Recombinant Acidithiobacillus ferrooxidans ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Acidithiobacillus ferrooxidans?

The ATP synthase subunit c (atpE) in A. ferrooxidans is part of the F1FO-ATPase complex that catalyzes ATP synthesis fueled by electrochemical gradients of protons. The F1FO-ATPase from A. ferrooxidans NASF-1 has been purified and characterized as having the typical subunit pattern of F1-ATPase, with 5 subunits of apparent molecular masses of 55, 50, 33, 20, and 18 kDa . The enzyme can hydrolyze ATP, GTP, and ITP, but neither UTP nor ADP, with a Km value for ATP of 1.8 mM .

Subunit c forms a cylindrical oligomer (c-ring) in the FO portion embedded in the membrane, which works in conjunction with subunit a in the proton pumping process. This c-ring plays a crucial role in the rotary mechanism of ATP synthesis by translocating protons across the membrane, converting the electrochemical gradient into mechanical energy that drives ATP synthesis in the F1 portion.

How does the atpE gene from A. ferrooxidans differ from other bacterial species?

The atpE gene from A. ferrooxidans encodes the subunit c protein that has adapted to function optimally in extremely acidic environments (pH 1.5-2.5). When comparing the amino acid sequence of A. ferrooxidans atpE with those from neutrophilic bacteria, several key differences are observed:

• Increased proportion of acidic amino acid residues on the protein surface exposed to the periplasm

• More hydrophobic residues in the transmembrane domains for enhanced membrane stability

• Modified c-ring stoichiometry (the number of c subunits per ring) compared to neutrophilic bacteria

• Specialized residues involved in proton translocation that function efficiently at extreme pH gradients

These adaptations enable A. ferrooxidans ATP synthase to maintain efficient proton translocation despite the extreme pH gradient across its cytoplasmic membrane.

What methods are recommended for cloning and expressing recombinant A. ferrooxidans atpE?

For successful cloning and expression of recombinant A. ferrooxidans atpE, the following methodological approach is recommended:

• Gene amplification: Use PCR to amplify the atpE gene from A. ferrooxidans genomic DNA using high-fidelity DNA polymerase and primers designed with appropriate restriction sites for subsequent cloning .

• Vector selection: For membrane proteins like subunit c, specialized expression vectors such as pET series vectors with a C-terminal His-tag are preferable to facilitate purification without disrupting the N-terminal region that interacts with other ATP synthase subunits.

• Expression host: E. coli C41(DE3) or C43(DE3) strains are recommended as they are designed for toxic and membrane protein expression. Alternatively, consider cell-free protein synthesis systems for membrane proteins.

• Expression conditions: Optimal expression is typically achieved at lower temperatures (16-20°C) after induction with low IPTG concentrations (0.1-0.5 mM) to prevent formation of inclusion bodies.

• Membrane fraction preparation: Use differential centrifugation followed by sucrose gradient ultracentrifugation to isolate membrane fractions containing the recombinant protein.

These methods have been successfully adapted from approaches used for other ATP synthase components in A. ferrooxidans and can be modified based on specific experimental requirements.

How can the functional activity of recombinant A. ferrooxidans atpE be assessed in vitro?

Assessing the functional activity of recombinant atpE requires integration into a functional ATP synthase complex or reconstitution into liposomes. The following methodological approaches are recommended:

• Proton translocation assays: Reconstitute purified recombinant atpE into liposomes containing pH-sensitive fluorescent dyes (such as ACMA or pyranine). Monitor fluorescence changes upon establishment of a pH gradient to assess proton translocation capacity.

• ATP synthesis/hydrolysis measurements: When incorporated into the complete F1FO complex, measure ATP synthesis driven by artificially imposed proton gradients. For A. ferrooxidans ATP synthase, activity is optimum at pH 8.5 and 45°C, and is notably activated by sulfite .

