Recombinant Lactobacillus casei ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) and what is its function in Lactobacillus casei?

ATP synthase subunit c (atpE) in Lactobacillus casei is a small, hydrophobic protein that forms the c-ring in the F₀ sector of ATP synthase. This 70-amino acid protein (sequence: MQFIAASIAAGIAAFGASIGNGMVISKTLEGMARQPEMAGTLRGTMFIGVGLIEAVPILSVVVAFMLMSR) is embedded in the membrane and plays a critical role in proton translocation . The c-ring functions as a rotary motor, converting the energy of a proton gradient across the membrane into mechanical energy that drives ATP synthesis in the F₁ sector. In bacterial systems like L. casei, this process is fundamental for energy production under various growth conditions. The c-subunit forms a ring-like structure with multiple copies arranged in a circle, creating a pathway for protons to move across the membrane, which ultimately drives the conformational changes necessary for ATP synthesis .

How does the structure of Lactobacillus casei atpE compare to ATP synthase subunit c from other bacterial species?

The ATP synthase subunit c from Lactobacillus casei shares structural similarities with other bacterial c-subunits but has species-specific variations. The L. casei atpE protein consists of 70 amino acids arranged primarily in α-helical structures that span the membrane . While the core function remains consistent across species, sequence variations affect specific interactions within the ATP synthase complex. For example, compared to the ATP synthase c-subunit from Mycobacterium tuberculosis, L. casei atpE may have different binding sites for potential inhibitors due to variations in amino acid composition .

Bacterial ATP synthases, including L. casei's, are simpler than their mitochondrial counterparts while performing the same core functions. The architecture of the membrane region in bacterial ATP synthases reveals the path of transmembrane proton translocation, which has been elucidated through structural studies of bacterial systems like Bacillus PS3 . These structural differences impact how researchers approach experimental design when studying L. casei atpE compared to other bacterial or eukaryotic ATP synthase components.

What expression systems are most effective for producing recombinant Lactobacillus casei atpE protein?

E. coli expression systems have proven most effective for recombinant production of Lactobacillus casei atpE. When expressing this hydrophobic membrane protein, several key factors must be considered. First, codon optimization is crucial—designing a synthetic atpE gene with codons optimized for E. coli expression significantly improves yield . Second, the choice of expression vector impacts success rates. Vectors containing strong promoters like T7 (pET systems) or tac (pMAL systems) coupled with appropriate fusion tags (particularly His-tags for subsequent purification) work effectively for L. casei atpE expression .

For optimal expression conditions, growth at 37°C until reaching an optical density of 0.6-0.7, followed by IPTG induction (typically at 1.0 mM concentration) has shown good results . Due to the hydrophobic nature of atpE, co-expression with chaperone proteins such as DnaK, DnaJ, and GrpE can substantially increase yields of properly folded protein. These chaperones help prevent aggregation of the hydrophobic subunit c during expression . Additionally, using E. coli strains designed for membrane protein expression (such as C41/C43 or T7 Express lysY/Iq) further improves success rates for this challenging protein.

What purification strategies yield the highest purity of recombinant Lactobacillus casei atpE?

Purification of recombinant L. casei atpE requires specialized approaches due to its hydrophobic nature and membrane association. The most effective purification strategy begins with proper cell lysis, typically using a combination of enzymatic (lysozyme) and mechanical (sonication) methods in a buffer containing protease inhibitors . For His-tagged L. casei atpE, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary purification step, with elution using an imidazole gradient .

Due to the protein's hydrophobicity, detergent selection is critical. Mild detergents like n-dodecyl β-D-maltoside (DDM) or digitonin effectively solubilize the protein while maintaining its native structure. Following IMAC, size exclusion chromatography provides further purification and allows buffer exchange into a stabilizing formulation. The final purified product typically achieves >90% purity as determined by SDS-PAGE . For long-term storage, the addition of 6% trehalose in Tris/PBS-based buffer at pH 8.0 provides stability, and the lyophilized protein should be stored at -20°C/-80°C . When reconstituting the protein, it's recommended to add 5-50% glycerol (final concentration) and aliquot for storage to avoid repeated freeze-thaw cycles.

