Recombinant Buchnera aphidicola subsp. Baizongia pistaciae ATP synthase subunit c (atpE)

How does atpE from Buchnera aphidicola compare structurally to ATP synthase subunit c from other bacteria?

While no crystal structure specifically for Buchnera aphidicola atpE has been reported in the search results, structural studies of ATP synthase subunit c from other bacteria, such as Mycobacteria, provide insight into likely structural features. The protein typically forms a ring structure (c-ring) in the membrane that constitutes part of the Fo domain of ATP synthase .

Compared to free-living bacteria, Buchnera's atpE likely retains core structural elements essential for function while potentially showing adaptations related to its endosymbiotic lifestyle. Given the extreme genome reduction observed in Buchnera (approximately 10% of its genome is devoted to amino acid biosynthesis), any conserved elements in atpE likely represent essential functional domains .

What expression systems are most effective for producing recombinant Buchnera aphidicola atpE protein?

Heterologous expression in E. coli is the established method for producing recombinant Buchnera aphidicola atpE protein. The highly hydrophobic nature of this membrane protein presents significant challenges for expression and requires optimization of several parameters:

• Vector selection: Vectors with strong but controllable promoters (like pET systems) are preferable.

• E. coli strain: BL21(DE3) or derivatives are commonly used for membrane protein expression.

• Induction conditions: Lower temperatures (16-20°C) and reduced IPTG concentrations often improve proper folding.

• Fusion tags: N-terminal His-tags facilitate purification while minimizing interference with protein folding .

For difficult membrane proteins like atpE, specialized E. coli strains designed for membrane protein expression (such as C41/C43) might improve yields by reducing toxicity associated with membrane protein overexpression.

What purification strategies yield the highest purity and functional integrity of recombinant atpE protein?

Purification of recombinant Buchnera aphidicola atpE requires specialized approaches due to its hydrophobic nature:

• Membrane extraction: Carefully optimized detergent solubilization is critical, typically using mild detergents like n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG).

• Immobilized metal affinity chromatography (IMAC): His-tagged proteins can be purified using Ni-NTA resin with detergent-containing buffers.

• Size exclusion chromatography: A final polishing step to achieve >90% purity and to separate monomeric from oligomeric forms.

• Storage considerations: The protein is typically stored in buffers containing 6% trehalose at pH 8.0 to maintain stability . Repeated freeze-thaw cycles should be avoided.

For functional studies, it's crucial to verify that the purified protein retains its native conformation, which can be assessed through circular dichroism spectroscopy to examine secondary structure elements.

How can researchers assess the functional integrity of recombinant Buchnera aphidicola atpE in vitro?

Assessing functional integrity of recombinant atpE requires specialized approaches since it functions as part of a multiprotein complex:

• Reconstitution into liposomes: The purified protein can be incorporated into lipid vesicles to assess its ability to form functional c-rings.

• Proton conductance assays: Measuring pH changes across liposome membranes containing reconstituted atpE can assess ion channel activity.

• Binding studies: Using techniques like isothermal titration calorimetry (ITC) to measure binding of known ATP synthase inhibitors can indirectly assess proper folding.

• ATP synthesis coupling: While challenging, reconstitution of the complete ATP synthase complex with recombinant components can provide direct functional assessment.

Methods developed for ATP synthase subunit c from other bacterial systems, such as those used for Mycobacterium tuberculosis, can be adapted for the Buchnera protein, taking into account differences in optimal lipid environments and buffer conditions .

What experimental approaches can be used to study protein-protein interactions between atpE and other ATP synthase subunits?

Several complementary techniques can elucidate protein-protein interactions within the ATP synthase complex:

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry can identify contact points between atpE and other subunits.

• Förster resonance energy transfer (FRET): With fluorescently labeled subunits, FRET can detect proximity and interactions in reconstituted systems.

• Surface plasmon resonance (SPR): Measuring binding kinetics between immobilized atpE and other subunits.

• Co-immunoprecipitation: Using antibodies against tags or the native protein to pull down interaction partners.

• Bacterial two-hybrid systems: Modified for membrane proteins, these can detect interactions in a cellular context.

When investigating these interactions, researchers should consider the unique evolutionary context of Buchnera's ATP synthase. The extreme genome reduction in this obligate symbiont may have led to specialized adaptations in subunit interactions compared to free-living bacteria .

How has atpE evolved in the context of Buchnera's reduced genome, and what does this reveal about essential ATP synthase functions?

