Recombinant Chloroflexus aurantiacus ATP synthase subunit beta (atpD)

What is the basic structure and function of Chloroflexus aurantiacus ATP synthase subunit beta (atpD)?

Chloroflexus aurantiacus ATP synthase subunit beta (atpD) is a critical component of the F-type ATP synthase (F₁F₀), comprising 471 amino acid residues as part of the catalytic complex that drives ATP synthesis in this ancient photosynthetic bacterium. The protein is classified under EC 7.1.2.2 and has UniProt ID A9WGS4 in Chloroflexus aurantiacus strain ATCC 29366 / DSM 635 / J-10-fl .

The beta subunit forms part of the F₁ catalytic head of the ATP synthase complex, which works in conjunction with the membrane-embedded F₀ domain to convert the proton-motive force into chemical energy in the form of ATP. Unlike typical bacterial ATP synthases, C. aurantiacus ATP synthase exhibits unique structural features, including a pair of peripheral stalks connecting to the F₁ head through a dimer of δ-subunits and two membrane-embedded a-subunits that asymmetrically position outside and clamp the c₁₀-ring . This unique architecture contributes to the specialized function of C. aurantiacus ATP synthase in utilizing the proton gradient generated during photosynthesis.

How does C. aurantiacus ATP synthase differ from other bacterial ATP synthases?

C. aurantiacus ATP synthase exhibits a previously unrecognized architecture with several unique features that distinguish it from other bacterial ATP synthases:

• Dual a-subunit structure: Unlike most bacterial ATP synthases that contain a single a-subunit, C. aurantiacus ATP synthase possesses two membrane-embedded a-subunits that are asymmetrically positioned around the c₁₀-ring .

• Enhanced proton translocation capacity: The dual a-subunit architecture creates two proton inlets on the periplasmic side and two proton outlets on the cytoplasmic side, allowing more protons to be translocated compared to single a-subunit ATP synthases .

• Specialized peripheral stalk arrangement: The enzyme features a unique pair of peripheral stalks that connect to the F₁ head through a dimer of δ-subunits .

• Evolutionary adaptation: These structural differences likely represent adaptations to the light-dependent photosynthetic lifestyle of C. aurantiacus, allowing it to efficiently utilize the proton gradient generated during photosynthesis under various environmental conditions .

This unique architecture makes C. aurantiacus ATP synthase particularly interesting for studying the evolution of energy transduction mechanisms in early photosynthetic organisms.

What are the optimal expression systems for producing recombinant C. aurantiacus ATP synthase subunit beta?

Based on current research methodologies, the optimal expression systems for recombinant C. aurantiacus ATP synthase subunit beta include:

• E. coli expression systems: These are commonly used for expressing thermophilic proteins due to their high yield and simplicity. For C. aurantiacus atpD, strains like BL21(DE3) with pET-based vectors have shown good expression levels. The expression can be optimized with the following parameters:

  • Induction with 0.5-1.0 mM IPTG

  • Expression temperature of 25-30°C (lower than standard 37°C to enhance proper folding)

  • Expression duration of 12-16 hours

  • Addition of rare codon tRNAs when necessary

• Cell-free expression systems: For functional studies requiring proper assembly with other ATP synthase subunits, cell-free systems can be advantageous.

Similar expression approaches have been successfully used for other C. aurantiacus proteins, such as malonyl-CoA reductase, which was produced as a recombinant protein and purified for enzymatic studies .

When expressing the beta subunit alone, it's important to consider that its native function depends on interaction with other ATP synthase subunits. For functional studies, co-expression with other components might be necessary.

What purification challenges are specific to C. aurantiacus ATP synthase subunit beta, and how can they be addressed?

Purification of recombinant C. aurantiacus ATP synthase subunit beta presents several challenges that require specific methodological approaches:

• Thermostability considerations: As C. aurantiacus is a thermophilic organism, its proteins often exhibit increased stability at higher temperatures. Take advantage of this property by incorporating heat treatment steps (60-65°C for 15-20 minutes) early in the purification process to precipitate E. coli host proteins while keeping the target protein soluble.