How can researchers verify the correct folding and functionality of recombinant Lactobacillus casei atpE?

Verifying correct folding and functionality of recombinant L. casei atpE requires multiple complementary approaches. First, circular dichroism (CD) spectroscopy should be employed to confirm the expected predominantly α-helical secondary structure characteristic of the c-subunit. Second, reconstitution experiments are essential—the purified protein must be incorporated into liposomes or nanodiscs to create a membrane-like environment where functionality can be assessed.

Functionality can be verified through proton translocation assays using pH-sensitive fluorescent dyes (like ACMA or pyranine) to monitor proton movement across the membrane of reconstituted liposomes. Additionally, when the c-subunit is reconstituted with other ATP synthase components, ATP synthesis/hydrolysis activity can be measured using standard enzyme assays that track either ATP production or phosphate release. Native-PAGE analysis helps determine whether the recombinant c-subunit can properly assemble into the characteristic c-ring structure, which is essential for its function . Finally, limited proteolysis tests can indicate proper folding, as correctly folded proteins typically show resistance to proteolytic digestion at specific sites compared to misfolded variants.

What are the critical factors affecting the assembly of recombinant Lactobacillus casei atpE into functional c-rings?

The assembly of recombinant L. casei atpE into functional c-rings is influenced by several critical factors. First, the lipid environment plays a crucial role—specific lipid compositions, particularly those containing cardiolipin, facilitate proper c-ring assembly. The lipid-to-protein ratio during reconstitution experiments must be carefully optimized, typically between 50:1 and 200:1 (w/w), to achieve functional assembly . Second, pH and ionic strength significantly impact assembly efficiency; typically, a pH range of 7.0-8.0 and moderate ionic strength (100-150 mM) provide optimal conditions.

The oligomerization process is also temperature-dependent, with most successful assemblies occurring at 25-30°C over 24-48 hours. Cross-linking studies have shown that the number of c-subunits in the ring can vary between bacterial species, affecting proton-to-ATP ratios and energy efficiency . Native-PAGE analysis reveals that improper ratios of c-subunit to other ATP synthase components can lead to free, unassembled c-subunit, as observed in certain experimental conditions . The presence of detergents during the assembly process must be carefully controlled—while necessary for solubilization, excessive detergent can disrupt protein-protein interactions essential for ring formation. Finally, metal ions, particularly Mg²⁺, at concentrations of 2-5 mM, stabilize the assembled structure through interactions with acidic residues in the c-subunit.

How does post-translational modification affect Lactobacillus casei atpE function, and how can these modifications be characterized?

Post-translational modifications (PTMs) of L. casei atpE can significantly impact its function and interaction with other ATP synthase components. While less extensively studied than in eukaryotic systems, bacterial c-subunits can undergo modifications including phosphorylation, acetylation, and lipidation. These modifications affect proton binding, c-ring stability, and interactions with the a-subunit during proton translocation.

To characterize these modifications, researchers should employ a multi-method approach. Mass spectrometry, particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS), provides the most comprehensive identification of PTMs. This technique can detect mass shifts corresponding to specific modifications and determine their exact locations within the protein sequence. Phosphorylation can be detected using phospho-specific antibodies in Western blot analysis, while specific staining methods (Pro-Q Diamond for phosphorylation or periodic acid-Schiff for glycosylation) can visualize modifications on gel-separated proteins. Site-directed mutagenesis of potentially modified residues, followed by functional assays, helps establish the physiological relevance of identified PTMs. Computational prediction tools can guide experimental approaches by identifying potential modification sites based on consensus sequences. When investigating the functional impact of PTMs, researchers should compare native L. casei atpE isolated from bacterial membranes with recombinantly expressed protein to identify differences in modification patterns that might affect function.

What methodologies are most effective for studying the interaction between Lactobacillus casei atpE and potential inhibitor compounds?

Studying interactions between L. casei atpE and potential inhibitors requires a comprehensive approach combining computational and experimental methods. Initially, homology modeling should be used to generate a 3D structural model of L. casei atpE based on known structures from related organisms, followed by refinement through molecular dynamics simulations . This model serves as the foundation for virtual screening against compound libraries like ZINC and PubChem using tools such as RASPD and PyRx to identify compounds with favorable binding energies .