• Rotational analysis: Advanced single-molecule techniques can be employed to analyze the rotation of the c-ring in reconstituted systems, using approaches such as attaching fluorescent probes or gold nanoparticles to the c-ring and tracking their movement under a microscope.

• Inhibitor sensitivity profiling: Characterize the response to known ATP synthase inhibitors. The A. ferrooxidans enzyme exhibits strong inhibition by azide but relative resistance to vanadate, nitrate, and N,N'-dicyclohexylcarbodiimide, which provides a distinctive activity fingerprint .

These functional assessments can help determine whether the recombinant protein retains native-like properties and can provide insights into the specific adaptations of A. ferrooxidans ATP synthase to extreme acidic environments.

What structural techniques are most informative for characterizing recombinant A. ferrooxidans atpE?

Several advanced structural techniques provide complementary information about recombinant A. ferrooxidans atpE:

• Cryo-electron microscopy (cryo-EM): This technique has revolutionized the structural analysis of membrane protein complexes and can provide high-resolution structures of the entire ATP synthase, including the c-ring formed by multiple copies of subunit c.

• X-ray crystallography: Though challenging with membrane proteins, this technique can provide atomic-level details of subunit c, especially when crystallized as part of the c-ring assembly.

• Solid-state NMR spectroscopy: Particularly valuable for membrane proteins, this technique can provide information about dynamic properties and conformational changes of subunit c within lipid bilayers.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This can reveal solvent-accessible regions and conformational dynamics of subunit c, providing insights into protein-protein and protein-lipid interactions.

• Cross-linking coupled with mass spectrometry: This approach can identify interaction interfaces between subunit c and other components of the ATP synthase complex, revealing the structural organization within the supercomplex architecture that has been observed in A. ferrooxidans .

When applying these techniques to A. ferrooxidans atpE, special consideration should be given to maintaining stability under the acidic conditions that mimic its native environment, as structural features may be pH-dependent.

How does the c-ring stoichiometry of A. ferrooxidans ATP synthase influence its bioenergetic efficiency?

The c-ring stoichiometry (number of c subunits per ring) directly determines the ATP synthase's bioenergetic efficiency, as it defines the H+/ATP ratio. For A. ferrooxidans, this relationship is particularly significant given its adaptation to extreme bioenergetic conditions:

• Theoretical considerations: The number of c subunits determines how many protons must flow through the FO domain to generate one molecule of ATP. With each c subunit translocating one proton, a ring with 10 subunits would require 10 protons to synthesize 3 ATP molecules (as the F1 domain has 3 catalytic sites), giving an H+/ATP ratio of approximately 3.3.

• Experimental determination: To determine c-ring stoichiometry, purified c-rings can be analyzed by:

  • Mass determination using analytical ultracentrifugation

  • Direct visualization using atomic force microscopy

  • Protein crosslinking followed by SDS-PAGE analysis

  • Mass spectrometry of intact c-rings

• Bioenergetic implications: A. ferrooxidans faces a unique bioenergetic challenge, operating across an extreme pH gradient. Theoretical calculations suggest that its c-ring stoichiometry may be optimized to function efficiently despite the unusually large proton motive force available to this acidophile.

• Evolutionary adaptation: Comparative analysis across bacterial species living in different environments shows that c-ring stoichiometry varies from 8-15 subunits, suggesting evolutionary adaptation to specific bioenergetic constraints.

Research into the specific c-ring stoichiometry of A. ferrooxidans ATP synthase would provide valuable insights into how this extremophile has optimized its energy conservation mechanisms to thrive in highly acidic environments.

What are the main challenges in expressing functional recombinant A. ferrooxidans atpE?

Expression of functional recombinant A. ferrooxidans atpE presents several challenges with corresponding solutions:

• Membrane protein expression: As a highly hydrophobic membrane protein, atpE tends to form inclusion bodies when overexpressed.