• Potential aggregation issues: ATP synthase subunits often have hydrophobic regions that can lead to aggregation. Addressing this challenge requires:

  • Using detergents like 0.05-0.1% Triton X-100 or CHAPS during lysis and early purification steps

  • Maintaining adequate ionic strength (150-300 mM NaCl) throughout purification

  • Including 5-10% glycerol as a stabilizing agent in all buffers

• Purification strategy: An effective multi-step purification approach would involve:

  • Initial immobilized metal affinity chromatography (IMAC) using His-tagged constructs

  • Intermediate ion exchange chromatography to remove contaminants

  • Final size exclusion chromatography to obtain pure, homogeneous protein

• Quality control assessment: Verify proper folding and activity through:

  • Circular dichroism to confirm secondary structure

  • ATPase activity assays (with reconstituted complexes or other ATP synthase subunits)

  • Thermofluor assays to assess stability under various buffer conditions

For studies requiring functional ATP synthase complexes, establishing a co-expression and co-purification strategy for multiple subunits would be necessary, potentially using the approaches that have been successful for similar multi-subunit protein complexes from thermophilic organisms.

What are the most effective methods for structural characterization of recombinant C. aurantiacus ATP synthase subunit beta?

Several complementary techniques have proven effective for structural characterization of C. aurantiacus ATP synthase components:

When designing structural studies, researchers should consider combining these methods for comprehensive characterization. For instance, high-resolution cryo-EM of the complete ATP synthase complex can be complemented with crystallographic studies of individual domains to obtain the most complete structural understanding.

How can researchers effectively analyze the interface between ATP synthase subunit beta and other complex components?

Analysis of the interfaces between ATP synthase subunit beta and other components requires specialized techniques focused on protein-protein interactions:

• Computational interface prediction and analysis:

  • Use structural bioinformatics tools to predict interface residues based on available structures

  • Calculate conservation scores across homologs to identify functionally important interface residues

  • Perform molecular dynamics simulations to understand the dynamics of interfaces

• Experimental interface mapping:

  • Cross-linking mass spectrometry (XL-MS) to identify residues in close proximity

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify protected regions upon complex formation

  • Site-directed mutagenesis of predicted interface residues followed by binding and functional assays

• Interface characterization in the context of ATP synthase function:

  • Analyze interfaces in different rotational states observed in cryo-EM structures

  • Compare interfaces with those in ATP synthases from other organisms to identify C. aurantiacus-specific features

  • Correlate interface characteristics with the unique functioning of C. aurantiacus ATP synthase, particularly its dual a-subunit architecture

• Functional validation of interface residues:

  • Generate recombinant mutants with alterations at key interface residues

  • Assess impact on complex formation using size-exclusion chromatography or analytical ultracentrifugation

  • Measure enzymatic activity to correlate structural changes with functional outcomes

The unique architecture of C. aurantiacus ATP synthase, with its dual a-subunits and distinctive peripheral stalk arrangement , makes interface analysis particularly important for understanding how this enzyme has adapted to function efficiently in an early photosynthetic organism.

What assays can be used to measure the ATPase activity of recombinant C. aurantiacus ATP synthase beta subunit?

Several complementary assays can be employed to measure the ATPase activity of recombinant C. aurantiacus ATP synthase components:

• Coupled enzyme assays: These are the gold standard for continuous monitoring of ATPase activity:

  • Pyruvate kinase/lactate dehydrogenase (PK/LDH) assay: This assay couples ATP hydrolysis to NADH oxidation, which can be monitored at 340 nm.

  • Composition: 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 50 mM KCl, 2.5 mM phosphoenolpyruvate, 0.2 mM NADH, 2-5 units of pyruvate kinase and lactate dehydrogenase, and varying concentrations of ATP (0.1-5 mM)

  • Analysis: Calculate activity based on the rate of NADH oxidation (ε₃₄₀ = 6,220 M⁻¹cm⁻¹)

• Malachite green phosphate assay: For endpoint measurements of released phosphate:

  • Procedure: Incubate enzyme with ATP, stop reaction at different timepoints with acid, and measure released phosphate using malachite green reagent

  • Sensitivity: Can detect nanomolar amounts of phosphate

  • Advantage: Less prone to interference from other components

• Luciferase-based ATP consumption assay: Measures remaining ATP after reaction:

  • Procedure: After the ATPase reaction, measure remaining ATP using a luciferase-based ATP detection kit

  • Advantage: Highly sensitive, can be used in high-throughput format

For meaningful functional analysis, consider these methodological aspects:

• Temperature considerations: As C. aurantiacus is thermophilic, perform assays at physiologically relevant temperatures (50-60°C)

• Reconstitution requirements: Isolated beta subunit may show minimal activity; reconstitution with alpha and gamma subunits may be necessary for significant activity

• Data analysis: Calculate kinetic parameters (Km, Vmax) under different conditions using appropriate software

Using these assays, researchers can characterize how mutations, inhibitors, or different environmental conditions affect the ATPase activity of C. aurantiacus ATP synthase components.

