Recombinant Lactobacillus johnsonii ATP synthase subunit c (atpE)

How does L. johnsonii atpE differ structurally and functionally from ATP synthase c subunits in other bacterial species?

Unlike some bacteria that employ different pathways for energy generation, L. johnsonii primarily uses homofermentative metabolism, which makes the ATP synthase complex particularly important for energy production . The atpE protein's sequence and structure are adapted to function optimally within the specific membrane environment and pH conditions encountered by L. johnsonii in its natural habitat.

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

E. coli expression systems have proven successful for recombinant production of L. johnsonii atpE. The available research indicates that expression with an N-terminal His-tag facilitates subsequent purification while maintaining protein functionality . The full-length protein (amino acids 1-70) can be successfully expressed in E. coli host strains optimized for membrane protein production.

The recommended methodology includes:

• Cloning the atpE gene into an expression vector with an N-terminal His-tag

• Transformation into E. coli expression strains

• Induction of protein expression under controlled conditions

• Cell lysis and membrane fraction isolation

• Solubilization using appropriate detergents

• Purification via immobilized metal affinity chromatography (IMAC)

What are the optimal storage conditions for maintaining stability of purified recombinant L. johnsonii atpE?

Purified recombinant L. johnsonii atpE protein requires specific storage conditions to maintain stability and functionality. Based on the available research, the following storage protocol is recommended:

• Short-term storage (up to one week): Store working aliquots at 4°C in Tris-based buffer.

• Long-term storage: Store at -20°C or preferably -80°C with 50% glycerol as a cryoprotectant.

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity.

• For lyophilized preparations: Store the lyophilized powder at -20°C/-80°C and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL just before use .

Research indicates that the addition of 6% trehalose to the storage buffer (Tris/PBS-based, pH 8.0) enhances stability during freeze-thaw cycles .

How can researchers assess the functional integrity of recombinant L. johnsonii atpE in reconstituted membrane systems?

Assessing functional integrity of recombinant L. johnsonii atpE requires specialized techniques that examine both structural incorporation and proton translocation activity:

• Liposome Reconstitution Assay: Incorporate purified atpE into liposomes containing pH-sensitive fluorescent dyes. Monitor proton translocation across the membrane upon addition of an electrochemical gradient.

• ATP Synthesis Measurement: When reconstituted with other ATP synthase components, measure ATP synthesis rates using luciferase-based luminescence assays.

• Circular Dichroism (CD) Spectroscopy: Assess secondary structure integrity of the recombinant protein in detergent micelles or reconstituted membrane systems.

• Oligomerization Analysis: Use crosslinking assays followed by SDS-PAGE to determine if atpE forms the expected c-ring structure essential for functionality.

• Membrane Potential Measurements: Employ potentiometric dyes to assess if reconstituted atpE can maintain membrane potential in artificial membrane systems.

These methodologies provide complementary information about both structural and functional aspects of the recombinant protein.

What is the relationship between atpE function and carbohydrate metabolism in L. johnsonii?

L. johnsonii, like other lactobacilli, primarily employs homofermentative metabolism to convert hexoses to lactic acid, making ATP synthesis crucial for energy homeostasis . The relationship between atpE function and carbohydrate metabolism can be understood through several key connections:

• Energy Coupling: ATP synthase (including atpE) utilizes the proton gradient generated during glycolysis to synthesize ATP, completing the energy production cycle in L. johnsonii.

• Metabolic Adaptation: Genome analysis of L. johnsonii strains reveals that 37.31% of carbohydrate-active enzyme genes belong to glycoside hydrolases (GHs), which play crucial roles in carbohydrate breakdown . This robust carbohydrate metabolism generates proton motive force that drives ATP synthase activity.

• pH Homeostasis: The atpE-containing ATP synthase complex helps maintain cytoplasmic pH by consuming protons during ATP synthesis, counterbalancing acidification from lactic acid production.

• Regulatory Networks: ATP levels influence carbohydrate uptake systems, creating a feedback loop between ATP synthase activity and substrate acquisition pathways.

Research techniques to investigate these relationships include metabolic flux analysis, gene expression studies during growth on different carbon sources, and phenotypic characterization of atpE mutants.

What advanced structural biology techniques are most suitable for characterizing the molecular structure of L. johnsonii atpE?