The evolution of atpE in Buchnera aphidicola provides fascinating insights into essential ATP synthase functions under extreme genome reduction:

• Sequence conservation: Comparative analysis reveals highly conserved residues that likely represent functionally essential sites. These conserved elements can be identified through multiple sequence alignment with atpE from diverse bacterial species.

• Reduced selective pressure: The strong within-host selection observed in Buchnera (as demonstrated for other genes) suggests that atpE may experience unusual selective pressures compared to free-living bacteria .

• Loss of regulatory elements: Given the general loss of transcriptional regulators in Buchnera, expression of atpE likely lacks the sophisticated regulation seen in other bacteria. This may be reflected in the gene's flanking regions and promoter structure .

The clonal nature of Buchnera transmission (lack of recombination) further shapes evolutionary patterns . Research approaches should include phylogenetic analysis comparing atpE sequences across different Buchnera strains and related symbionts to identify lineage-specific adaptations.

What genomic context surrounds the atpE gene in Buchnera aphidicola, and how does this differ from free-living bacteria?

The genomic organization around atpE in Buchnera aphidicola reflects its evolutionary history as an endosymbiont with a reduced genome:

• Operon structure: In most bacteria, ATP synthase genes are organized in the highly conserved atp operon. Analysis should determine whether Buchnera maintains this organization or shows rearrangements.

• Regulatory elements: Buchnera has lost many transcriptional regulators, suggesting atpE expression may rely on simplified regulatory mechanisms compared to free-living bacteria .

• Codon usage: Examination of synonymous codon usage in atpE can reveal adaptations to the symbiotic lifestyle and AT-rich genomic context typical of Buchnera.

How can recombinant atpE be used to study energy metabolism in the aphid-Buchnera symbiotic relationship?

Recombinant atpE provides valuable tools for investigating energy metabolism in this obligate symbiotic relationship:

• Metabolic flux analysis: Using recombinant atpE in reconstituted systems to measure ATP production rates under conditions mimicking the aphid cellular environment.

• Host-symbiont interface studies: Developing labeled atpE variants to track localization and potential interactions with host proteins or membranes.

• Comparative energy efficiency: Assessing whether Buchnera's ATP synthase shows adaptations for altered efficiency compared to free-living bacteria, using recombinant systems with defined subunit composition.

• Nutrient dependency: Investigating how varying ion gradients and metabolite availability affect ATP synthase function, potentially reflecting adaptations to the nutrient-rich but specialized aphid cellular environment.

These approaches can help elucidate how Buchnera has adapted its energy production to support essential functions like amino acid biosynthesis for the host while operating within the constraints of an extremely reduced genome .

What insights can functional studies of Buchnera atpE provide about the evolution of obligate endosymbiosis?

Functional characterization of Buchnera atpE can yield important insights into endosymbiont evolution:

• Functional constraints: Comparing enzymatic parameters (substrate affinity, catalytic efficiency) between Buchnera atpE and homologs from free-living bacteria can reveal whether adaptation to the host environment has altered functional constraints.

• Host dependency: Assessment of whether Buchnera ATP synthase has evolved dependency on host-derived factors would illuminate co-evolutionary processes.

• Selective pressures: Analysis of within-host selection on atpE variants can reveal whether energy production efficiency is under strong selection in the symbiotic context .

• Metabolic integration: Examining potential adaptations that might coordinate ATP production with host metabolic needs or the provision of essential amino acids to the host.

These studies contribute to understanding fundamental evolutionary processes in endosymbiosis, potentially revealing parallel adaptations across different symbiotic systems and illuminating paths toward organellogenesis.

What are the main technical challenges in working with recombinant Buchnera atpE, and how can they be addressed?

Working with recombinant Buchnera atpE presents several technical challenges:

• Low expression yields: As a highly hydrophobic membrane protein, atpE often expresses poorly in heterologous systems.

  • Solution: Optimize codon usage for the expression host, use specialized strains (C41/C43), and explore fusion partners that enhance membrane protein expression.

• Protein aggregation: Improper folding can lead to inclusion body formation.

  • Solution: Express at lower temperatures (16-20°C), use milder induction conditions, and consider membrane-mimetic environments during purification.

• Maintaining functional conformation: Detergent solubilization can disrupt native protein structure.

  • Solution: Screen multiple detergents, include stabilizing agents like glycerol and trehalose, and validate protein folding using biophysical techniques .

