Recombinant Chloroflexus aggregans ATP synthase subunit c (atpE)

What is Chloroflexus aggregans ATP synthase subunit c (atpE) and what are its structural characteristics?

ATP synthase subunit c (atpE) from Chloroflexus aggregans is a 76-amino acid membrane protein that forms part of the F0 sector of ATP synthase, a critical enzyme in cellular energy production. The protein has a UniProt ID of B8G6H1 and is encoded by the atpE gene (locus name: Cagg_0990). The amino acid sequence is MEGLNLVATALAVGLGAIGPGVGIGIIVSGAVQAIGRNPEIENRVVTYMFIGIAFTEALAI FGLVIAFLIGFGVLQ, which reveals its highly hydrophobic nature suitable for membrane integration .

The protein serves as a crucial component of the proton-translocating portion of ATP synthase, where it forms a membrane-embedded oligomeric ring structure. This c-ring plays a vital role in the rotary mechanism of ATP synthesis by facilitating proton movement across the membrane, which drives the conformational changes necessary for ATP production.

What is the taxonomic and ecological context of Chloroflexus aggregans?

Chloroflexus aggregans is a filamentous phototrophic bacterium belonging to the phylum Chloroflexi. It forms dense cell aggregates through active gliding movement . This organism is typically found in thermal springs where it contributes to microbial mat formations, often growing alongside cyanobacteria and other thermophilic microorganisms.

C. aggregans is a facultative autotroph that can switch between phototrophic growth using light energy and heterotrophic metabolism. This metabolic versatility contributes to its ecological success in thermal environments where nutrient availability may fluctuate. The bacterium plays an important role in carbon cycling within these specialized ecosystems, utilizing a unique carbon fixation pathway called the 3-hydroxypropionate bi-cycle .

How does the atpE protein function within the context of Chloroflexi metabolism?

The atpE protein functions as part of the ATP synthase complex, which plays a central role in energy conservation across all domains of life. In Chloroflexus aggregans, this protein is particularly interesting because it operates within the context of a versatile metabolism that includes both the ability to perform photosynthesis and to assimilate organic carbon compounds.

Chloroflexus species, including C. aggregans, possess all genes required for the complete 3-hydroxypropionate bi-cycle, which serves as their primary carbon fixation pathway . This pathway is energetically more demanding than the Calvin-Benson cycle used by plants and cyanobacteria, requiring more ATP per fixed carbon. The ATP synthase complex containing the atpE subunit is therefore critical in providing the necessary energy currency for this metabolic process.

The ATP synthase operates at the convergence point of the proton gradient generated either through photosynthetic electron transport or respiratory electron transport, making it a versatile energy conversion system that supports the organism's adaptability to different growth conditions.

What are the optimal conditions for expressing recombinant Chloroflexus aggregans ATP synthase subunit c in E. coli?

Successful expression of recombinant Chloroflexus aggregans ATP synthase subunit c in E. coli requires careful optimization of several parameters:

• Expression system selection: BL21(DE3) or similar E. coli strains designed for membrane protein expression are recommended. Consider using C41(DE3) or C43(DE3) strains specifically developed for toxic or membrane proteins.

• Vector design: The protein has been successfully expressed with an N-terminal His-tag as documented in commercially available products . For optimal expression, consider using vectors with tightly controlled inducible promoters (T7 or tac) and appropriate fusion tags to aid solubility and purification.

• Culture conditions:

  • Growth temperature: 18-25°C after induction (lower temperatures often improve membrane protein folding)

  • Media: Enriched media such as Terrific Broth supplemented with glucose (0.4%) to prevent leaky expression

  • Induction: Use lower IPTG concentrations (0.1-0.5 mM) and induce at mid-log phase (OD600 of 0.6-0.8)

  • Post-induction growth: 12-16 hours at reduced temperature

• Codon optimization: Consider codon optimization for E. coli, as Chloroflexus has different codon usage patterns that may affect translation efficiency.

These conditions should be systematically optimized through small-scale expression trials before scaling up to larger cultures for protein purification.