Top candidates from virtual screening should be evaluated experimentally using binding assays such as isothermal titration calorimetry (ITC), which provides thermodynamic parameters (ΔH, ΔS, and Kd) of the interaction, or surface plasmon resonance (SPR), which measures association and dissociation kinetics. For membrane proteins like atpE, detergent-solubilized protein or reconstituted proteoliposomes are typically used in these assays.

Functional validation of binding can be performed using ATP synthesis/hydrolysis assays with reconstituted ATP synthase complexes containing L. casei atpE. Compounds showing binding should demonstrate inhibition of enzymatic activity, with IC50 values determined through dose-response experiments. Structural confirmation of binding modes can be obtained using techniques such as hydrogen-deuterium exchange mass spectrometry (HDX-MS) or, ideally, X-ray crystallography or cryo-EM of the protein-inhibitor complex. Molecular dynamics simulations and MM-GBSA (Molecular Mechanics Generalized Born and Surface Area) analyses provide insights into the stability of protein-inhibitor complexes and accurate binding free energy calculations . The table below summarizes binding energies for potential inhibitors identified for bacterial ATP synthase subunit c:

How can site-directed mutagenesis be used to investigate the functional domains of Lactobacillus casei atpE?

Site-directed mutagenesis represents a powerful approach for investigating functional domains within L. casei atpE. Key residues for mutation should be selected based on sequence conservation analysis, structural modeling, and comparison with better-characterized ATP synthase c-subunits. Critical targets include the proton-binding site (typically an acidic residue in transmembrane helix 2), residues at the c-c interface involved in ring stability, and residues that interact with other ATP synthase subunits.

The mutagenesis workflow begins with designing primers containing the desired mutation, followed by PCR amplification of the mutated gene. After verification by sequencing, the mutant atpE should be expressed using the same optimized E. coli system used for wild-type expression . Both conservative (maintaining similar physicochemical properties) and non-conservative mutations should be created to fully understand residue function. Purification of mutant proteins follows the same protocol as wild-type, allowing direct comparison of properties.

Functional characterization involves reconstitution into liposomes and measurement of proton translocation efficiency using fluorescent probes. ATP synthesis/hydrolysis assays with reconstituted systems containing mutant c-subunits quantify the impact on enzymatic activity. Structural integrity can be assessed through circular dichroism to detect any secondary structure changes, and thermal stability assays to identify destabilizing mutations. Assembly competence should be evaluated through native-PAGE analysis to determine if mutants can form proper c-rings . Cross-linking studies help identify residues involved in c-c interactions or interactions with other subunits. Finally, in vivo complementation assays, where mutant atpE genes are expressed in ATP synthase-deficient bacterial strains, provide physiological relevance to the observed effects.

What approaches can resolve contradictions in experimental data regarding Lactobacillus casei atpE stoichiometry and assembly?

Resolving contradictions in experimental data regarding L. casei atpE stoichiometry and assembly requires a multi-technique approach and careful consideration of experimental conditions. First, researchers should recognize that different methods have inherent limitations—mass measurements might not distinguish between functional and non-functional assemblies, while functional assays may not detect all assembled structures.

A comprehensive approach begins with protein quantification using multiple methods (Bradford, BCA, and amino acid analysis) to establish accurate protein concentrations for stoichiometric calculations. Native mass spectrometry provides precise mass measurements of intact c-rings, allowing determination of the number of c-subunits per ring. Cross-linking combined with mass spectrometry helps identify specific interaction interfaces between subunits. Cryo-electron microscopy and single-particle analysis can directly visualize c-ring structure and determine subunit count through symmetry analysis .

Experimental conditions significantly impact results—detergent type, concentration, and lipid content can alter observed stoichiometry. Researchers should systematically test multiple conditions and report all parameters in publications. Functional correlation is essential—proton/ATP ratios measured in ATP synthesis assays should be consistent with the determined c-ring stoichiometry. When contradictions persist, data from multiple independent methods should be integrated, with greater weight given to results obtained under near-native conditions. Finally, comparing data from different research groups using standardized protocols on identical protein constructs can help resolve laboratory-specific variables contributing to contradictory results.

