Recombinant Thiobacillus ferrooxidans ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Thiobacillus ferrooxidans?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of F-type ATP synthase in Thiobacillus ferrooxidans (also classified as Acidithiobacillus ferrooxidans). This integral membrane protein forms the c-ring structure within the F0 domain that is essential for proton translocation across the membrane during ATP synthesis. Similar to other F-type ATPases, the c subunits form a ring-like oligomeric structure within the membrane that rotates during catalysis, converting the energy of proton flow into mechanical energy used for ATP synthesis in the F1 sector. In acidophilic organisms like T. ferrooxidans, ATP synthase has adapted to function optimally in low pH environments, making it particularly interesting for bioenergetics research.

The functional significance of ATP synthase is demonstrated by research on similar ATP synthase subunits in other organisms. For instance, in mammals, three isoforms of ATP synthase subunit c exist, differing only in their mitochondrial targeting peptides, yet showing non-redundant functions in respiratory chain maintenance .

How does ATP synthase contribute to energy metabolism in T. ferrooxidans?

ATP synthase plays a pivotal role in the unique energy metabolism of T. ferrooxidans, which can derive energy from the oxidation of ferrous iron and reduced sulfur compounds. The bacterium generates a proton gradient across its membrane during these oxidation processes, and ATP synthase harnesses this gradient for ATP production.

The energy metabolism of T. ferrooxidans is closely tied to its ability to oxidize ferrous ions to ferric ions, a process that can be studied under controlled redox conditions . The resulting proton motive force drives ATP synthesis through the ATP synthase complex. This energy transduction mechanism is crucial for T. ferrooxidans to survive in extremely acidic environments with pH values as low as 1.5-2.0, where other energy sources may be limited.

T. ferrooxidans also possesses ATP sulfurylase (ATPS), which catalyzes ATP and sulfate synthesis from adenosine phosphosulfate (APS) and pyrophosphate (PPi), representing the final stage of sulfite oxidation for energy acquisition . This dual energy generation system highlights the metabolic versatility of this organism in harsh environmental conditions.

What is the relationship between ATP synthase and the acidophilic nature of T. ferrooxidans?

T. ferrooxidans thrives in highly acidic environments (pH 1.5-2.0) where its ATP synthase operates under extreme conditions. The ATP synthase complex, including the c subunit, has evolved specific adaptations to function efficiently in these conditions. These adaptations likely include:

• Modified proton-binding sites in the c-ring that remain functional at low pH

• Structural features that maintain protein stability in acidic conditions

• Specialized regulatory mechanisms that optimize ATP synthesis in fluctuating pH environments

The acidophilic nature of T. ferrooxidans creates a naturally steep proton gradient across the cytoplasmic membrane (external pH ~2, internal pH ~6.5), which the ATP synthase exploits for energy generation. This unusual bioenergetic situation makes the ATP synthase of T. ferrooxidans particularly interesting for studying extreme bioenergetics and could potentially inspire the design of acid-stable energy-generating systems.

What are the optimal expression systems for recombinant T. ferrooxidans ATP synthase subunit c?

Based on successful approaches with related proteins, several expression systems are suitable for recombinant production of T. ferrooxidans ATP synthase subunit c:

• E. coli Expression System: This is the most commonly used approach, as demonstrated with the ATP sulfurylase of A. ferrooxidans, which was successfully expressed in E. coli BL21 StarTM (DE3) . For ATP synthase subunit c, the pET expression system with IPTG induction has proven effective for membrane proteins.

• Mammalian Cell Expression: For proteins requiring complex folding or post-translational modifications, mammalian cell expression systems may be advantageous, as demonstrated for the ATP synthase subunit delta (atpH) from T. ferrooxidans .

Expression Protocol Table for E. coli System:

The protocol should be optimized through small-scale expression trials varying temperature, IPTG concentration, and expression duration to maximize yield and solubility of the target protein.

What purification strategies are most effective for recombinant ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c requires specialized approaches due to its hydrophobic nature as a membrane protein:

• Detergent Extraction: The first critical step involves solubilizing the membrane fraction with appropriate detergents (DDM, LDAO, or C12E8) at concentrations just above their critical micelle concentration.