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

Purification of hydrophobic membrane proteins like ATP synthase subunit c requires specialized approaches:

• Cell lysis and membrane preparation:

  • Use mechanical disruption methods (French press or sonication) in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and protease inhibitors

  • Isolate membranes by ultracentrifugation (100,000 × g for 1 hour)

  • Solubilize membranes using appropriate detergents

• Detergent selection and optimization:

  • Primary solubilization: n-Dodecyl β-D-maltoside (DDM, 1-2%) or n-Octyl β-D-glucopyranoside (OG, 2-3%)

  • For functional studies, consider milder detergents like digitonin (1-2%)

  • Test detergent concentration ranges for optimal solubilization without denaturation

• Affinity chromatography:

  • For His-tagged protein: Ni-NTA resin with imidazole gradient elution (20-250 mM)

  • Include detergent (0.05-0.1%) in all purification buffers

  • Consider on-column detergent exchange if needed for downstream applications

• Additional purification steps:

  • Size exclusion chromatography to remove aggregates and achieve higher purity

  • Ion exchange chromatography as a polishing step

• Storage considerations:

  • Store in buffer containing 50% glycerol at -20°C/-80°C

  • Avoid repeated freeze-thaw cycles

  • For short-term storage, keep aliquots at 4°C for up to one week

The purified protein should be reconstituted in appropriate buffer systems depending on the intended downstream applications, with recommended concentrations of 0.1-1.0 mg/mL in Tris/PBS-based buffer (pH 8.0) with 6% trehalose .

How can researchers assess the functional integrity of purified recombinant atpE protein?

Assessing the functional integrity of ATP synthase subunit c requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm proper secondary structure (predominantly α-helical)

  • Thermal stability assays to determine protein melting temperature and stability

  • Size exclusion chromatography to verify oligomeric state

• Membrane incorporation:

  • Reconstitution into liposomes using a lipid mixture that mimics the native environment

  • Freeze-fracture electron microscopy to visualize c-ring formation in membranes

  • Fluorescence recovery after photobleaching (FRAP) to assess mobility in membranes

• Functional assays:

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Binding assays with other ATP synthase subunits (particularly subunit a)

  • ATP hydrolysis/synthesis activity when reconstituted with other ATP synthase components

• Biophysical interaction studies:

  • Isothermal titration calorimetry (ITC) to measure binding of known inhibitors (e.g., oligomycin)

  • Surface plasmon resonance (SPR) to quantify interactions with other subunits

These multiple lines of evidence provide a comprehensive assessment of whether the purified recombinant protein maintains its native structure and functional properties.

How does the structure and function of Chloroflexus aggregans ATP synthase subunit c compare to homologs in other extremophiles?

The ATP synthase subunit c from Chloroflexus aggregans presents several interesting structural adaptations compared to homologs in other extremophiles:

Functionally, these structural adaptations reflect evolutionary responses to different environmental pressures. The C. aggregans ATP synthase operates at moderately high temperatures but without the extreme pH or salt conditions faced by other extremophiles. The protein likely represents an intermediate adaptation state between mesophilic and hyperthermophilic organisms.

The amino acid sequence (MEGLNLVATALAVGLGAIGPGVGIGIIVSGAVQAIGRNPEIENRVVTYMFIGIAFTEALAI FGLVIAFLIGFGVLQ) shows characteristic features that allow it to function optimally in microbial mats of thermal springs . The positioning of glycine residues and the distribution of polar and non-polar residues suggest a functional adaptation to maintain proper folding and oligomerization at elevated temperatures.

What role does ATP synthase subunit c play in the adaptation of Chloroflexus aggregans to its thermal environment?

ATP synthase subunit c plays a critical role in the thermal adaptation of Chloroflexus aggregans through several mechanisms:

The atpE protein thus represents an important component in a network of adaptations that allow C. aggregans to thrive in thermal environments where few other phototrophs can survive, making it an excellent model for studying thermal adaptation of membrane proteins and bioenergetic systems.

How does the 3-hydroxypropionate bi-cycle in Chloroflexus species relate to ATP synthase function?

The 3-hydroxypropionate bi-cycle and ATP synthase function are intricately connected in Chloroflexus species, including C. aggregans, through energetic and metabolic integration:

• Energetic demands: The 3-hydroxypropionate bi-cycle requires significant ATP input compared to other carbon fixation pathways. For each pyruvate molecule produced, the pathway consumes:

  • 7 ATP molecules directly in carboxylation and activation reactions

  • Additional ATP for downstream biosynthetic processes

• Metabolic flexibility support: Chloroflexus aggregans possesses all the genes required for the complete 3-hydroxypropionate bi-cycle , which enables:

  • Photoautotrophic growth using light energy and CO₂

  • Mixotrophic growth incorporating both CO₂ fixation and organic carbon utilization

  • Heterotrophic growth on acetate and other organic acids

• Integrated regulation: ATP synthase activity and carbon metabolism regulation are coordinated:

  • ATP:ADP ratios influence the activity of key enzymes in the 3-hydroxypropionate bi-cycle

  • Enzyme activities of the 3-hydroxypropionate bi-cycle are "marginally affected when cells were grown heterotrophically with such organic substrates" , suggesting constitutive expression

• Evolutionary significance: The unique metabolic strategy of Chloroflexus was likely "fostered in an environment in which traces of organic compounds can be coassimilated" . This suggests that ATP synthase function evolved to support a flexible metabolism capable of utilizing various energy sources available in thermal microbial mats.