How can recombinant Lactobacillus casei atpE be utilized in structural biology studies?

For cryo-electron microscopy studies, the protein can be studied either as individual c-subunits or, more informatively, as assembled c-rings. The sample preparation typically involves vitrification of the protein solution on glyoxal-plasma treated grids. Recent advances in cryo-EM have enabled visualization of ATP synthase complexes in different rotational states, revealing crucial structural details about subunit arrangements and interactions . For nuclear magnetic resonance (NMR) studies, isotope labeling (¹⁵N, ¹³C) of the recombinant protein is necessary. This can be achieved by expressing the protein in minimal media containing ¹⁵N-ammonium chloride and ¹³C-glucose as sole nitrogen and carbon sources.

The recombinant protein also serves as an excellent model for computational structural biology, including molecular dynamics simulations to study protein flexibility, proton translocation mechanisms, and interactions with other ATP synthase components or inhibitors . These structural studies collectively provide insights into the molecular mechanisms of ATP synthesis and how variations in the c-subunit structure contribute to functional differences between bacterial species.

What potential biotechnological applications exist for engineered variants of Lactobacillus casei atpE?

Engineered variants of L. casei atpE offer several promising biotechnological applications. In bioenergy research, modified c-subunits with altered proton binding sites can create ATP synthases with customized proton-to-ATP ratios, potentially enhancing ATP production efficiency in biotechnological processes. For nanomotor development, the c-ring's natural rotary function makes it an ideal component for constructing molecular machines, with engineered versions providing controlled motion at the nanoscale for applications in nanorobotics and drug delivery systems.

In biosensing, c-rings functionalized with recognition elements can detect proton gradient changes in response to specific analytes, creating sensitive biosensors for environmental monitoring or diagnostic applications. For antimicrobial development, since the c-subunit is essential for bacterial survival, engineered atpE variants that maintain function in beneficial bacteria while being resistant to ATP synthase inhibitors could protect probiotic strains during antimicrobial therapy .

Modified c-subunits can also serve as platforms for studying membrane protein folding and assembly mechanisms, providing insights applicable to other membrane protein systems. In synthetic biology, engineered c-subunits could be incorporated into minimal cells or artificial organelles to provide energy-generating capabilities. For protein purification technologies, c-rings with added affinity tags at specific locations could serve as detergent-resistant membrane protein purification tags, helping to solubilize and purify other challenging membrane proteins. These applications demonstrate the versatility of engineered L. casei atpE in addressing various biotechnological challenges.

How does the research on Lactobacillus casei atpE contribute to our understanding of bacterial bioenergetics?

Research on L. casei atpE significantly advances our understanding of bacterial bioenergetics in several ways. First, it provides insights into energy conservation mechanisms in probiotic bacteria, as L. casei is a key probiotic species with industrial importance. Studies on its ATP synthase components help explain how these bacteria maintain energy homeostasis under various environmental conditions encountered in fermentation processes and the human gut.

The c-subunit's role in determining the proton-to-ATP ratio directly impacts cellular energetic efficiency. By comparing L. casei atpE structure and function with those from other bacteria, researchers can understand how different species have evolved varied energy conservation strategies. This comparative approach reveals adaptations to specific ecological niches, such as acidic environments where L. casei naturally thrives .

Investigation of L. casei atpE also contributes to understanding regulatory mechanisms of bacterial ATP synthesis. Post-translational modifications and protein-protein interactions involving the c-subunit provide insights into how bacteria modulate energy production in response to environmental changes. The molecular details of proton translocation through the c-ring illuminate fundamental aspects of chemiosmotic energy conversion—the core process of biological energy generation across all domains of life.

Additionally, research on recombinant expression and assembly of functional L. casei atpE advances methods for studying membrane protein complexes in general . These methodological developments have broader applications in membrane protein research beyond ATP synthases. Finally, structure-function studies of L. casei atpE contribute to understanding the evolutionary relationships between bacterial and eukaryotic ATP synthases, highlighting conserved mechanisms and specialized adaptations that have emerged through evolution.

