Recombinant Methylacidiphilum infernorum ATP synthase subunit b (atpF)

What is the structural role of subunit b (atpF) in Methylacidiphilum infernorum ATP synthase?

Subunit b (atpF) in Methylacidiphilum infernorum is a critical component of the peripheral stalk of the F1FO-ATP synthase complex. Based on homology with other bacterial ATP synthases, it functions as part of the stator structure (peripheral stalk) that connects the membrane-embedded FO domain with the catalytic F1 domain. This peripheral stalk prevents rotation of the α3β3-headpiece during catalysis and serves to smoothen transmission of power between the rotary c-ring and the α3:β3:γ:ε domain . In thermoacidophiles like Methylacidiphilum infernorum, which grow optimally at 50-60°C under acidic conditions, the subunit b likely has evolved specific structural adaptations to maintain stability and function under these extreme conditions .

How does the atpF subunit contribute to ATP synthesis in Methylacidiphilum infernorum?

The atpF subunit doesn't directly participate in the catalytic reaction of ATP synthesis but plays an essential structural role. By forming part of the peripheral stalk (along with subunit δ), it resists the rotational torque generated during proton translocation through the FO domain. This structural stability enables efficient energy transfer from the proton motive force to the catalytic sites where ATP is synthesized. In Methylacidiphilum as a methanotroph, the ATP synthase is crucial for energy production during both methane oxidation and potential hydrogen-based autotrophic growth under microaerobic conditions .

What are the predicted domains and functional regions of Methylacidiphilum infernorum atpF?

While specific structural data for Methylacidiphilum infernorum atpF is limited, comparative analysis with other bacterial ATP synthases suggests it likely contains:

• A transmembrane N-terminal domain that anchors it to the membrane

• A coiled-coil domain that extends from the membrane to interact with the F1 domain

• Potential interaction sites with other peripheral stalk components (subunit δ)

• Specialized temperature and pH adaptation regions appropriate for a thermoacidophile

These domains would enable it to function at the optimal growth conditions of Methylacidiphilum species (50-60°C under acidic conditions) .

What expression systems are most effective for producing recombinant Methylacidiphilum infernorum atpF?

E. coli-based expression systems are commonly used for recombinant production of Methylacidiphilum infernorum atpF . The thermoacidophilic nature of the source organism may present challenges for proper folding in mesophilic expression hosts. Researchers should consider the following approaches:

• Using E. coli strains optimized for membrane protein expression (C41/C43)

• Expression with fusion tags that enhance solubility (MBP, SUMO)

• Codon optimization for the expression host

• Low-temperature induction protocols (16-18°C) to aid proper folding

• Co-expression with chaperones if misfolding occurs

The recombinant protein can be produced through in vitro E. coli expression systems, though yield optimization may require significant protocol adjustments .

What purification strategy yields the highest purity and stability for recombinant atpF?

A multi-step purification approach is recommended for obtaining high-purity recombinant Methylacidiphilum infernorum atpF:

• Initial capture using affinity chromatography (His-tag or other fusion tags)

• Intermediate purification using ion exchange chromatography

• Final polishing step with size exclusion chromatography

For optimal stability during purification, consider these parameters:

• Buffer pH: 5.0-6.0 (reflecting the acidophilic nature of the source organism)

• Temperature: 4°C for purification steps while maintaining thermostability

• Addition of stabilizing agents: glycerol (10-20%), specific lipids, or mild detergents

• Inclusion of reducing agents to prevent oxidation of cysteine residues

After purification, the protein can be stored at -20°C/-80°C with a shelf life of approximately 6 months in liquid form or 12 months in lyophilized form .

How can recombinant atpF be used to study ATP synthase assembly mechanisms?

Recombinant Methylacidiphilum infernorum atpF can serve as a valuable tool for investigating ATP synthase assembly through several experimental approaches:

• In vitro reconstitution experiments:

  • Express and purify individual subunits (including atpF)

  • Combine under controlled conditions to monitor assembly

  • Use analytical techniques (native PAGE, analytical ultracentrifugation) to characterize assembly intermediates

• Interaction studies with partner subunits:

  • Surface plasmon resonance (SPR) or microscale thermophoresis (MST) to quantify binding affinities

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Yeast two-hybrid or bacterial two-hybrid systems to screen for interaction partners

• Structural studies:

  • Cryo-EM analysis of partially assembled complexes

  • X-ray crystallography of subcomplexes containing atpF

  • NMR studies of isolated domains

These approaches can reveal unique assembly characteristics related to the thermoacidophilic adaptations of Methylacidiphilum infernorum ATP synthase .

What methods are recommended for studying the interaction between atpF and other ATP synthase subunits?