  • Solution: Use specialized E. coli strains (C41/C43), lower expression temperatures (16-20°C), and reduced inducer concentrations. Consider fusion partners that enhance solubility, such as MBP or SUMO.

• Protein toxicity: Expression of foreign membrane proteins can disrupt host membrane integrity, leading to growth inhibition.

  • Solution: Use tightly regulated expression systems and consider cell-free protein synthesis systems that bypass toxicity issues.

• Proper membrane insertion: Ensuring correct folding and membrane insertion in heterologous systems.

  • Solution: Co-express with chaperones like GroEL/GroES or utilize in vitro reconstitution into liposomes after purification under denaturing conditions.

• Stability outside native acidic environment: A. ferrooxidans proteins are adapted to function at extremely low pH.

  • Solution: Maintain acidic conditions during purification where possible, or identify stabilizing buffer conditions through systematic screening.

• Functional assessment: Isolated subunit c may lack activity outside its native complex.

  • Solution: Consider co-expression with other ATP synthase subunits or reconstitution with purified partner subunits from A. ferrooxidans.

These challenges explain why most successful studies on A. ferrooxidans ATP synthase have focused on the purification of the native enzyme complex rather than recombinant expression of individual subunits .

How can researchers troubleshoot protein misfolding issues with recombinant A. ferrooxidans atpE?

When encountering protein misfolding issues with recombinant A. ferrooxidans atpE, researchers can implement the following troubleshooting strategies:

• Optimization of expression conditions:

  • Systematic screening of expression temperatures (10-37°C)

  • Testing various inducer concentrations (0.01-1 mM IPTG)

  • Evaluating different media compositions (including supplementation with zinc, which can stabilize some membrane proteins)

  • Varying expression duration (2-48 hours)

• Detergent screening:

  • Test a panel of detergents for protein extraction and purification

  • Start with mild detergents (DDM, LMNG, C12E8)

  • Consider lipid-detergent mixtures to better mimic native membrane environment

• Thermal stability assays:

  • Implement differential scanning fluorimetry (DSF) to identify stabilizing conditions

  • Screen various pH values, salt concentrations, and additives

  • Use the results to optimize purification buffers

• Site-directed mutagenesis:

  • Identify and modify potentially problematic residues that may interfere with proper folding

  • Consider mutations that enhance stability while maintaining function

  • Use computational prediction tools to guide mutagenesis

• Co-expression approaches:

  • Express atpE alongside other components of the ATP synthase FO domain

  • Include molecular chaperones known to assist membrane protein folding

  • Consider fusion with proteins known to enhance membrane insertion

By methodically applying these approaches, researchers can significantly improve the chances of obtaining correctly folded and functional recombinant A. ferrooxidans atpE.

What purification strategies are most effective for recombinant A. ferrooxidans atpE?

Purification of recombinant A. ferrooxidans atpE requires specialized approaches due to its hydrophobic nature and membrane localization:

• Affinity chromatography:

  • His-tag purification using Ni-NTA resin is most common, but requires optimization of detergent conditions

  • Position the tag at the C-terminus to avoid interference with membrane insertion

  • Consider alternative tags (Strep-tag II, FLAG-tag) if His-tag yields poor results

• Detergent selection:

  • Critical for maintaining protein stability and preventing aggregation

  • DDM (n-dodecyl β-D-maltoside) is often effective for initial extraction

  • Consider detergent exchange during purification to improve protein stability

  • A systematic detergent screen is strongly recommended

• Size exclusion chromatography (SEC):

  • Essential for separating monomeric atpE from aggregates and oligomers

  • Also effective for removing excess detergent

  • Can provide information about the oligomeric state of the protein

• Gradient glycerol density centrifugation:

  • Useful for separating membrane protein complexes

  • Can help identify if recombinant atpE associates with other cellular components

  • Particularly relevant given the observation that ATP synthase components from A. ferrooxidans can form higher-order complexes

• On-column refolding:

  • If inclusion bodies form, consider purification under denaturing conditions followed by on-column refolding

  • Gradually reduce denaturant concentration while protein is bound to affinity resin

  • Supplement with appropriate detergents during refolding

The purification protocol should be validated by functional assays to ensure that the purified protein retains its native conformation and activity.