• Affinity Chromatography: If expressed with a His-tag, immobilized metal affinity chromatography (IMAC) can be used for initial purification, as is common practice for other recombinant proteins from T. ferrooxidans .

• Size Exclusion Chromatography (SEC): This technique helps separate the properly folded protein from aggregates and can also be used for detergent exchange.

• Ion Exchange Chromatography: This may be useful as a polishing step, particularly if the protein has a distinctive isoelectric point.

Purification Workflow:

• Cell lysis by sonication or French press in buffer containing protease inhibitors

• Membrane fraction isolation by ultracentrifugation

• Detergent solubilization of membrane proteins

• IMAC purification

• SEC for further purification and detergent exchange

• Concentration using centrifugal filters designed for membrane proteins

For storage, it's advisable to follow practices similar to those used for ATP synthase subunit delta, which recommends avoiding repeated freeze-thaw cycles and storing working aliquots at 4°C for up to one week, with long-term storage at -20°C in the presence of 50% glycerol .

How can I verify the expression and activity of recombinant ATP synthase subunit c?

Verification of successful expression and functional activity of recombinant ATP synthase subunit c can be accomplished through multiple complementary approaches:

• Expression Verification:

  • SDS-PAGE analysis to confirm the presence of a protein band at the expected molecular weight

  • Western blotting using antibodies against the fusion tag or the protein itself

  • Mass spectrometry to confirm protein identity and integrity

• Structural Integrity Assessment:

  • Circular dichroism (CD) spectroscopy to analyze secondary structure content

  • Limited proteolysis to evaluate proper folding

  • Thermal shift assays to assess protein stability

• Functional Activity Assays:

  • ATP synthesis/hydrolysis assays in reconstituted proteoliposomes

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • Complementation studies in ATP synthase-deficient bacterial strains

The bioluminescence assay technique used for ATP sulfurylase activity determination could be adapted for ATP synthase activity analysis, measuring ATP formation through light emission by the luciferase enzyme . When designing functional assays, it's important to consider the natural acidic environment of T. ferrooxidans and implement appropriate pH controls.

What structural features distinguish T. ferrooxidans ATP synthase subunit c from those of neutrophilic bacteria?

The ATP synthase subunit c from T. ferrooxidans likely possesses unique structural adaptations that enable it to function in extremely acidic environments. Although specific structural data for T. ferrooxidans ATP synthase subunit c is limited in the provided search results, comparative analysis with known structures suggests several key distinguishing features:

• Modified Proton-Binding Site: The conserved carboxylate residue (typically Asp or Glu) in the c-ring that binds protons likely has a modified microenvironment that alters its pKa, allowing protonation/deprotonation cycles to occur efficiently at low pH.

• Enhanced Structural Stability: Increased number of salt bridges, hydrophobic interactions, or disulfide bonds that maintain structural integrity in acidic conditions.

• Specialized Inter-Subunit Interfaces: The interface between c subunits and other components of the ATP synthase complex (particularly subunit a) may contain adaptations that optimize proton transfer under acidic conditions.

By analyzing the available sequence information from related T. ferrooxidans proteins, such as ATP synthase subunit delta (atpH) which has been characterized , researchers can identify potential unique motifs or residues that might confer acid stability.

Structural studies using techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy would be necessary to fully elucidate these distinguishing features.

How does the oligomeric assembly of ATP synthase c-ring differ in T. ferrooxidans compared to other species?

The c-ring of ATP synthase typically consists of 8-15 subunit c monomers, with the exact number varying between species. While specific information about the T. ferrooxidans c-ring stoichiometry is not provided in the search results, several considerations for research in this area include:

• Ring Size Determination: Techniques such as native mass spectrometry, atomic force microscopy, or electron microscopy could determine the number of c subunits in the T. ferrooxidans c-ring.

• Functional Implications: The c-ring size affects the proton-to-ATP ratio and thus the bioenergetic efficiency of ATP synthesis. In acidophiles like T. ferrooxidans, this ratio may be optimized for the naturally large pH gradient.