What are common challenges in expressing recombinant Lactobacillus casei atpE and how can they be overcome?

Researchers frequently encounter several challenges when expressing recombinant L. casei atpE. Protein toxicity often limits expression, as the hydrophobic c-subunit can disrupt E. coli membrane integrity. This can be addressed by using tightly regulated expression systems, reducing induction temperature to 18-25°C, and employing specialized E. coli strains like C41/C43 designed for toxic membrane proteins . Inclusion body formation is another common issue due to protein aggregation. Co-expression with chaperone proteins (DnaK, DnaJ, GrpE) significantly reduces aggregation by assisting proper folding . Using fusion partners like maltose-binding protein (MBP) can also enhance solubility.

Low expression yield is frequently reported, which can be improved through codon optimization for E. coli, using high-copy-number plasmids with strong promoters, and optimizing culture conditions (media composition, induction timing). For protein degradation issues, incorporating protease inhibitor cocktails during all purification steps and working at reduced temperatures (4°C) minimizes proteolytic activity . Western blotting with specific antibodies helps monitor protein integrity throughout the purification process.

Improper membrane insertion can occur when overexpressing membrane proteins. Using E. coli strains with enhanced membrane protein insertion machinery and optimizing the signal sequence, if used, improves proper membrane localization. Finally, difficulties in detecting the expressed protein due to its small size (70 amino acids) can be addressed by using specialized gel systems (Tricine-SDS-PAGE) designed for small proteins and applying sensitive detection methods like silver staining or immunoblotting with anti-His antibodies for tagged versions .

What strategies can improve the stability of purified Lactobacillus casei atpE during storage and handling?

Maintaining stability of purified L. casei atpE requires careful attention to buffer composition, storage conditions, and handling procedures. The optimal buffer composition includes 20 mM Tris-HCl at pH 8.0 with 150 mM NaCl as a base. Adding 6% trehalose significantly improves stability by preventing protein denaturation during freeze-thaw cycles . For detergent selection, mild non-ionic detergents like DDM (0.05-0.1%) or digitonin (0.1-0.3%) effectively maintain protein solubility while preserving structure. Including 10-20% glycerol in storage buffers provides further protection against freeze-induced denaturation.

The purified protein should be stored as aliquots at -80°C for long-term storage to avoid repeated freeze-thaw cycles. For daily handling, maintaining the protein at 4°C rather than room temperature significantly extends its usable lifetime . Lyophilization offers an alternative storage method, especially for shipping or long-term storage, with reconstitution in the appropriate buffer containing the same detergent used during purification .

Prior to experiments, centrifugation at 20,000×g for 15 minutes removes any aggregated protein. Stability can be monitored through various techniques: circular dichroism for secondary structure maintenance, dynamic light scattering for aggregation detection, and functional assays to confirm retained activity. Adding reducing agents (1-5 mM DTT or 2 mM β-mercaptoethanol) prevents oxidative damage to cysteine residues if present. Finally, avoiding extreme pH conditions (maintain pH 7.0-8.5) and using low-binding plastic tubes for storage prevents protein loss through surface adsorption.

How can researchers troubleshoot issues in reconstitution experiments with Lactobacillus casei atpE?

Reconstitution experiments with L. casei atpE present several potential challenges that researchers must address systematically. When facing poor incorporation into liposomes, the lipid composition should be optimized by testing mixtures containing phosphatidylcholine, phosphatidylethanolamine, and cardiolipin at different ratios. The protein:lipid ratio should be varied (typically testing 1:50 to 1:200 w/w) to identify optimal conditions. The detergent removal method significantly impacts reconstitution efficiency—comparing gradual removal using Bio-Beads or dialysis against rapid dilution helps identify the most effective approach for L. casei atpE.

For non-functional reconstituted protein, researchers should verify protein orientation in liposomes using protease protection assays or antibody binding to exposed epitopes. The proton leakiness of liposomes should be assessed using pH-sensitive dyes in control experiments without protein. Different reconstitution buffers (varying pH, ionic strength, and divalent cation concentration) should be tested to identify conditions that maintain protein function.