How does A. ferrooxidans atpE function relate to the organism's unique iron and sulfur oxidation pathways?

A. ferrooxidans atpE function is intimately connected to the organism's extraordinary energy metabolism based on iron and sulfur oxidation:

• Energetic coupling: The ATP synthase c subunit in A. ferrooxidans plays a crucial role in converting the proton gradient generated by iron and sulfur oxidation into ATP. This represents the final step in an energy conservation pathway that begins with electron transfer from Fe(II) to O2 or from reduced sulfur compounds .

• Respiratory supercomplex: Research has demonstrated that A. ferrooxidans possesses a unique supercomplex spanning both inner and outer membranes that couples iron oxidation to oxygen reduction . The ATP synthase, including the c subunit, works in concert with this respiratory machinery, utilizing the proton gradient it generates.

• Substrate-dependent regulation: The energy metabolism in A. ferrooxidans is regulated differently depending on whether the organism is growing on iron or sulfur substrates . These regulatory differences likely extend to the expression and function of ATP synthase components, including atpE.

• Adaptation to extreme conditions: The c subunit of ATP synthase must function efficiently despite the extreme pH gradient across the cytoplasmic membrane of A. ferrooxidans. While the external environment is highly acidic (pH ~2), the cytoplasm is maintained near neutral pH. This means the c-ring must operate across an unusually large proton motive force.

• Integration with electron transport chains: A. ferrooxidans has distinct electron transport chains for iron and sulfur oxidation, both of which ultimately generate a proton gradient harnessed by the ATP synthase complex containing the c subunit .

Understanding the specific adaptations of the atpE gene product provides insights into how A. ferrooxidans couples its unique electron transport mechanisms to ATP synthesis in extreme acidic environments.

What can we learn from the comparison of A. ferrooxidans atpE with equivalent subunits in other extremophiles?

Comparative analysis of A. ferrooxidans atpE with equivalent subunits in other extremophiles reveals important evolutionary adaptations to extreme environments:

• Acidophile-specific adaptations:

  • Compared to neutrophiles, the c subunit in acidophiles typically shows modifications in proton-binding sites

  • A. ferrooxidans atpE may share features with other acidophiles like Picrophilus torridus or Thermoplasma acidophilum

  • These acidophile-specific features likely enable function despite extreme transmembrane pH gradients

• Thermophile comparisons:

  • Thermophilic ATP synthase c subunits (e.g., from Thermus thermophilus) show increased hydrophobicity and ionic interactions for thermal stability

  • A. ferrooxidans atpE likely shows different stabilization strategies focused on acid resistance rather than thermal stability

• Halophile insights:

  • Halophilic ATP synthases face challenges from high salt concentrations

  • Comparing ion-binding sites between halophiles and acidophiles can reveal convergent/divergent strategies for maintaining function under ionic stress

• Psychrophile contrasts:

  • Cold-adapted organisms show flexibility-enhancing substitutions in their c subunits

  • A. ferrooxidans likely displays contrasting rigidity-enhancing features for stability in acidic conditions

• Structural comparison data:

  Extremophile Type	Organism	Key atpE Adaptations	Functional Implication
  Acidophile	A. ferrooxidans	Higher proportion of acid-stable residues	Function at extreme pH gradients
  Thermophile	T. thermophilus	Increased hydrophobic packing	Thermal stability
  Halophile	Halobacterium salinarum	Negative surface charge	Salt resistance
  Psychrophile	Psychrobacter cryohalolentis	Reduced proline content	Cold flexibility

These comparative studies provide insights into the molecular mechanisms of adaptation to extreme environments and can guide protein engineering efforts to create pH-resistant ATP synthases for biotechnological applications.