Determining the molecular structure of L. johnsonii atpE requires specialized approaches for membrane proteins:

• Cryo-Electron Microscopy (Cryo-EM): Particularly effective for membrane protein complexes like ATP synthase. This technique can visualize the c-ring assembly of multiple atpE subunits and their arrangement within the complex.

• X-ray Crystallography: While challenging for membrane proteins, this technique can provide high-resolution structural data when atpE is crystallized with appropriate detergents or lipid cubic phase methods.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: Solution NMR or solid-state NMR can provide atomic-level structural information, especially useful for determining dynamic properties of atpE.

• Molecular Dynamics Simulations: Computational approaches complement experimental techniques by predicting protein behavior in membrane environments and interactions with other ATP synthase components.

• Cross-linking Mass Spectrometry: This technique can identify interaction interfaces between atpE subunits and with other ATP synthase components.

The methodological workflow typically combines multiple approaches, starting with homology modeling based on related structures, followed by experimental validation and refinement using the techniques listed above.

How does the structure of recombinant L. johnsonii atpE influence its functional properties in different membrane environments?

The structure-function relationship of L. johnsonii atpE is strongly influenced by membrane composition and properties:

• Lipid Interaction: The highly hydrophobic sequence of atpE (MKYLAAAIAAGLAALAASYGNGKVISKTIEGMARQPESANNLRATMFIGVGLIEAVPILAIVIGFLILFL) suggests extensive interaction with membrane phospholipids . These interactions stabilize the protein and influence its proton-binding properties.

• Oligomeric Assembly: Multiple atpE subunits assemble into a c-ring structure, with the number of subunits potentially varying based on membrane thickness and composition. This oligomeric structure creates the proton translocation pathway.

• pH Adaptation: The structure of atpE contains specific protonatable residues that function optimally within the pH range encountered by L. johnsonii in its natural environment (often acidic due to lactic acid production).

Experimental approaches to study these relationships include:

• Reconstitution in liposomes of varying lipid composition

• Site-directed mutagenesis of key residues followed by functional assays

• Hydrogen-deuterium exchange mass spectrometry to identify membrane-exposed regions

• Lipid nanodiscs to study protein function in controlled membrane environments

What is the potential role of atpE in the probiotic properties of L. johnsonii?

Although atpE's primary function relates to energy metabolism, research suggests potential connections to L. johnsonii's probiotic properties:

• Acid Tolerance: L. johnsonii strains like GJ231 demonstrate tolerance to acidic conditions, which is important for survival in gastrointestinal environments . ATP synthase activity, including atpE function, contributes to pH homeostasis and may enhance acid resistance.

• Energy Production for Beneficial Metabolites: L. johnsonii produces short-chain fatty acids, bacteriocins, and hydrogen peroxide that inhibit pathogenic bacteria . Efficient energy production via ATP synthase supports the synthesis of these beneficial compounds.

• Adhesion and Colonization: L. johnsonii strains demonstrate adhesion capabilities to intestinal cells and auto-aggregation properties . These functions require energy for expression of adhesion factors and cell envelope maintenance.

• Oxidative Stress Resistance: Some L. johnsonii strains show antioxidant properties . ATP synthase function may indirectly support antioxidant mechanisms by providing energy for detoxification systems.

Experimental approaches to investigate these connections include:

• Comparative studies of ATP synthase activity in probiotic vs. non-probiotic strains

• Correlation of ATP levels with probiotic-associated functions

• Gene expression analysis of atpE during host-microbe interactions

How does atpE expression change in L. johnsonii under different environmental conditions relevant to its probiotic applications?

Understanding the regulation of atpE expression provides insights into L. johnsonii's adaptation to different environments:

• Gastrointestinal Transit: During passage through the gastrointestinal tract, L. johnsonii encounters varying pH levels, bile concentrations, and oxygen tensions. Research indicates that probiotic lactobacilli like L. johnsonii can thrive in weak acids, 0.3% bile salts, and artificial gastrointestinal fluid models . The expression of energy-generating components, including atpE, likely adjusts to maintain ATP production under these stressful conditions.

• Carbon Source Availability: The genome of L. johnsonii contains numerous carbohydrate-active enzymes responsible for carbohydrate degradation . ATP synthase expression, including atpE, may vary depending on available carbon sources to optimize energy harvest.