How can researchers effectively reconstitute functional C. aurantiacus ATP synthase complexes for in vitro studies?

Reconstitution of functional C. aurantiacus ATP synthase complexes requires careful methodological consideration:

• Co-expression strategy:

  • Design a multi-cistronic expression system (e.g., using pET-Duet or pACYC-Duet vectors) to simultaneously express multiple ATP synthase subunits

  • Include at minimum the alpha, beta, and gamma subunits for F₁-ATPase activity

  • For complete F₁F₀ complex, include membrane subunits with appropriate membrane targeting sequences

• Membrane protein handling:

  • Extract membrane proteins using mild detergents (DDM, LMNG, or Amphipol A8-35)

  • Maintain detergent above critical micelle concentration throughout purification

  • Consider nanodiscs, liposomes, or amphipols for final reconstitution to maintain native-like environment

• Reconstitution into liposomes for proton-pumping studies:

  • Prepare liposomes from E. coli polar lipids or synthetic lipids (DOPC/DOPE/DOPG mixture)

  • Detergent-mediated reconstitution followed by detergent removal using Bio-Beads

  • Verify orientation using accessibility assays (e.g., protease protection)

• Functional validation methods:

  • ATP synthesis assay using acid-base transition

  • Proton pumping assay using pH-sensitive dyes (ACMA or pyranine)

  • ATP hydrolysis assays as described in previous question

• Quality control criteria:

  • Homogeneity assessment by size exclusion chromatography

  • Negative stain EM to verify complex integrity

  • Proteoliposome characterization (size, protein:lipid ratio, orientation)

The unique dual a-subunit architecture of C. aurantiacus ATP synthase presents special challenges for reconstitution. Researchers should verify that both a-subunits are properly incorporated and that the asymmetric positioning around the c-ring is maintained in the reconstituted system.

How does C. aurantiacus ATP synthase subunit beta differ evolutionarily from other bacterial and chloroplast ATP synthases?

C. aurantiacus ATP synthase subunit beta occupies a unique evolutionary position that provides insight into the early divergence of photosynthetic ATP synthases:

• Phylogenetic position:

  • C. aurantiacus represents one of the earliest branches of photosynthetic organisms

  • Its ATP synthase shares features with both bacterial and chloroplast ATP synthases, potentially representing an evolutionary intermediate

• Structural comparisons:

  • C. aurantiacus ATP synthase contains unique structural elements not found in other bacterial ATP synthases, most notably the dual a-subunit architecture

  • While the beta subunit core structure is conserved (as expected for this catalytically critical component), it shows specific adaptations to interact with C. aurantiacus-specific peripheral components

• Functional adaptations:

  • The ATP synthase has evolved to function efficiently in phototrophic conditions, with evidence that C. aurantiacus continuously synthesizes ATP by photophosphorylation even in the presence of O₂

  • This dual capability (functioning under both aerobic and anaerobic conditions) represents an important evolutionary adaptation

• Comparative sequence analysis:

  • Sequence alignment of ATP synthase beta subunits across different organisms reveals conservation of catalytic residues but divergence in regions that interact with other subunits

  • These differences likely reflect adaptation to the specific architecture of C. aurantiacus ATP synthase, particularly its unique peripheral stalk and dual a-subunit arrangement

• Co-evolution with carbon fixation pathways:

  • C. aurantiacus utilizes the 3-hydroxypropionate bi-cycle for autotrophic carbon fixation rather than the Calvin cycle

  • The ATP synthase likely co-evolved with this alternative carbon fixation pathway to meet its specific energy requirements

Understanding these evolutionary differences provides valuable insights into the adaptation of energy conservation mechanisms during the early evolution of photosynthesis.

What insights does the structure of C. aurantiacus ATP synthase provide about the evolution of bioenergetic systems in early photosynthetic organisms?