To investigate interactions between Methylacidiphilum infernorum atpF and other ATP synthase subunits, consider these methodological approaches:

• Biophysical interaction analysis:

  • Isothermal titration calorimetry (ITC): For thermodynamic parameters of binding

  • Förster resonance energy transfer (FRET): For real-time interaction monitoring

  • Analytical ultracentrifugation: For complex stoichiometry determination

• Structural methods:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To identify interaction surfaces

  • Cryo-EM: To visualize entire complex architecture

  • Disulfide cross-linking: To confirm proximity of specific residues

• Mutational analysis:

  • Alanine scanning of predicted interaction interfaces

  • Deletion constructs to identify minimal binding domains

  • Chimeric constructs with homologous proteins from other species

These approaches can help elucidate how the peripheral stalk components coordinate with other subunits under the thermoacidophilic conditions relevant to Methylacidiphilum infernorum .

How does the structure-function relationship of atpF in Methylacidiphilum infernorum compare to other extremophiles?

The ATP synthase subunit b (atpF) from Methylacidiphilum infernorum likely exhibits specialized adaptations compared to mesophilic bacteria and other extremophiles:

Research approaches to investigate these differences include:

• Comparative sequence analysis across phylogenetic groups

• Structural modeling and molecular dynamics simulations under varying conditions

• Thermal stability assays comparing recombinant proteins from different sources

• Functional complementation experiments in heterologous systems

• Directed evolution studies to identify critical residues

Understanding these adaptations can provide insights into how ATP synthases maintain function under extreme conditions and guide engineering efforts for biotechnological applications .

What role might atpF play in the bioenergetic adaptation of Methylacidiphilum to thermoacidophilic environments?

The atpF subunit likely contributes significantly to bioenergetic adaptation of Methylacidiphilum infernorum to extreme environments through:

• Structural integrity maintenance:

  • Enhanced stability of the peripheral stalk at high temperatures

  • Acid-resistant protein-protein interfaces

  • Specialized electrostatic interactions maintaining assembly

• Energy coupling optimization:

  • Efficient transfer of rotational energy under extreme conditions

  • Potentially modified elastic properties to accommodate thermally-induced structural fluctuations

  • Specialized interfaces with both membrane and soluble components

• Integration with unique metabolic pathways:

  • Coordination with methane oxidation pathways specific to Methylacidiphilum

  • Adaptation to varying electron transport chains based on substrate availability

  • Possible roles in reverse electron flow required for autotrophic growth on hydrogen

Exploring these adaptations requires integrative approaches combining structural biology, biophysics, and metabolic engineering. As a thermoacidophilic methanotroph, Methylacidiphilum infernorum's ATP synthase has likely evolved unique features to support growth on methane and potentially hydrogen under microaerobic conditions at high temperatures and low pH .

How can site-directed mutagenesis of atpF be used to investigate the thermostability mechanisms of Methylacidiphilum infernorum ATP synthase?

Site-directed mutagenesis of Methylacidiphilum infernorum atpF provides a powerful approach to understand thermostability mechanisms through targeted modifications:

Experimental Design Strategy:

• Identify candidate residues through:

  • Sequence alignment with mesophilic homologs to identify divergent positions

  • Structural prediction to locate stabilizing interactions

  • Computational analysis of charged residue networks

  • Hydrophobic core analysis

• Create systematic mutations:

  • Thermostability-conferring to thermolability-inducing substitutions

  • Introduction of mesophilic equivalents at key positions

  • Charge-neutralizing or charge-reversing mutations at surface positions

  • Disruption of predicted salt bridges or hydrogen bonding networks

• Analyze effects through:

  • Thermal stability assays (differential scanning calorimetry, thermal shift assays)

  • Functional reconstitution to assess activity at different temperatures

  • Structural characterization of mutants compared to wild-type

  • In silico molecular dynamics simulations to predict conformational changes

This systematic approach can reveal specific adaptations that enable function at the high temperature optimum (50-60°C) characteristic of Methylacidiphilum species .

How does the structure and function of atpF in Methylacidiphilum infernorum compare to that in mycobacteria?

While specific structural data for Methylacidiphilum infernorum atpF is limited, comparison with the better-characterized mycobacterial ATP synthases reveals important differences and similarities:

The peripheral stalk in mycobacteria, which includes the b subunit (atpF), plays a critical role in smoothening power transmission between the rotary c-ring and the α3:β3:γ:ε domain . Whether Methylacidiphilum infernorum employs similar structural mechanisms adapted to thermoacidophilic conditions remains to be fully characterized.

What can comparative genomics reveal about the evolution of atpF in thermoacidophilic methanotrophs?

Comparative genomics approaches can provide valuable insights into atpF evolution in thermoacidophilic methanotrophs like Methylacidiphilum infernorum:

• Phylogenetic analysis can:

  • Trace the evolutionary history of atpF relative to other ATP synthase components

  • Identify signatures of positive selection at specific residues

  • Reveal potential horizontal gene transfer events

  • Map coevolution with other ATP synthase subunits

• Structural prediction based on sequence conservation can:

  • Identify highly conserved regions critical for function

  • Highlight thermoacidophilic-specific adaptations

  • Predict subunit interaction interfaces

• Genomic context analysis can reveal:

  • Organization of ATP synthase genes in different methanotrophs

  • Potential regulatory elements specific to thermoacidophiles

  • Co-evolution with metabolic pathways unique to Methylacidiphilum

Research approaches should integrate these computational analyses with experimental validation using recombinant proteins. This may reveal how Methylacidiphilum's ATP synthase has adapted to support both methanotrophic metabolism and potential hydrogen-based autotrophic growth under thermoacidophilic conditions .

