• Carbon catabolite repression (CCR) mechanisms involving catabolite-responsive elements (cre) sites may influence the expression of genes encoding energy production systems, including ATP synthase, during growth on different carbon sources .

Methodologically, researchers investigating these relationships should:

• Perform comparative transcriptomics of L. sakei grown on different carbon sources

• Analyze ATP levels and ATP synthase activity under various growth conditions

• Conduct targeted mutagenesis of key regulatory genes to assess their impact on ATP synthase expression and function

What structural and functional differences exist between L. sakei atpE and homologous proteins in other Lactobacillus species?

Comparative analysis of L. sakei atpE with homologous proteins in other Lactobacillus species reveals several important differences:

• Sequence comparison: The 70-amino acid sequence of L. sakei atpE (MNFLAAAIAAGLAAFAASYGNGKVISKTIESMARQPELSAQLRSTMFIGVGLIEAVPILSIVVSFLILFS) contains species-specific variations in key functional regions .

• Structural implications: These sequence variations may affect:

  • Proton-binding sites efficiency

  • Oligomerization properties in the c-ring formation

  • Interactions with other ATP synthase subunits

  • Membrane insertion and stability

• Functional adaptations: Differences likely reflect adaptations to:

  • L. sakei's specific ecological niches (meat and fish environments)

  • Temperature sensitivity relevant to fermentation conditions

  • pH tolerance during fermentation processes

  • Energy efficiency mechanisms specific to L. sakei metabolism

To methodically investigate these differences, researchers should:

• Perform comprehensive phylogenetic analysis of atpE sequences across Lactobacillus species

• Use homology modeling and molecular dynamics simulations to predict structural differences

• Conduct site-directed mutagenesis to identify functionally critical residues

• Apply complementation studies using heterologous expression systems

How can recombinant L. sakei atpE be used as a tool to study bioenergetics in lactic acid bacteria?

Recombinant L. sakei atpE can serve as a valuable research tool for investigating bioenergetics in lactic acid bacteria through several methodological approaches:

• Reconstitution studies:

  • Incorporate purified recombinant atpE into liposomes

  • Measure proton translocation rates under varying conditions

  • Assess the impact of membrane composition on activity

• Interaction analysis:

  • Use recombinant atpE as bait in pull-down assays to identify interaction partners

  • Perform crosslinking experiments to capture transient interactions

  • Employ FRET-based approaches to study dynamic assembly of ATP synthase complexes

• Inhibitor screening:

  • Develop high-throughput assays using recombinant atpE

  • Screen for compounds that specifically target ATP synthase in lactic acid bacteria

  • Characterize binding mechanisms through biochemical and biophysical approaches

• Structure-function relationships:

  • Generate site-directed mutants to identify critical residues

  • Correlate structural features with proton translocation efficiency

  • Map the topology of the protein within membrane environments

When designing such experiments, researchers should control for potential artifacts introduced by the recombinant expression system and ensure that the His-tag does not interfere with the protein's native function.

What are the experimental challenges in studying the oligomerization properties of atpE in native membrane environments?

Investigating atpE oligomerization in native membrane environments presents several methodological challenges:

• Extraction difficulties:

  • The highly hydrophobic nature of atpE requires specialized detergents

  • Maintaining the oligomeric state during extraction is technically challenging

  • Native oligomerization may be disrupted by conventional solubilization methods

• Analytical limitations:

  • Standard size-exclusion chromatography may not accurately resolve membrane protein oligomers

  • Light scattering techniques require careful control of detergent micelles

  • Native PAGE conditions must be optimized for membrane protein complexes

• Structural characterization barriers:

  • Crystallization of membrane protein oligomers is notoriously difficult

  • Cryo-EM sample preparation may destabilize native oligomeric states

  • Distinguishing between functional oligomers and aggregates requires multiple complementary approaches

To address these challenges, researchers should consider these methodological approaches:

• Use mild solubilization conditions with detergents like digitonin or amphipols

• Apply native mass spectrometry optimized for membrane protein complexes

• Employ chemical crosslinking followed by mass spectrometry (XL-MS)

• Utilize fluorescence-based techniques like FRET or single-molecule tracking in reconstituted systems

• Implement advanced microscopy techniques such as high-speed atomic force microscopy (HS-AFM)

How does prophage integration in L. sakei genomes potentially affect atpE expression and ATP synthase function?