The ATP synthase c-subunit (atpE) thus plays a central role in converting the proton gradient generated through photosynthesis or respiration into the ATP needed to drive the organism's distinctive carbon metabolism, representing a pivotal adaptation to the ecological niche occupied by these bacteria.

What are common challenges in recombinant expression of Chloroflexus aggregans atpE and how can they be addressed?

Researchers frequently encounter several challenges when expressing recombinant Chloroflexus aggregans atpE:

• Poor expression yields:

  • Problem: Membrane proteins often express at low levels in heterologous systems

  • Solutions:

    • Screen multiple E. coli expression strains (BL21, C41/C43, Rosetta)

    • Test different induction parameters (temperature, IPTG concentration, induction time)

    • Consider fusion partners that enhance expression (SUMO, MBP, Mistic)

    • Use specialized media formulations with enhanced buffering capacity

• Protein aggregation and inclusion body formation:

  • Problem: Hydrophobic membrane proteins often misfold and aggregate

  • Solutions:

    • Lower induction temperature to 15-18°C

    • Add chemical chaperones to culture media (glycerol 5%, sorbitol 0.5M)

    • Co-express molecular chaperones (GroEL/ES, DnaK/J)

    • For recovery from inclusion bodies, optimize denaturation and refolding protocols with different detergents

• Poor solubilization efficiency:

  • Problem: Incomplete extraction from membranes

  • Solutions:

    • Screen multiple detergents beyond standard options (DDM, OG)

    • Test newer amphipathic polymers like SMA (styrene-maleic acid) copolymers

    • Optimize detergent:protein ratios systematically

    • Consider sequential extraction with increasing detergent concentrations

• Loss of protein during purification:

  • Problem: Protein precipitation or aggregation during purification steps

  • Solutions:

    • Maintain critical micelle concentration (CMC) of detergent in all buffers

    • Add stabilizing agents (glycerol 10%, specific lipids)

    • Optimize buffer pH and ionic strength

    • Consider on-column detergent exchange to more stable alternatives

Systematic troubleshooting using small-scale expression trials and purification optimization can address most of these challenges, leading to improved yields of functional protein for downstream applications.

What analytical techniques provide the most informative structural insights into Chloroflexus aggregans ATP synthase subunit c?

Multiple complementary analytical techniques can provide comprehensive structural insights into the atpE protein:

A multi-technique approach combining these methods provides the most comprehensive understanding of atpE structure and function in different environmental conditions.

How can researchers design effective mutagenesis studies to investigate key functional residues in Chloroflexus aggregans atpE?

Designing effective mutagenesis studies for atpE requires systematic planning and consideration of the protein's structure-function relationships:

• Target residue selection strategies:

  • Evolutionary conservation analysis: Align atpE sequences across diverse species to identify invariant residues

  • Structural prediction: Focus on residues in predicted functional regions (ion-binding site, subunit interfaces)

  • Homology-based targeting: Select residues corresponding to functionally important positions in better-characterized homologs

  • Chemical logic: Target residues capable of participating in proton transfer (Asp, Glu, His)

• Mutation design principles:

  • Conservative substitutions: Replace residues with chemically similar amino acids (e.g., Asp→Glu) to probe subtle effects

  • Non-conservative substitutions: Replace with dramatically different residues (e.g., Asp→Ala) to abolish function

  • Charge inversions: Replace acidic residues with basic ones and vice versa to test electrostatic hypotheses

  • Special cases: Consider introducing cysteine residues for subsequent labeling studies

• Functional assay selection:

  • Proton translocation: Fluorescence-based assays using pH-sensitive dyes in reconstituted systems

  • ATP synthesis: Coupled enzyme assays when reconstituted with other ATP synthase components

  • Structural integrity: CD spectroscopy and thermal stability assays to confirm proper folding

  • Assembly competence: Co-purification assays with other subunits to test complex formation

• Experimental controls:

  • Wild-type protein expressed and purified in parallel

  • Mutations known to abolish function (negative control)

  • Mutations predicted to preserve function (positive control)

  • Multiple biological replicates with independent protein preparations

By systematically investigating the effects of mutations on protein function, researchers can develop a detailed understanding of the structure-function relationships in Chloroflexus aggregans ATP synthase subunit c, potentially revealing unique adaptations related to its thermophilic lifestyle.

What are the best approaches for reconstituting Chloroflexus aggregans ATP synthase components for functional studies?