What are common challenges in working with recombinant Methylacidiphilum infernorum atpF and how can they be addressed?

Researchers working with recombinant Methylacidiphilum infernorum atpF may encounter several challenges, each requiring specific troubleshooting approaches:

• Low expression yields:

  • Optimize codon usage for expression host

  • Test different promoter systems

  • Evaluate various E. coli strains (BL21(DE3), C41/C43, Rosetta)

  • Optimize induction conditions (temperature, IPTG concentration, induction time)

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

• Protein aggregation/insolubility:

  • Express at lower temperatures (16-20°C)

  • Include solubilizing agents in lysis buffer (mild detergents, higher salt)

  • Try different buffer systems reflecting acidic preference of source organism

  • Co-express with chaperones

  • Consider refolding from inclusion bodies

• Protein instability:

  • Store at appropriate temperature (-20°C/-80°C)

  • Include stabilizing agents (glycerol, specific lipids)

  • Consider lyophilization for longer storage (12 months stability)

  • Test different buffer compositions for pH stability

• Functional assays:

  • Ensure proper reconstitution with partner subunits

  • Test activity under conditions mimicking thermoacidophilic environment

  • Verify protein folding through circular dichroism or limited proteolysis

These approaches should be systematically evaluated to optimize work with this challenging protein from an extremophilic organism.

How can researchers verify the correct folding and functionality of recombinant Methylacidiphilum infernorum atpF?

Verifying proper folding and functionality of recombinant Methylacidiphilum infernorum atpF requires several complementary approaches:

• Structural integrity assessment:

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

  • Intrinsic fluorescence to evaluate tertiary structure

  • Size exclusion chromatography to assess oligomeric state

  • Limited proteolysis to confirm compact folding

  • Thermal shift assays to determine stability profiles

• Functional verification:

  • Binding assays with known interaction partners (other ATP synthase subunits)

  • Co-purification experiments with partner subunits

  • Assembly into larger subcomplexes

  • Complementation studies in heterologous systems

• Activity reconstitution:

  • Assembly of minimal functional units with other purified subunits

  • Liposome reconstitution for membrane-associated functionality tests

  • ATP synthesis/hydrolysis assays of reconstituted complexes

• Thermal stability verification:

  • Activity retention after heat treatment (appropriate for a thermophile)

  • Comparing stability at different pH values reflecting acidophilic nature

  • Long-term storage stability tests

These methods should be performed under conditions relevant to the thermoacidophilic nature of Methylacidiphilum infernorum, which grows optimally at 50-60°C under acidic conditions .

How might understanding atpF structure contribute to engineering ATP synthases for biotechnological applications?

The study of Methylacidiphilum infernorum atpF provides insights that could advance ATP synthase engineering for biotechnology:

• Thermal stability engineering:

  • Identifying thermostability determinants from Methylacidiphilum atpF

  • Transferring these features to mesophilic ATP synthases

  • Developing ATP synthases functional at industrial temperatures

  • Creating chimeric proteins with enhanced stability profiles

• pH tolerance optimization:

  • Understanding acidophilic adaptations in peripheral stalk components

  • Engineering broader pH optima for industrial applications

  • Developing ATP synthases resistant to pH fluctuations

• Metabolic engineering applications:

  • Integration with artificial photosynthetic systems

  • Coupling to biofuel production pathways

  • Development of ATP-generating systems for cell-free biotechnology

  • Creation of modular bioenergetic components for synthetic biology

These applications could leverage the natural adaptations of Methylacidiphilum infernorum ATP synthase to extreme conditions, potentially enabling more robust bioenergetic systems for industrial biotechnology .

What insights could structural studies of Methylacidiphilum infernorum atpF provide about ATP synthase adaptation to extreme environments?

Structural studies of Methylacidiphilum infernorum atpF could reveal several important adaptations to extreme environments:

• Thermostability mechanisms:

  • Specialized salt bridge networks

  • Enhanced hydrophobic core packing

  • Reduced flexibility in loop regions

  • Strategic placement of proline residues

• Acidophilic adaptations:

  • Modified surface charge distribution

  • Specialized proton-handling residues

  • pH-dependent conformational stability

  • Acid-resistant subunit interfaces

• Integration with methanotrophic metabolism:

  • Potential structural adaptations related to energy coupling

  • Interface adaptations with other subunits optimized for thermoacidophilic conditions

  • Specialized regulatory mechanisms

• Comparative insights:

  • Structural features distinct from human ATP synthase (potential drug targets)

  • Convergent evolution with other extremophilic ATP synthases

  • Unique adaptations specific to thermoacidophilic methanotrophs