Recent genomic analyses of L. sakei strains have revealed complex relationships between prophage integration and bacterial gene expression that may impact atpE:

• Genomic context effects:

  • Analysis of 43 Latilactobacillus sakei genomes identified 26 intact, 11 questionable, and 52 incomplete prophage sequences

  • The presence of 1-5 prophage sequences per strain suggests potential impacts on genome organization and gene expression

  • Prophage integration sites may disrupt operons or regulatory regions affecting energy metabolism genes

• Transcriptional interference:

  • Prophage-encoded transcriptional regulators may cross-regulate bacterial genes

  • Insertion near atpE or other ATP synthase genes could alter their expression patterns

  • Changes in DNA topology due to prophage integration might affect local transcription efficiency

• Metabolic burden considerations:

  • Maintenance of prophage DNA imposes energetic demands

  • Spontaneous prophage induction diverts cellular resources

  • These metabolic burdens may indirectly affect ATP synthase expression and activity

• Experimental approaches:

  • Compare ATP synthase expression and activity in isogenic strains with and without specific prophages

  • Analyze transcriptome data to identify correlations between prophage presence and ATP synthase gene expression

  • Perform targeted deletion of prophage elements to assess their impact on energy metabolism

This complex relationship between prophages and host metabolism requires careful experimental design with appropriate controls for strain background and growth conditions.

What are the optimal buffer conditions for maintaining recombinant L. sakei atpE stability and function?

Buffer optimization is critical for maintaining recombinant L. sakei atpE stability and functional integrity:

• Recommended buffer composition:

  • Base buffer: Tris/PBS-based buffer at pH 8.0

  • Stabilizing agent: 6% Trehalose

  • For long-term storage: Addition of 5-50% glycerol (optimally 50%)

• Critical parameters to consider:

  • pH: Maintain at pH 8.0 for optimal stability

  • Ionic strength: Moderate ionic strength helps prevent aggregation

  • Detergent selection: Critical for membrane protein stability

  • Reducing agents: May be necessary to prevent oxidation of cysteine residues

  • Metal ions: Some membrane proteins require specific metal ions for stability

• Stability assessment methods:

  • Circular dichroism to monitor secondary structure integrity

  • Dynamic light scattering to detect aggregation

  • Thermal shift assays to determine buffer effects on protein stability

  • Activity assays to confirm functional preservation

Researchers should systematically test buffer variations and document stability profiles under different storage conditions to establish optimal protocols for their specific experimental needs.

How can recombinant L. sakei atpE be incorporated into functional assays to study ATP synthesis?

To effectively study ATP synthesis using recombinant L. sakei atpE, several functional assay systems can be implemented:

• Reconstituted proteoliposome systems:

  • Purified recombinant atpE can be incorporated into liposomes along with other ATP synthase subunits

  • Establish a proton gradient using valinomycin/K+ or acidification methods

  • Measure ATP synthesis rates using luciferase-based luminescence assays

  • Control experiments should include protonophore controls to confirm gradient dependence

• Membrane vesicle preparations:

  • Express recombinant atpE in E. coli or other hosts

  • Prepare inverted membrane vesicles containing the protein

  • Establish proton gradients and measure ATP synthesis

  • Comparative analysis with vesicles lacking the recombinant protein

• Hybrid complex assembly:

  • Combine recombinant L. sakei atpE with ATP synthase subunits from model organisms

  • Assess functionality of hybrid complexes compared to homogeneous complexes

  • Identify species-specific functional characteristics through systematic subunit exchanges

• Single-molecule approaches:

  • Fluorescently label recombinant atpE

  • Monitor rotation of the c-ring using high-resolution fluorescence microscopy

  • Correlate rotational dynamics with ATP synthesis rates

These methodological approaches require careful controls for orientation of the protein in membranes and validation of functional integration.