• Co-culture with Other Microorganisms: When L. johnsonii is grown in mixed cultures with other bacteria (as would occur in the gut microbiome), significant changes in gene expression may occur. Studies with other lactobacilli have shown that bacterial components in mixed culture supernatants can influence functional properties .

Research methodologies to examine these adaptations include:

• Quantitative PCR to measure atpE expression under different conditions

• Proteomics to assess ATP synthase protein levels

• Reporter gene constructs to visualize expression dynamics in real-time

• Physiological measurements of ATP production rates in various environments

How can recombinant L. johnsonii atpE be used as a model system for studying membrane protein interactions and assembly?

Recombinant L. johnsonii atpE offers valuable opportunities as a model for membrane protein research:

• c-Ring Assembly Studies: The oligomerization of multiple atpE subunits into a functional c-ring makes it an excellent model for studying principles of membrane protein assembly and stability.

• Protein-Lipid Interactions: The highly hydrophobic nature of atpE allows investigation of specific protein-lipid interactions that govern membrane protein folding and function.

• Chimeric Protein Construction: Creating chimeric proteins with domains from atpE proteins of different bacterial species can reveal structure-function relationships and species-specific adaptations.

• Interaction Mapping: As part of the larger ATP synthase complex, atpE interacts with other subunits. These interactions can be mapped using techniques like cross-linking mass spectrometry and co-immunoprecipitation.

Methodological approaches include:

• In vitro translation systems with defined membrane environments

• Fluorescence resonance energy transfer (FRET) to study subunit interactions

• Single-molecule force spectroscopy to examine stability and unfolding

• Native mass spectrometry of membrane protein complexes

What computational approaches are most effective for predicting functional properties of L. johnsonii atpE variants?

Computational biology offers powerful tools for studying atpE variants:

• Homology Modeling: Based on structures of related ATP synthase c subunits, models of L. johnsonii atpE can be built and refined. These models provide a framework for understanding structure-function relationships.

• Molecular Dynamics Simulations: Simulations of atpE in membrane environments can predict how sequence variations affect protein dynamics, stability, and proton translocation.

• Quantum Mechanics/Molecular Mechanics (QM/MM): This approach is particularly valuable for studying the proton binding and translocation mechanism in atpE, which involves quantum effects.

• Evolutionary Analysis: Comparing atpE sequences across bacterial species can identify conserved functional residues and lineage-specific adaptations.

• Machine Learning Approaches: Trained on experimental data, these methods can predict functional consequences of mutations in atpE.

The computational workflow typically includes:

• Sequence alignment and conservation analysis

• Structural modeling and validation

• Energy minimization in membrane environment

• Simulation of dynamics and functional properties

• Experimental validation of key predictions

What methods are available for studying the interaction between recombinant L. johnsonii atpE and other ATP synthase subunits?

Studying interactions between atpE and other ATP synthase components requires specialized techniques:

• Co-expression Systems: Expressing atpE together with other ATP synthase subunits in heterologous systems allows for the study of complex assembly and stability.

• Pull-down Assays: His-tagged atpE can be used in pull-down experiments to identify interacting partners from L. johnsonii lysates or when co-expressed with other subunits.

• Bimolecular Fluorescence Complementation (BiFC): This technique can visualize protein-protein interactions in living cells by fusing complementary fragments of fluorescent proteins to potential interaction partners.

• Surface Plasmon Resonance (SPR): SPR can quantify binding affinities between purified atpE and other ATP synthase components.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach identifies regions of atpE that change in solvent accessibility upon binding to other subunits.

Implementation strategy:

• Begin with in silico prediction of interaction interfaces

• Validate using biochemical methods like cross-linking

• Quantify interactions with biophysical techniques

• Confirm biological relevance through functional assays

How does L. johnsonii atpE compare structurally and functionally with equivalent proteins from other Lactobacillus species?

Comparative analysis of atpE across Lactobacillus species reveals evolutionary adaptations and functional differences:

• Sequence Conservation: While the core functional domains of atpE are conserved across Lactobacillus species, specific variations in amino acid sequence reflect adaptations to particular niches. In L. johnsonii, the 70-amino acid sequence (MKYLAAAIAAGLAALAASYGNGKVISKTIEGMARQPESANNLRATMFIGVGLIEAVPILAIVIGFLILFL) contains characteristic hydrophobic regions essential for membrane integration .

