Recombinant Methylibium petroleiphilum ATP synthase subunit c (atpE)

What is Methylibium petroleiphilum and why is its ATP synthase subunit c of research interest?

Methylibium petroleiphilum is a methylotrophic bacterium belonging to the Comamonadaceae family of beta-Proteobacteria. It has gained significant research attention due to its unique ability to completely metabolize methyl tert-butyl ether (MTBE), a fuel oxygenate and environmental contaminant . The ATP synthase subunit c (atpE) is a critical component of the F1F0-ATP synthase complex responsible for ATP production via oxidative phosphorylation. This protein functions as part of the membrane-embedded F0 portion of the complex, specifically forming a cylindrical oligomer that participates in proton translocation across the membrane. Research interest in this protein stems from both its fundamental role in bioenergetics and potential applications in bioremediation research related to M. petroleiphilum's metabolic capabilities .

What are the structural characteristics of Methylibium petroleiphilum ATP synthase subunit c?

The Methylibium petroleiphilum ATP synthase subunit c (atpE) is a relatively small protein consisting of 82 amino acids with the sequence: MEHVLGFVALAAGLIIGLGAIGACIGIGIMGSKYLESAARQPELMNELQTKMFLLAGLIDAAFLIGVGIAMMFAFANPFVLK . The protein exhibits characteristic hydrophobic regions consistent with its membrane-spanning function, containing multiple glycine and alanine residues that facilitate tight packing within the membrane environment. As with other ATP synthase c subunits, it likely forms a cylindrical oligomeric structure within the membrane component of the ATP synthase complex. The protein contains transmembrane helices that create the proton channel essential for the chemiosmotic coupling mechanism of ATP synthesis .

How is recombinant Methylibium petroleiphilum ATP synthase subunit c typically expressed and purified?

The recombinant Methylibium petroleiphilum ATP synthase subunit c (atpE) protein is typically expressed in E. coli expression systems using a vector that introduces an N-terminal His-tag . This approach facilitates purification while maintaining protein functionality.

Methodological approach:

• Clone the atpE gene from Methylibium petroleiphilum into an expression vector with an N-terminal His-tag

• Transform into competent E. coli cells

• Induce protein expression with IPTG or similar inducer

• Harvest cells and lyse under denaturing conditions (due to the hydrophobic nature of the protein)

• Purify using nickel affinity chromatography

• Perform dialysis to remove denaturants and allow refolding

• Concentrate and lyophilize the purified protein

The resulting product has >90% purity as determined by SDS-PAGE and is typically supplied as a lyophilized powder in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 .

What are the optimal storage and reconstitution conditions for recombinant ATP synthase subunit c?

For optimal research outcomes with recombinant Methylibium petroleiphilum ATP synthase subunit c, proper storage and reconstitution are crucial:

Storage recommendations:

• Store lyophilized protein at -20°C to -80°C upon receipt

• Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

• Aliquot for storage at -20°C/-80°C

Researchers should note that repeated freeze-thaw cycles significantly reduce protein activity and should be avoided. For membrane protein studies requiring functional reconstitution into lipid bilayers, additional steps including detergent removal and controlled incorporation into liposomes may be necessary.

How does the structure and function of Methylibium petroleiphilum ATP synthase subunit c compare to other bacterial species and eukaryotic homologs?

Understanding the evolutionary and functional relationships between ATP synthase subunit c from various organisms provides valuable research insights:

In mammals, ATP synthase subunit c is encoded by three different nuclear genes (ATP5G1, ATP5G2, and ATP5G3) that produce identical mature proteins but with different mitochondrial targeting peptides . Research has demonstrated that these targeting peptides serve functions beyond protein import, including maintenance of respiratory chain structure and function. Knockdown experiments have shown that the isoforms are not functionally redundant despite having identical mature sequences .

The bacterial ATP synthase c subunits, including that from M. petroleiphilum, lack these targeting sequences but may have evolved specific adaptations to their ecological niches. When designing comparative studies, researchers should consider these structural and functional differences that may impact experimental outcomes.

What experimental challenges exist when working with recombinant ATP synthase subunit c, and how can they be addressed?

Working with ATP synthase subunit c presents several significant research challenges due to its highly hydrophobic nature and membrane localization:

• Solubility and aggregation issues:

  • Challenge: The protein's hydrophobic domains promote aggregation in aqueous solutions

  • Solution: Utilize appropriate detergents (DDM, CHAPS, or Triton X-100) at concentrations above their critical micelle concentration

  • Methodological approach: Screen multiple detergents at various concentrations; consider protein:detergent ratios of 1:50 to 1:200 by weight

• Maintaining native structure:

  • Challenge: Detergents may disrupt the native oligomeric state

  • Solution: Consider amphipols or nanodiscs for structural studies; use circular dichroism to verify secondary structure maintenance

• Functional reconstitution:

  • Challenge: Achieving proper orientation in artificial membranes

  • Solution: Controlled, slow detergent removal during liposome incorporation; consider using a pH gradient during reconstitution

• Protein-lipid interactions:

  • Challenge: Specific lipid requirements for optimal function

  • Solution: Test reconstitution with different lipid compositions; consider including cardiolipin for bacterial ATP synthase studies

When designing experiments, researchers should consider implementing control experiments with well-characterized ATP synthase c subunits from model organisms alongside the M. petroleiphilum protein to benchmark results and identify organism-specific characteristics.

How can researchers effectively study the role of ATP synthase subunit c in Methylibium petroleiphilum's metabolic capabilities?

Investigating the relationship between ATP synthase function and M. petroleiphilum's unique metabolic capabilities, particularly MTBE degradation, requires integrated methodological approaches:

• Gene knockout/knockdown studies:

  • Generate atpE knockout or conditional knockdown strains

  • Assess impact on growth, ATP production, and MTBE degradation

  • Complement with wild-type or mutant versions to confirm specificity

• Metabolic flux analysis:

  • Use 13C-labeled substrates to trace metabolic pathways with and without functional ATP synthase

  • Quantify flux changes in central carbon metabolism and MTBE degradation pathways

  • Correlate ATP production efficiency with metabolic pathway activities

• Transcriptomic and proteomic integration:

  • Perform RNA-Seq and proteomics under different growth conditions

  • Analyze co-expression patterns between ATP synthase components and MTBE degradation enzymes

  • Identify potential regulatory links between energy metabolism and xenobiotic degradation

• Bioenergetic measurements:

  • Measure membrane potential, proton motive force, and ATP synthesis rates

  • Compare energy parameters during growth on different carbon sources including MTBE

  • Evaluate how energy conservation efficiency affects degradation capabilities

M. petroleiphilum's genome contains multiple biodegradation pathways and has both chromosomal and plasmid-encoded functions related to its metabolic versatility . Research indicates possible connections between cobalamin (vitamin B12) availability and MTBE/TBA degradation efficiency . This suggests complex regulatory networks connecting energy metabolism, cofactor availability, and xenobiotic degradation that warrant thorough investigation.

What structural and functional analysis techniques are most appropriate for characterizing ATP synthase subunit c?

Comprehensive characterization of M. petroleiphilum ATP synthase subunit c requires multiple complementary techniques:

When implementing these methods, researchers should consider the following:

• Begin with sequence-based structural predictions and comparative modeling based on known c-subunit structures

• Design strategic mutations to probe key functional residues, particularly those involved in proton binding and translocation

• Combine in vitro reconstitution studies with in vivo functional assays to connect structural insights with physiological relevance

• Consider the native lipid environment and its impact on structure and function

Advances in native mass spectrometry and hydrogen-deuterium exchange techniques have recently improved the accessibility of structural information for membrane proteins like ATP synthase subunit c, providing new opportunities for detailed characterization .

How does ATP synthase subunit c function relate to Methylibium petroleiphilum's environmental adaptations?

M. petroleiphilum has evolved as a specialized methylotroph capable of degrading environmental pollutants like MTBE. The ATP synthase complex plays a critical role in this adaptation through several interconnected mechanisms:

• Energy conservation efficiency:

  • The ATP synthase c-ring composition (number of c subunits per ring) directly affects the H+/ATP ratio

  • Efficient energy conservation would be advantageous when growing on challenging carbon sources like MTBE

  • Research suggests potential adaptations in the c subunit that optimize ATP production under the specific bioenergetic constraints of MTBE metabolism

• Stress response integration:

  • Environmental stressors (pH, temperature, toxic compounds) affect proton motive force

  • ATP synthase structure and regulation must be optimized to maintain function under stress conditions

  • The atpE gene may have co-evolved with other stress response systems relevant to contaminated environments

• Metabolic network integration:

  • M. petroleiphilum contains multiple biodegradation pathways with complex regulation

  • Genome analysis reveals clusters of cob genes for cobalamin synthesis near MTBE degradation genes

  • The ATP synthase likely participates in regulatory networks connecting energy status with expression of degradation pathways

The bacteria's genome structure, with both chromosomal and plasmid components, suggests relatively recent acquisition of some metabolic capabilities . This raises interesting research questions about whether the ATP synthase components have undergone selection pressure for optimal function with these newly acquired pathways.

What are the current gaps in understanding ATP synthase subunit c function in non-model organisms like Methylibium petroleiphilum?

Despite significant advances in ATP synthase research, several knowledge gaps persist regarding ATP synthase subunit c in organisms like M. petroleiphilum:

• Species-specific regulatory mechanisms:

  • How is atpE expression regulated in response to different carbon sources?

  • Are there specific transcription factors or small RNAs that control ATP synthase composition?

  • Does growth on MTBE alter ATP synthase stoichiometry or regulation?

• Structural adaptations:

  • Does the M. petroleiphilum c-ring structure differ from model organisms?

  • Are there specific amino acid changes that optimize function for this organism's ecological niche?

  • How do lipid-protein interactions in M. petroleiphilum compare to well-studied systems?

• Integration with unique metabolic pathways:

  • How does ATP synthase function coordinate with the specialized MTBE degradation pathways?

  • Is there direct regulation between energy status and expression of xenobiotic degradation genes?

  • Do specific metabolites or cofactors (like vitamin B12) influence ATP synthase assembly or function?

• Evolution and horizontal gene transfer:

  • Did components of the ATP synthase co-evolve with the plasmid-encoded metabolic capabilities?

  • Is there evidence of selective pressure on ATP synthase genes in MTBE-degrading populations?

Addressing these gaps requires integrated research approaches combining molecular genetics, biochemistry, structural biology, and systems biology. The development of genetic tools specifically for M. petroleiphilum would significantly advance this research area.

What emerging technologies might advance understanding of ATP synthase subunit c structure and function?

Several cutting-edge technologies show promise for advancing ATP synthase subunit c research:

• High-resolution cryoEM approaches:

  • Recent advances allow structural determination of membrane proteins at near-atomic resolution

  • Time-resolved cryoEM could potentially capture different conformational states during the catalytic cycle

  • Application to M. petroleiphilum ATP synthase could reveal unique structural adaptations

• Single-molecule techniques:

  • FRET-based approaches to monitor conformational changes during function

  • Magnetic tweezers or optical traps to measure mechanical forces during ATP synthesis

  • Single-particle tracking in live cells to understand dynamic assembly processes

• Synthetic biology approaches:

  • Designer ATP synthases with modified c-rings to test structure-function hypotheses

  • Minimal synthetic cells incorporating purified ATP synthase components

  • Creation of chimeric systems combining components from different species

• Advanced computational methods:

  • Molecular dynamics simulations incorporating realistic membrane environments

  • Machine learning approaches to predict functional impacts of sequence variations

  • Systems biology models integrating ATP synthase function with whole-cell metabolism

These technologies, applied to M. petroleiphilum ATP synthase research, could provide unprecedented insights into how this enzyme system contributes to the organism's unique metabolic capabilities and environmental adaptations.

How might research on M. petroleiphilum ATP synthase contribute to bioremediation applications?

Understanding ATP synthase function in M. petroleiphilum has several potential applications for advancing bioremediation technologies:

• Optimizing bioremediation efficiency:

  • Knowledge of bioenergetic constraints could help design optimal conditions for MTBE degradation

  • Engineering ATP synthase for improved energy conservation might enhance degradation rates

  • Understanding the link between energy metabolism and degradation pathways could inform nutrient supplementation strategies

• Biomarker development:

  • ATP synthase expression patterns could serve as indicators of metabolic activity in environmental samples

  • Monitoring energy metabolism genes alongside degradation genes could provide more comprehensive assessment of bioremediation progress

• Synthetic biology applications:

  • Transferring optimized energy conservation systems to other biodegrading organisms

  • Creating synthetic consortia with complementary energy and degradation capabilities

  • Engineering regulatory links between sensing of pollutants and enhanced ATP synthesis