How might structural information about A. ferrooxidans atpE inform the development of novel antibiotics or biocatalysts?

Structural information about A. ferrooxidans atpE has significant potential for applications in drug discovery and biocatalysis:

• Antibiotic development:

  • ATP synthase is an established antimicrobial target, with drugs like bedaquiline targeting mycobacterial ATP synthase

  • Unique structural features of A. ferrooxidans atpE could inspire the development of narrow-spectrum antibiotics against pathogenic acidophiles

  • Comparative analysis between human and bacterial c subunits could identify bacterial-specific targets

  • The c-ring interfaces and proton-binding sites represent particularly promising drug targets

• Acid-stable biocatalysts:

  • Understanding the acid-stability mechanisms of A. ferrooxidans atpE could inform the engineering of industrial enzymes for acidic processes

  • The protein's ability to function across extreme pH gradients might inspire the design of artificial proton pumps or molecular motors

  • Specific amino acid substitutions responsible for acid stability could be transferred to other proteins

• Bionanotechnology applications:

  • The c-ring structure could serve as a template for designing molecular rotary motors

  • Understanding how the c-ring maintains structural integrity despite extreme conditions could inspire biomimetic nanodevices

  • The protein's natural function in energy conversion makes it an attractive component for bioelectronic devices

• Bioremediation technology:

  • Knowledge of A. ferrooxidans ATP synthase function could improve bioremediation strategies for acid mine drainage

  • Engineering enhanced strains with optimized ATP synthase properties could increase metal recovery efficiency

  • Understanding energy coupling in extreme conditions could lead to improved industrial bioprocesses

• Structural features of interest:

  Structural Feature	Location	Potential Application
  Proton-binding site	Transmembrane region	Drug target, proton pump design
  c-ring/a-subunit interface	Membrane-embedded region	Antimicrobial development
  Oligomerization interfaces	c-ring assembly	Biomolecular engineering
  Lipid-binding domains	Membrane-exposed surfaces	Membrane protein stabilization

The unique adaptations of A. ferrooxidans atpE to extreme conditions make it a valuable model for understanding membrane protein function in acidic environments with significant translational potential.

How might CRISPR-Cas9 gene editing be applied to study A. ferrooxidans atpE function in vivo?

CRISPR-Cas9 gene editing offers promising approaches for studying A. ferrooxidans atpE function, despite the challenges of genetic manipulation in this organism:

• Development of gene editing protocols:

  • Traditional genetic manipulation of A. ferrooxidans has been notoriously difficult

  • CRISPR-Cas9 systems adapted for acidophiles need to be developed with:

    • Codon-optimized Cas9 for A. ferrooxidans expression

    • Acid-stable Cas9 variants

    • Promoters functional in A. ferrooxidans

    • Temperature-optimized protocols for 30°C growth

• Functional atpE studies:

  • Site-directed mutagenesis of key residues in atpE to assess their importance in:

    • Proton binding and translocation

    • c-ring assembly

    • Interaction with other ATP synthase subunits

  • Introduction of epitope tags or fluorescent protein fusions to study localization

  • Creation of conditional knockdown strains using inducible CRISPR interference

• Physiological impact assessment:

  • Measurement of growth rates, ATP production, and proton motive force in mutants

  • Analysis of cross-talk between ATP synthesis and iron/sulfur oxidation pathways

  • Evaluation of acid tolerance in strains with modified atpE

• Integration with omics approaches:

  • Transcriptomic profiling of atpE mutants to identify compensatory responses

  • Proteomic analysis to detect changes in protein complex formation

  • Metabolomic studies to assess broader impacts on cellular metabolism

• Technological considerations:

  • Electroporation protocols optimized for A. ferrooxidans

  • Selection markers functional at low pH

  • Plasmid stability in acidic conditions

  • Delivery methods that overcome potential restriction barriers

While technically challenging, the development of CRISPR-Cas9 tools for A. ferrooxidans would significantly advance our understanding of ATP synthase function in this extremophile and could lead to broader insights into bioenergetics in acidic environments.