• Reconstitution challenges: Assembling functional ATP synthase complexes from recombinant components is technically demanding.

  • Solution: Develop stepwise reconstitution protocols, validate subcomplexes before full assembly, and use fluorescent or activity-based assays to confirm functionality.

How can researchers verify that their recombinant atpE preparation retains native-like structure and function?

Verification of native-like structure and function requires multiple complementary approaches:

• Structural assessment:

  • Circular dichroism spectroscopy to confirm secondary structure content

  • Size exclusion chromatography to verify oligomeric state

  • Limited proteolysis to assess proper folding (correctly folded proteins show characteristic proteolytic patterns)

• Functional validation:

  • Reconstitution into liposomes and proton conductance assays

  • Binding studies with known ATP synthase ligands or inhibitors

  • Assembly assays with other ATP synthase subunits

• Comparative analysis:

  • Side-by-side testing with related, better-characterized ATP synthase subunit c proteins

  • Verification that the protein responds similarly to pH, ionic strength, and lipid environment as expected for ATP synthase subunit c

• Activity correlation:

  • Correlation between structural integrity measures and functional assays to identify critical quality attributes

Given the absence of native Buchnera protein for direct comparison (due to difficulties in culturing this obligate symbiont), researchers must rely on indirect validation approaches and comparison with homologous proteins from other bacteria .

What are promising research avenues for understanding atpE function in the context of Buchnera-aphid symbiosis?

Several promising research directions can advance understanding of atpE's role in this symbiotic system:

• Cryo-EM structural studies: Determining the structure of Buchnera ATP synthase complexes using single-particle cryo-electron microscopy would reveal potential adaptations to the symbiotic lifestyle.

• In vivo imaging: Developing fluorescently tagged versions for visualization within aphid bacteriocytes to understand subcellular localization and potential host interactions.

• Host factor identification: Screens for aphid proteins that interact with Buchnera ATP synthase components could reveal host-symbiont co-evolution at the molecular level.

• Comparative bioenergetics: Systematic comparison of ATP synthase efficiency across free-living bacteria, facultative symbionts, and obligate symbionts like Buchnera to identify patterns in bioenergetic adaptation during symbiosis evolution.

• Metabolic modeling: Integration of experimental data on ATP synthase function into whole-cell metabolic models of the Buchnera-aphid system to understand energy flux in this symbiotic relationship.

These approaches could illuminate how energy production in Buchnera has adapted to support essential functions like amino acid biosynthesis for the host .

How might systems biology approaches integrate atpE function into broader understanding of the Buchnera-aphid metabolic partnership?

Systems biology offers powerful frameworks for understanding atpE within the broader symbiotic system:

These approaches could reveal emergent properties of the symbiotic system not apparent from studying individual components, potentially identifying energy metabolism as a key regulated interface between host and symbiont .

What protocols need to be adapted when designing experiments with Buchnera proteins compared to proteins from cultivable bacteria?

Working with Buchnera proteins requires specific methodological adaptations:

Additionally, researchers should consider the evolutionary context of Buchnera's reduced genome when interpreting experimental results, as protein function may be affected by the loss of interacting partners or regulatory elements present in free-living bacteria .

What are best practices for setting up reconstitution experiments to study ATP synthase function using recombinant Buchnera components?

Successful reconstitution of ATP synthase function using recombinant Buchnera components requires careful experimental design:

• Component preparation:

  • Express and purify individual subunits under conditions that maintain native-like folding

  • Verify oligomeric state and structural integrity of each component before assembly

  • Consider co-expression of interacting subunits to enhance proper folding

• Membrane mimetic selection:

  • Screen different lipid compositions to identify optimal environments

  • Consider lipid mixtures that mimic the unique composition of Buchnera membranes

  • Test both liposomes and nanodiscs as reconstitution platforms

• Assembly protocol:

  • Develop stepwise assembly protocols starting with stable subcomplexes

  • Monitor assembly using techniques like fluorescence anisotropy or FRET

  • Optimize protein:lipid ratios for efficient incorporation without aggregation

• Functional validation:

  • Establish multiple complementary assays (proton translocation, ATP synthesis)

  • Include positive controls using well-characterized ATP synthase components

  • Verify orientation of incorporated complexes in membrane mimetics

The experimental design should acknowledge limitations in directly comparing to native Buchnera ATP synthase (due to cultivation challenges) and instead focus on internal controls and comparison with homologous systems from other bacteria .