Functional reconstitution of ATP synthase components requires careful consideration of membrane mimetics and experimental conditions:

• Lipid selection and optimization:

  • Composition considerations:

    • Synthetic lipids: POPC/POPE mixtures (70:30) provide good baseline performance

    • Natural lipid extracts: E. coli polar lipids closely match bacterial membrane properties

    • Specialized additions: Include cardiolipin (5-10%) to enhance activity

  • Physical properties:

    • Adjust acyl chain length and saturation to match thermal conditions of C. aggregans

    • Consider using archaeal-type lipids with ether linkages for enhanced thermal stability

• Reconstitution methods:

  • Liposome preparation:

    • Thin film hydration followed by extrusion through polycarbonate filters (100-400 nm)

    • Detergent removal techniques: Bio-Beads, dialysis, or gel filtration

    • Protein:lipid ratios: Test range from 1:50 to 1:200 (w/w)

  • Nanodiscs formation:

    • Select appropriate membrane scaffold protein (MSP) variants

    • Optimize MSP:lipid:protein ratios through small-scale screening

  • Polymer-based systems:

    • Amphipols: A8-35 or PMAL-C8 for enhanced stability

    • SMALPs: Direct extraction from membranes while preserving native lipid environment

• Activity verification methods:

  • ATP synthesis:

    • Luciferase-based ATP detection assays

    • Establish proton gradient using acid-base transition or valinomycin-induced K⁺ diffusion

  • Proton pumping:

    • ACMA fluorescence quenching assays

    • Potentiometric dyes for membrane potential measurements

  • Rotation assays:

    • Single-molecule FRET for monitoring conformational changes

    • Direct observation using gold nanoparticle labeling and high-speed microscopy

• Thermostability considerations:

  • Perform functional assays at physiologically relevant temperatures (50-60°C)

  • Include compatible solutes (trehalose, glycine betaine) to enhance stability

  • Consider thermostable fluorescent probes for high-temperature measurements

These approaches allow researchers to investigate the functional properties of Chloroflexus aggregans ATP synthase components under conditions that reflect their native environment, providing insights into thermoadaptation mechanisms.

How does Chloroflexus aggregans ATP synthase subunit c compare with homologs across the bacterial domain?

ATP synthase subunit c shows interesting evolutionary patterns across bacteria, with Chloroflexus aggregans demonstrating distinctive features:

The comparison reveals that while ATP synthase subunit c is highly conserved in its core function across bacteria, there are subtle adaptations in sequence and structure that likely reflect ecological niches and bioenergetic requirements. The Chloroflexus aggregans ATP synthase subunit c shows characteristics consistent with its thermophilic lifestyle while maintaining the core functional elements required for proton translocation.

Interestingly, the ATP synthase genes in Chloroflexi appear to have evolved alongside their unique carbon fixation pathway, the 3-hydroxypropionate bi-cycle, which is energetically more demanding than other carbon fixation pathways . This suggests co-evolution of energy-generating and energy-consuming pathways in these bacteria.

What bioinformatic tools are most suitable for analyzing evolutionary relationships of Chloroflexus aggregans ATP synthase?

Researchers investigating the evolutionary relationships of Chloroflexus aggregans ATP synthase can employ several specialized bioinformatic approaches:

• Sequence analysis tools:

  • Multiple sequence alignment: MAFFT or T-Coffee with specific parameters for membrane proteins

  • Visualization: Jalview with hydrophobicity coloring to highlight membrane-spanning regions

  • Conservation analysis: ConSurf server to map evolutionary conservation onto structural models

• Phylogenetic analysis frameworks:

  • Maximum likelihood methods: IQ-TREE or RAxML with appropriate substitution models for membrane proteins

  • Bayesian inference: MrBayes or PhyloBayes for more robust statistical support

  • Tools for testing evolutionary hypotheses: PAML for detecting sites under selection

• Specialized analyses for ATP synthase:

  • Structure-guided sequence analysis: Partition analysis based on functional domains

  • Coevolution analysis: Looking for correlated mutations between subunits using methods like GREMLIN

  • Horizontal gene transfer detection: Phylogenetic incongruence analysis between ATP synthase genes and species trees

• Recommended analysis workflow:

  • Gather homologous sequences across diverse bacterial phyla

  • Perform careful sequence alignment with manual curation of transmembrane regions

  • Construct phylogenetic trees using multiple methods for robustness

  • Map sequence changes onto structural models to identify functional implications

  • Correlate evolutionary patterns with ecological niches and metabolic strategies

This comprehensive bioinformatic approach can reveal how the ATP synthase c-subunit has evolved in Chloroflexus aggregans compared to other bacteria, potentially identifying adaptive changes that facilitate function in thermal environments or integration with the 3-hydroxypropionate bi-cycle .