• Assembly Mechanism: The assembly of c-rings in acidophiles may involve specialized chaperones or assembly factors that ensure proper oligomerization under extreme pH conditions.

What is the role of ATP synthase subunit c in the adaptation of T. ferrooxidans to extreme environments?

ATP synthase subunit c likely plays a crucial role in T. ferrooxidans' adaptation to extreme acidic environments. This adaptation involves:

• pH Resistance: The c subunit must maintain its structure and function at extremely low external pH values while participating in proton translocation from the acidic exterior to the more neutral cytoplasm.

• Energy Efficiency: In energy-limited extreme environments, the c-ring structure may be optimized to maximize ATP yield from the available proton gradient.

• Redox Integration: The ATP synthase in T. ferrooxidans must function in coordination with the organism's unique redox metabolism involving iron and sulfur oxidation, as studied in controlled redox experiments .

• Stress Response: The ATP synthase complex may play a role in the organism's response to environmental stresses beyond pH, such as heavy metal exposure common in acidic mine drainage where these bacteria thrive.

Understanding the specific adaptations of ATP synthase subunit c could provide insights into the fundamental mechanisms of bioenergetic systems under extreme conditions and potentially inform the design of robust energy-generating systems for biotechnological applications.

What mutagenesis approaches are most effective for studying structure-function relationships in T. ferrooxidans ATP synthase subunit c?

Site-directed mutagenesis is a powerful tool for investigating structure-function relationships in ATP synthase subunit c. When designing mutagenesis studies for T. ferrooxidans atpE, consider the following methodological approaches:

• Target Selection Strategy:

  • Conserved proton-binding residues (e.g., the essential carboxylate)

  • Residues at subunit interfaces within the c-ring

  • Residues unique to acidophilic organisms, identified through multiple sequence alignment

  • Residues potentially involved in acid stability

• Mutagenesis Protocol:
  The PCR-based mutagenesis approach used for studying the ATP sulfurylase gene in A. ferrooxidans can be adapted for atpE:

  • Design primers containing the desired mutation

  • Amplify the gene using a high-fidelity polymerase

  • Clone the mutated gene into an expression vector (e.g., pET system)

  • Verify the mutation by sequencing

  • Express in E. coli BL21(DE3) or another suitable host

• Functional Analysis of Mutants:

  • Assess protein expression and stability through SDS-PAGE and western blotting

  • Analyze ATP synthesis activity in reconstituted systems

  • Measure proton translocation efficiency

  • Evaluate acid stability of mutant proteins

  • Determine structural changes using spectroscopic methods

By systematically mutating key residues and characterizing the resulting phenotypes, researchers can map the functional domains of ATP synthase subunit c and identify residues critical for its adaptation to acidic environments.

How can I study protein-protein interactions between ATP synthase subunit c and other components of the complex?

Understanding the protein-protein interactions within the ATP synthase complex is crucial for elucidating its assembly and function. Several methodological approaches can be employed:

• In vitro Interaction Studies:

  • Pull-down assays using tagged recombinant proteins

  • Surface Plasmon Resonance (SPR) to measure binding kinetics

  • Isothermal Titration Calorimetry (ITC) for thermodynamic parameters

  • Chemical cross-linking followed by mass spectrometry to identify interaction interfaces

• In vivo Interaction Studies:

  • Bacterial two-hybrid systems adapted for membrane proteins

  • Co-immunoprecipitation from native or recombinant systems

  • FRET-based approaches using fluorescently labeled subunits

• Structural Studies of Subcomplexes:

  • Cryo-electron microscopy of partially assembled complexes

  • X-ray crystallography of purified subcomplexes

  • NMR studies of interaction domains

• Computational Approaches:

  • Molecular docking simulations

  • Coevolution analysis to predict interaction surfaces

  • Molecular dynamics simulations of the c-ring with neighboring subunits

These approaches can reveal how ATP synthase subunit c interacts with other components, particularly the a subunit, which forms the critical interface for proton translocation, and how these interactions may be specialized in acidophilic organisms like T. ferrooxidans.