When encountering heterogeneous proteoliposome populations, size extrusion through polycarbonate membranes (100-200 nm pores) can create more uniform vesicles. Freeze-fracture electron microscopy provides visual confirmation of proper protein incorporation and distribution. For aggregation during reconstitution, stepwise addition of protein to preformed liposomes often reduces aggregation compared to mixing protein and lipids prior to liposome formation. Finally, functional verification should employ multiple complementary assays, such as proton pumping measurements using ACMA fluorescence quenching and ATP synthesis activity when the c-subunit is co-reconstituted with other ATP synthase components.

What considerations are important when designing experiments to study Lactobacillus casei atpE interactions with other ATP synthase subunits?

When studying interactions between L. casei atpE and other ATP synthase subunits, several key considerations ensure reliable and interpretable results. First, expression system compatibility is crucial—researchers should establish expression systems that produce all interacting subunits in functional form, potentially using polycistronic constructs for co-expression of multiple subunits . Tag placement requires careful consideration, as improper positioning can sterically hinder interactions. Creating multiple constructs with tags at different positions (N-terminal, C-terminal, or internal loops) helps identify optimal configurations that don't disrupt native interactions.

Detergent selection significantly impacts protein-protein interactions—different detergents should be screened, as some may disrupt specific interactions while preserving others. Mild detergents like digitonin, amphipols, or nanodisc systems often better preserve subunit interactions compared to harsher detergents. Purification strategies should be designed to maintain subunit associations, potentially using tandem affinity purification with tags on different subunits to isolate intact subcomplexes.

Interaction detection methods should include multiple complementary techniques. Co-immunoprecipitation with antibodies against specific subunits can identify interacting partners. Pull-down assays using affinity-tagged subunits followed by mass spectrometry identify interaction components. For quantitative interaction analysis, microscale thermophoresis or surface plasmon resonance provides binding affinity measurements. Structural techniques like chemical cross-linking followed by mass spectrometry map specific interaction interfaces, while native mass spectrometry determines subunit stoichiometry within complexes.

Control experiments are essential—using unrelated membrane proteins to confirm specificity of observed interactions and systematically testing individual subunit omissions to identify essential components for complex formation. Finally, functional validation ensures that observed interactions are physiologically relevant, typically through reconstitution of subunit combinations in liposomes and measurement of activity (proton translocation, ATP synthesis) to correlate structural interactions with function.

How should researchers interpret contradictory results between in vitro and in vivo studies of Lactobacillus casei atpE?

When faced with contradictory results between in vitro and in vivo studies of L. casei atpE, researchers should adopt a systematic approach to reconcile these differences. First, consider the experimental context differences: in vitro systems lack the complex cellular environment, including interacting proteins, metabolites, and native membrane composition, which may be essential for proper function. In contrast, in vivo systems present challenges in controlling variables and isolating specific effects attributable to atpE.

Researchers should examine protein modification differences—post-translational modifications present in vivo may be absent in recombinant systems, affecting protein function . Mass spectrometry comparison of native and recombinant protein can identify such differences. The lipid environment significantly impacts membrane protein function; in vitro reconstitution experiments should be conducted with lipid compositions mimicking the native L. casei membrane to minimize this variable.

When studying protein-protein interactions, recognize that in vitro studies may miss transient or weak interactions that occur in the cellular context. Conversely, overexpression in vivo may create non-physiological interactions. Cross-validation using complementary techniques helps address these limitations—for example, confirming in vitro binding studies with in vivo co-immunoprecipitation or fluorescence resonance energy transfer (FRET) experiments.

For functional studies, differences in energy state and ion gradients between in vitro and in vivo conditions can lead to contradictory results. Researchers should carefully control and match electrochemical parameters when possible. When contradictions persist, developing intermediate systems like spheroplasts or right-side-out membrane vesicles can bridge the gap between fully isolated protein and intact cells. Finally, genetic approaches such as site-directed mutagenesis followed by both in vitro characterization and in vivo complementation assays provide powerful tools to reconcile contradictory results by linking specific molecular features to observed phenotypes.