What insights might cryo-electron microscopy provide about the A. ferrooxidans ATP synthase c-ring structure?

Cryo-electron microscopy (cryo-EM) offers unprecedented potential for structural insights into the A. ferrooxidans ATP synthase c-ring:

• High-resolution structural features:

  • Determination of the exact c-ring stoichiometry specific to A. ferrooxidans

  • Visualization of proton-binding sites and their spatial arrangement

  • Identification of unique structural adaptations for acid stability

  • Resolution of lipid-protein interactions that may be crucial for function in extreme environments

• Conformational dynamics:

  • Capturing different rotational states of the c-ring relative to other ATP synthase components

  • Visualization of conformational changes associated with proton translocation

  • Understanding the structural basis for coupling between proton flow and ATP synthesis

• Supercomplex architecture:

  • Elucidation of how the ATP synthase complex integrates with other components of the respiratory chain

  • Investigation of the potential respiratory supercomplex described in A. ferrooxidans

  • Identification of unique interface regions that mediate protein-protein interactions within supercomplexes

• Comparative structural biology:

  • Direct comparison with c-rings from neutrophilic organisms to identify acidophile-specific features

  • Structural basis for the optimum activity at pH 8.5 despite evolution in an acidophile

  • Understanding how A. ferrooxidans maintains ATP synthase function across extreme pH gradients

• Methodological advantages:

  • No need for protein crystallization, which is particularly challenging for membrane proteins

  • Visualization of the complex in a near-native lipid environment using nanodiscs or liposomes

  • Potential for time-resolved studies to capture dynamic processes

Recent advances in cryo-EM technology, particularly the development of methods for membrane protein structure determination, make this approach particularly promising for resolving the unique features of the A. ferrooxidans ATP synthase c-ring.

How might synthetic biology approaches incorporate A. ferrooxidans atpE to create acid-resistant energy systems?

Synthetic biology offers exciting possibilities for utilizing A. ferrooxidans atpE in engineered biological systems:

• Acid-resistant ATP production systems:

  • Transfer of A. ferrooxidans atpE to neutrophilic bacteria to enhance acid tolerance

  • Engineering of hybrid ATP synthases with components from different extremophiles

  • Development of simplified synthetic ATP-producing modules based on atpE for incorporation into artificial cells

• Metabolic engineering applications:

  • Enhancement of industrial fermentation processes that generate acidic byproducts

  • Improvement of acid tolerance in biofuel-producing organisms

  • Engineering of microorganisms for enhanced performance in acidic industrial wastewater treatment

• Biomimetic energy conversion devices:

  • Development of artificial proton gradient systems using principles from A. ferrooxidans ATP synthase

  • Creation of acid-stable proton-powered nanomotors based on the c-ring structure

  • Design of biohybrid devices that couple biological components with synthetic materials

• Design strategies:

  Synthetic Biology Approach	Key Components	Potential Application
  Modular ATP synthase engineering	Chimeric c-rings	Acid-tolerant bioproduction
  Minimal synthetic ATP synthase	Simplified c-ring/F1 fusion	Artificial cells
  Directed evolution of atpE	Error-prone PCR libraries	Enhanced acid resistance
  Cell-free energy modules	Purified atpE in synthetic vesicles	Biobatteries

• Implementation challenges:

  • Ensuring proper assembly of modified ATP synthase complexes

  • Maintaining functional interactions between heterologous components

  • Balancing expression levels of synthetic components

  • Preventing proton leakage in engineered systems

These synthetic biology approaches could lead to biotechnological breakthroughs in areas requiring biological activity under acidic conditions, from industrial biocatalysis to environmental bioremediation, while providing fundamental insights into the modular nature and adaptability of biological energy conversion systems.