What are the best approaches for reconstituting functional T. ferrooxidans ATP synthase in artificial membrane systems?

Reconstitution of functional ATP synthase in artificial membrane systems is essential for detailed biophysical and biochemical studies. For T. ferrooxidans ATP synthase, the following methodological approaches are recommended:

• Liposome Preparation:

  • Use lipid compositions that mimic the native membrane of T. ferrooxidans or are optimized for stability at low pH

  • Prepare liposomes by extrusion through polycarbonate membranes to control size

  • Consider incorporating pH-sensitive fluorescent dyes for monitoring proton translocation

• Protein Incorporation Methods:

  • Detergent-mediated reconstitution with controlled detergent removal using Bio-Beads or dialysis

  • Direct incorporation during liposome formation for the c-ring

  • Step-wise reconstitution of subcomplexes to ensure proper assembly

• Functional Verification:

  • ATP synthesis assays under an artificial pH gradient

  • ATP hydrolysis coupled to proton pumping

  • Rotational assays using fluorescently labeled subunits

  • Patch-clamp electrophysiology to measure proton currents

• Environmental Parameter Optimization:

  • Test functionality across a range of pH values (pH 1.5-7.0)

  • Evaluate the effects of different ion concentrations, particularly iron ions which are relevant to T. ferrooxidans' natural environment

  • Assess temperature dependence of activity

This reconstitution approach allows for controlled studies of ATP synthase function under defined conditions, facilitating the investigation of how this enzyme complex operates in the extreme environments where T. ferrooxidans thrives.

How does ATP synthase subunit c from T. ferrooxidans compare to analogous proteins in other extremophiles?

ATP synthase subunit c from T. ferrooxidans likely shares some adaptations with those from other extremophiles while possessing unique features specific to acidophily. A comparative analysis would include:

• Sequence Alignment Analysis:

  • Compare primary sequences from acidophiles, thermophiles, alkaliphiles, and mesophiles

  • Identify conserved motifs and clade-specific variations

  • Analyze amino acid composition patterns that correlate with environmental adaptation

• Structural Comparison:

  • Examine differences in the proton-binding site architecture

  • Compare oligomeric ring sizes and interface designs

  • Analyze stabilizing elements (salt bridges, hydrophobic interactions) across different extremophiles

• Functional Comparison:

  • Compare proton-to-ATP ratios across species

  • Analyze pH optima and activity ranges

  • Assess thermal stability profiles

• Evolutionary Analysis:

  • Construct phylogenetic trees to trace the evolution of adaptations

  • Identify cases of convergent evolution in different extremophile lineages

  • Analyze horizontal gene transfer events that may have contributed to extremophile adaptations

Such comparative studies can reveal general principles of protein adaptation to extreme environments and identify features that are specifically related to acid tolerance in T. ferrooxidans.

Are there isoforms of ATP synthase subunit c in T. ferrooxidans similar to those observed in mammals?

• Genomic Analysis:

  • Search for multiple atpE genes in the T. ferrooxidans genome

  • Analyze regulatory regions for environmental response elements

  • Compare with the mammalian system where three isoforms exist with identical mature peptides but different targeting sequences

• Transcriptomic Analysis:

  • Perform RNA-Seq under various growth conditions to detect differential expression of potential isoforms

  • Analyze transcript start sites to identify alternative promoters or splice variants

  • Quantify expression levels in response to environmental stresses

• Proteomic Analysis:

  • Use mass spectrometry to identify potential isoforms at the protein level

  • Analyze post-translational modifications that might create functional diversity

  • Examine protein localization patterns

If isoforms exist, they may serve specialized functions similar to the non-redundant roles observed for mammalian ATP synthase subunit c isoforms in respiratory chain maintenance , potentially helping T. ferrooxidans adapt to fluctuating environmental conditions in its extreme habitat.

What are the future research directions for T. ferrooxidans ATP synthase subunit c?

Future research on T. ferrooxidans ATP synthase subunit c should focus on several promising directions: