Recombinant Methylococcus capsulatus ATP synthase epsilon chain 2 (atpC2)

What is ATP synthase epsilon chain 2 (atpC2) in Methylococcus capsulatus?

ATP synthase epsilon chain 2 (atpC2) is a protein subunit of the F-type ATP synthase complex in Methylococcus capsulatus, a methanotrophic bacterium capable of utilizing methane as its sole carbon and energy source. The epsilon subunit is part of the F1 catalytic domain of ATP synthase and plays a crucial role in regulating ATP synthesis by coupling the rotation of the central stalk to the catalytic activity of the enzyme. In M. capsulatus, genome analysis has revealed the presence of two copies of genes encoding ATP synthase components, including the epsilon chain . This duplication may reflect adaptation to different energy demands or environmental conditions faced by this specialized bacterium.

The presence of two ATP synthase gene sets in M. capsulatus is particularly notable as this organism needs to efficiently manage energy production while oxidizing methane, which is a distinctive metabolic feature of methanotrophs. The epsilon chain functions as part of the rotary motor mechanism that drives ATP synthesis, connecting the proton-translocating FO portion with the catalytic F1 portion of the ATP synthase complex.

How does atpC2 differ from atpC1 in Methylococcus capsulatus?

The M. capsulatus genome contains two copies each of the genes necessary to assemble functional ATP synthase complexes . The differences between atpC1 and atpC2 likely reflect functional specialization that allows the bacterium to adapt to varying energy requirements under different growth conditions. While specific sequence comparison data for the epsilon chains is not detailed in available research, the pattern observed with other ATP synthase subunits suggests these paralogs may have diverged to optimize function in different cellular contexts.

Similar to what has been observed with the ATP synthase subunit a 2 (atpB2), which has been characterized in detail , atpC2 likely possesses unique amino acid sequences that contribute to specialized functions. These differences may affect interactions with other ATP synthase subunits, influence the efficiency of energy coupling, or modify regulatory properties of the complex. Research comparing the expression patterns of these paralogs under various growth conditions would provide valuable insights into their specialized roles.

What is the role of atpC2 in the energy metabolism of M. capsulatus?

In M. capsulatus, the ATP synthase complex plays a critical role in energy production, particularly in relation to the organism's unique methane oxidation pathways. The epsilon chain 2 (atpC2) contributes to this process by helping regulate the ATP synthesis activity of the complex in response to cellular energy demands. M. capsulatus has evolved sophisticated electron transfer mechanisms to support methane oxidation, and the ATP synthase complex is an integral part of this energy conversion system .

The genome-scale metabolic model for M. capsulatus Bath has identified three possible modes of electron transfer that support energy generation: the redox-arm, direct coupling, and uphill electron transfer . Each of these modes likely places different demands on the ATP synthase complex. The epsilon subunit, as a regulatory component, may help optimize ATP synthesis efficiency under these varying conditions. Since M. capsulatus possesses two sets of ATP synthase genes, it is possible that each set is preferentially expressed or activated depending on which electron transfer mode is dominant under specific growth conditions.

What expression systems are optimal for producing recombinant M. capsulatus atpC2?

Based on successful expression strategies for other ATP synthase subunits from M. capsulatus, E. coli-based expression systems appear to be effective for recombinant production of atpC2. For instance, the ATP synthase subunit a 2 (atpB2) has been successfully expressed in E. coli with an N-terminal His-tag . A similar approach could be applied to atpC2 production.

For optimal expression of M. capsulatus atpC2, researchers should consider the following methodological approach:

• Vector selection: pET-based expression vectors under the control of T7 promoter are recommended for high-level expression.

• Affinity tag placement: An N-terminal His-tag facilitates purification while minimizing interference with protein folding.

• Host strain selection: E. coli BL21(DE3) or Rosetta strains are preferred, especially if the target protein contains rare codons.

• Expression conditions: Induction with 0.5-1.0 mM IPTG at lower temperatures (16-25°C) over extended periods (16-24 hours) often improves soluble protein yield.

• Protein extraction: Gentle lysis methods using lysozyme combined with mild detergents help preserve protein structure.

Purification can be achieved using immobilized metal affinity chromatography (IMAC) with a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, similar to the conditions used for atpB2 . The purified protein should be stored at -20°C/-80°C with the addition of 5-50% glycerol to prevent freeze-thaw damage.

How can researchers study interactions between atpC2 and other ATP synthase subunits?

Investigating protein-protein interactions between atpC2 and other ATP synthase subunits requires a multi-faceted approach that combines structural, biochemical, and genetic techniques:

• Co-immunoprecipitation (Co-IP): Using antibodies specific to atpC2 or other ATP synthase subunits to pull down protein complexes, followed by Western blot analysis or mass spectrometry to identify interacting partners.

• Bacterial two-hybrid system: This genetic approach can screen for interactions between atpC2 and other ATP synthase subunits in vivo, providing insights into the assembly of the complex.

• Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC): These techniques can quantify binding affinities between purified atpC2 and other subunits under controlled conditions.

• Cross-linking coupled with mass spectrometry: This approach can identify specific residues involved in subunit interactions, providing structural insights into the ATP synthase assembly.