The unique structure of C. aurantiacus ATP synthase provides several profound insights into the evolution of bioenergetic systems:

• Dual proton pathway innovation:

  • The presence of two a-subunits creating two proton inlets and two outlets represents a novel solution for enhancing proton translocation efficiency

  • This suggests that early photosynthetic organisms evolved specialized adaptations to maximize energy capture from limited light resources

• Adaptation to fluctuating environments:

  • C. aurantiacus can grow via both photosynthesis and aerobic respiration

  • Its ATP synthase structure reveals adaptations that allow it to function efficiently under varying redox conditions, suggesting evolutionary optimization for environmental flexibility

• Intermediate evolutionary features:

  • The unique peripheral stalk arrangements and interaction with the F₁ head through a dimer of δ-subunits represents a previously unrecognized architecture

  • These features may represent evolutionary intermediates between ancestral bacterial ATP synthases and more specialized photosynthetic versions

• Energy conservation optimization:

  • The structure suggests adaptations for maximizing ATP production efficiency under the unique energetic constraints of anoxygenic photosynthesis

  • This provides insight into how early photosynthetic organisms overcame energetic challenges before the evolution of oxygenic photosynthesis

• Integration with carbon fixation:

  • The ATP synthase likely co-evolved with C. aurantiacus' 3-hydroxypropionate bi-cycle for carbon fixation

  • This suggests that the evolution of bioenergetic systems was tightly coupled to the evolution of carbon fixation pathways

These insights highlight how structural analysis of ATP synthases from early-branching photosynthetic organisms like C. aurantiacus can illuminate the evolutionary trajectory of bioenergetic systems, providing a window into the adaptations that enabled the success of photosynthetic life.

How can structural insights from C. aurantiacus ATP synthase inform the engineering of more efficient bioenergetic systems?

The unique structural features of C. aurantiacus ATP synthase offer several promising avenues for engineering enhanced bioenergetic systems:

• Dual proton translocation pathway implementation:

  • The dual a-subunit architecture of C. aurantiacus ATP synthase, which creates two proton inlets and two outlets, could serve as a template for engineering ATP synthases with enhanced proton translocation efficiency

  • This approach could potentially increase the ATP production capacity in synthetic biological systems

• Thermal stability engineering:

  • As a thermophilic organism, C. aurantiacus proteins exhibit enhanced thermal stability

  • Identifying the structural elements that contribute to this stability could inform the design of robust ATP synthases for biotechnological applications under harsh conditions

• Environmental adaptability transfer:

  • C. aurantiacus can continuously synthesize ATP by photophosphorylation even in the presence of O₂

  • Engineering this functional flexibility into other systems could create bioenergetic components that operate efficiently across varying environmental conditions

• Integration with alternative carbon fixation pathways:

  • C. aurantiacus utilizes the 3-hydroxypropionate bi-cycle rather than the Calvin cycle

  • Co-engineering ATP synthases with alternative carbon fixation pathways could optimize energy utilization in synthetic biological systems

• Proton-pumping efficiency improvement:

  • Detailed understanding of the proton translocation mechanism in C. aurantiacus ATP synthase could inform modifications to increase the proton:ATP ratio in engineered systems

  • This could lead to more energy-efficient biological production systems

Methodologically, these engineering efforts would require:

• Precise structure-guided mutagenesis

• Hybrid ATP synthase construction with components from different organisms

• Directed evolution approaches targeting specific performance parameters

• Comprehensive functional validation in reconstituted systems

What are the most promising research directions for further understanding the unique features of C. aurantiacus ATP synthase?

Based on current knowledge, several high-priority research directions would advance understanding of C. aurantiacus ATP synthase:

• Mechanistic studies of dual a-subunit function:

  • Investigate whether the two a-subunits operate synchronously or asynchronously

  • Determine if both proton pathways contribute equally to ATP synthesis

  • Develop single-molecule approaches to track rotational dynamics with two a-subunits

• Regulatory mechanisms under changing environmental conditions:

  • Study how C. aurantiacus ATP synthase activity is regulated during transitions between photosynthetic and respiratory metabolism

  • Investigate post-translational modifications that might modulate ATP synthase function

  • Examine expression regulation under different growth conditions

• Interaction with other bioenergetic components:

  • Characterize interactions between ATP synthase and photosynthetic reaction centers

  • Investigate potential supercomplexes that might coordinate electron transport and ATP synthesis

  • Study membrane organization and potential specialized membrane domains

• Comparative analysis across Chloroflexus species:

  • Perform comprehensive comparative analysis of ATP synthase components across multiple Chloroflexus species and related photosynthetic bacteria

  • Identify conserved unique features versus species-specific adaptations

  • Reconstruct the evolutionary trajectory of this unique ATP synthase architecture

• Integration with systems biology:

  • Develop comprehensive metabolic models incorporating the unique features of C. aurantiacus ATP synthase

  • Study how ATP synthase function is coordinated with the 3-hydroxypropionate bi-cycle

  • Investigate energy budget allocation under different environmental conditions

These research directions would benefit from methodological innovations including:

• Advanced cryo-EM approaches to capture additional functional states

• Single-molecule techniques to study rotational dynamics

• In situ structural studies in native-like membrane environments

• Development of genetic tools for C. aurantiacus to enable in vivo studies

What are common pitfalls when working with recombinant C. aurantiacus ATP synthase components, and how can they be addressed?

Researchers working with recombinant C. aurantiacus ATP synthase components frequently encounter several challenges:

• Expression yield and solubility issues:

  • Problem: Low expression yield or formation of inclusion bodies

  • Solutions:

    • Optimize codon usage for expression host

    • Reduce expression temperature (16-25°C)

    • Use solubility-enhancing fusion tags (SUMO, MBP)

    • Test different E. coli strains (C41/C43 for membrane proteins)

• Protein stability challenges:

  • Problem: Protein degradation or aggregation during purification

  • Solutions:

    • Include protease inhibitors throughout purification

    • Maintain appropriate ionic strength (150-300 mM NaCl)

    • Add stabilizing agents (5-10% glycerol, 1-5 mM ATP)

    • Leverage thermostability by performing purification steps at elevated temperatures (40-50°C)

• Complex assembly difficulties:

  • Problem: Failure to form proper multi-subunit complexes

  • Solutions:

    • Co-expression of multiple subunits rather than separate expression and mixing

    • Gradually remove denaturing agents for controlled refolding

    • Include molecular chaperones during expression (GroEL/ES)

    • Verify correct complex formation by native PAGE and analytical SEC

• Functional inconsistency:

  • Problem: Variable or low enzymatic activity

  • Solutions:

    • Ensure complete removal of inhibitory compounds (imidazole, high salt)

    • Verify presence of essential cofactors (Mg²⁺)

    • Test activity at physiologically relevant temperatures (50-60°C)

    • For complete F₁F₀ complexes, ensure proper reconstitution into membrane mimetics

• Reconstitution challenges:

  • Problem: Poor incorporation into liposomes or nanodiscs

  • Solutions:

    • Optimize protein:lipid ratios

    • Test different detergents for solubilization (DDM, LMNG)

    • Adjust reconstitution temperature and incubation time

    • Verify orientation and functionality after reconstitution

Table 1: Troubleshooting guide for common issues with C. aurantiacus ATP synthase components

What methodological considerations are important when investigating the dual a-subunit architecture of C. aurantiacus ATP synthase?

Investigating the unique dual a-subunit architecture of C. aurantiacus ATP synthase requires specialized methodological approaches:

• Structural analysis considerations:

  • Challenge: Capturing the asymmetric arrangement of dual a-subunits

  • Approaches:

    • High-resolution cryo-EM with extensive particle classification

    • Cross-linking mass spectrometry to map spatial relationships

    • Site-directed spin labeling combined with EPR to measure distances

    • Focused refinement techniques to resolve membrane regions at higher resolution

• Functional analysis of individual a-subunits:

  • Challenge: Determining the contribution of each a-subunit to proton translocation

  • Approaches:

    • Site-directed mutagenesis of key residues in each a-subunit separately

    • Selective labeling of each a-subunit with different probes

    • Development of hybrid complexes with only one functional a-subunit

    • Measurement of proton translocation using pH-sensitive fluorescent dyes

• Expression and assembly verification:

  • Challenge: Ensuring proper incorporation of both a-subunits

  • Approaches:

    • Differential tagging of a-subunits for verification

    • Quantitative mass spectrometry to confirm stoichiometry

    • Antibody-based detection of specific a-subunit epitopes

    • Sequential purification using different affinity tags

• Probing proton pathways:

  • Challenge: Mapping the dual proton translocation pathways

  • Approaches:

    • Molecular dynamics simulations with explicit membrane and water

    • Identification of key residues through conservation analysis and mutagenesis

    • pH-dependent structural or functional studies

    • Proton accessibility studies using chemical probes

• Evolutionary analysis considerations:

  • Challenge: Understanding the evolutionary origin of dual a-subunits

  • Approaches:

    • Comprehensive phylogenetic analysis of a-subunits across diverse species

    • Identification of potential gene duplication events

    • Ancestral sequence reconstruction and characterization

    • Comparative genomics across Chloroflexi phylum

Researchers investigating this unique architecture should combine these methodological approaches to build a comprehensive understanding of how dual a-subunits contribute to the function and efficiency of C. aurantiacus ATP synthase .