• Species-Specific Adaptations: Differences in atpE may correspond to the diverse habitats of Lactobacillus species. L. johnsonii is primarily found in the gastrointestinal tract , while other species inhabit dairy, plant, or other animal-associated environments . These ecological differences may be reflected in subtle structural variations that optimize function in specific conditions.

• Energy Generation Strategy: While L. johnsonii primarily employs homofermentative metabolism for energy production , other Lactobacillus species may use heterofermentative pathways. These metabolic differences could influence the role and regulation of ATP synthase components like atpE.

Research methodologies for comparative analysis include:

• Multiple sequence alignment of atpE across Lactobacillus species

• Phylogenetic analysis to trace evolutionary relationships

• Homology modeling to visualize structural differences

• Heterologous expression and functional comparison of atpE variants

What can comparative genomic analysis reveal about the evolution and adaptation of atpE in different L. johnsonii strains?

Comparative genomics provides insights into the evolutionary history and adaptive significance of atpE:

• Conservation Across Strains: Analysis of different L. johnsonii strains (such as CNCM I-12250/La1/NCC 533 and GJ231) can reveal the degree of atpE conservation within the species . High conservation would suggest crucial functional constraints.

• Horizontal Gene Transfer: Examination of genomic context around atpE can identify potential horizontal gene transfer events that might have contributed to adaptive evolution in certain lineages.

• Selection Pressure: Calculating the ratio of non-synonymous to synonymous substitutions (dN/dS) in atpE sequences across strains can identify regions under positive or purifying selection.

• Adaptive Correlations: Correlating atpE sequence variations with strain-specific phenotypes (such as acid tolerance, colonization ability, or metabolic capabilities) can reveal functional significance of specific mutations.

Research approach:

• Whole genome sequencing of multiple L. johnsonii strains

• Identification and extraction of atpE and surrounding genomic regions

• Multiple sequence alignment and calculation of conservation metrics

• Correlation of sequence variations with phenotypic differences

• Experimental validation of predicted functional effects

Table 1: Comparison of Key Properties of L. johnsonii atpE with Other Bacterial ATP Synthase c Subunits

Table 2: Recommended Experimental Protocols for L. johnsonii atpE Research

What emerging technologies hold the most promise for advancing our understanding of L. johnsonii atpE function and regulation?

Several cutting-edge technologies are poised to transform research on L. johnsonii atpE:

• Cryo-Electron Tomography: This technique can visualize ATP synthase complexes in their native membrane environment at near-atomic resolution, providing insights into the physiological arrangement and dynamics of atpE.

• Single-Molecule FRET: By labeling specific sites on atpE, this approach can monitor real-time conformational changes during proton translocation and ATP synthesis.

• Nanopore Recording: Electrical recording of single c-rings incorporated into lipid membranes can provide unprecedented insights into proton conduction mechanisms.

• CRISPR-Cas9 Genome Editing: Precise modification of atpE in the native L. johnsonii genome allows for in vivo functional studies that were previously challenging.

• Advanced Computational Methods: Integration of machine learning with molecular simulation can predict functional consequences of atpE variations with increasing accuracy.

Implementation strategy:

• Collaborate across disciplines (structural biology, biophysics, computational biology)

• Develop L. johnsonii-specific genetic tools

• Combine in vitro and in vivo approaches

• Integrate data across multiple scales (from atomic to cellular)

How might engineered variants of L. johnsonii atpE contribute to the development of next-generation probiotics?

Engineered atpE variants offer intriguing possibilities for enhanced probiotic applications:

• Improved Acid Tolerance: Modifying atpE to optimize ATP synthase function at low pH could enhance L. johnsonii survival during gastrointestinal transit.

• Enhanced Energy Efficiency: Engineering atpE variants with altered c-ring stoichiometry might increase ATP production efficiency, potentially boosting the production of beneficial metabolites.

• Temperature Adaptation: Modifications to improve thermal stability could enhance survival during product manufacturing and storage.

• Controlled Colonization: Engineering energy metabolism through atpE modifications could help regulate growth rates and persistence in the gut.

• Biosensor Development: Fusion of reporter domains to atpE could create strains that respond to specific gastrointestinal conditions by modulating ATP synthase activity.

Research pathway:

• Rational design based on structural understanding

• Directed evolution for desired properties

• In vitro validation of variant function

• Animal studies to assess colonization and beneficial effects

• Safety assessment of engineered strains