• Cryo-electron microscopy: This technique can visualize the entire ATP synthase complex and determine the position and interactions of atpC2 within the assembled complex.

For studying the functional consequences of these interactions, ATP synthesis assays using reconstituted proteoliposomes containing wild-type or mutant versions of atpC2 could reveal how specific interactions affect enzyme activity and regulation. The genome-scale metabolic model for M. capsulatus Bath provides a framework for interpreting these experimental results in the context of the organism's energy metabolism .

What structural features distinguish the epsilon chain of ATP synthase in methanotrophs like M. capsulatus?

The structural features of ATP synthase epsilon chains in methanotrophs like M. capsulatus likely reflect adaptations to their unique energy metabolism. While specific structural data for M. capsulatus atpC2 is not directly reported in the available research, several distinctive features can be inferred:

• N-terminal domain: Likely contains a β-sandwich motif that interacts with the γ subunit of ATP synthase.

• C-terminal domain: Probably consists of two α-helices that can adopt different conformations, functioning as a molecular switch to regulate ATP synthesis activity.

• Methanotroph-specific residues: May contain unique amino acid substitutions that optimize function in the context of methane oxidation energy pathways.

These structural features would enable atpC2 to respond to the energy state of the cell and regulate ATP synthase activity accordingly. In M. capsulatus, which has evolved to utilize methane as its primary energy source, the regulatory functions of atpC2 are likely optimized to coordinate with the three electron transfer modes identified in the genome-scale metabolic model: redox-arm, direct coupling, and uphill electron transfer .

Comparative structural analysis between atpC1 and atpC2 would be particularly valuable for identifying specific adaptations. Researchers could utilize homology modeling based on crystal structures of epsilon subunits from related bacteria, coupled with molecular dynamics simulations to predict functional differences.

What techniques are recommended for studying the role of atpC2 in different electron transfer modes?

To investigate the role of atpC2 in the different electron transfer modes of M. capsulatus, researchers should employ a combination of genetic, biochemical, and systems biology approaches:

The implementation of these approaches should be guided by the three electron transfer modes described in the literature: (1) the redox-arm, where pMMO draws electrons from the quinone pool; (2) direct coupling, where electrons from methanol oxidation are transferred directly to pMMO; and (3) uphill electron transfer, where electrons from methanol dehydrogenase feed back into the ubiquinol pool . Each mode likely places different demands on the ATP synthase complex, and atpC2 may play a role in adapting the enzyme's activity to these varying conditions.

How can researchers accurately measure the activity of recombinant atpC2 in vitro?

Accurately measuring the activity of recombinant atpC2 requires assessing both its intrinsic properties and its functional impact on ATP synthesis. The following methodological approaches are recommended:

• ATP synthase reconstitution assay:

  • Purify the individual ATP synthase subunits, including recombinant atpC2.

  • Reconstitute the complete ATP synthase complex in liposomes.

  • Measure ATP synthesis rates upon generation of a proton gradient.

  • Compare results with complexes containing native atpC2 or atpC1.

• Conformational change analysis:

  • Use fluorescence resonance energy transfer (FRET) with labeled atpC2 to monitor conformational changes in response to ATP, ADP, or membrane potential.

  • Employ circular dichroism (CD) spectroscopy to assess structural changes under various conditions.

• Binding affinity measurement:

  • Determine binding kinetics between atpC2 and other ATP synthase subunits using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC).

  • Compare binding properties with those of atpC1 to identify functional differences.

• ATPase inhibition assay:

  • The epsilon subunit can inhibit ATP hydrolysis activity, which can be measured using a coupled enzyme assay that monitors phosphate release or NADH oxidation.

  • Compare the inhibitory capacity of atpC2 versus atpC1 under various conditions.

For all these assays, it's crucial to maintain appropriate buffer conditions similar to those used for other M. capsulatus ATP synthase subunits. A Tris/PBS-based buffer with 6% trehalose at pH 8.0 has been successfully used for the ATP synthase subunit a 2 and may be suitable for atpC2 as well. Temperature control is also critical, as M. capsulatus is a thermotolerant organism that grows optimally around 45°C.

What are the best approaches for analyzing the evolutionary conservation of atpC2 among methanotrophs?

To analyze the evolutionary conservation of atpC2 among methanotrophs, researchers should employ a comprehensive phylogenetic approach combined with functional analysis:

• Sequence retrieval and alignment:

  • Collect atpC2 sequences from diverse methanotrophs using BLAST searches against genomic databases.

  • Include sequences from non-methanotrophic bacteria as outgroups.

  • Perform multiple sequence alignment using MUSCLE or MAFFT algorithms with refinement options.

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood (RAxML or IQ-TREE) and Bayesian inference (MrBayes) methods.

  • Apply appropriate evolutionary models based on model testing (ProtTest).

  • Use bootstrap analysis or posterior probabilities to assess node support.

• Synteny analysis:

  • Examine the genomic context of atpC2 across methanotrophs to identify conserved gene clusters.

  • Compare with the synteny of atpC1 to understand duplication patterns.

• Selection pressure analysis:

  • Calculate dN/dS ratios to identify sites under positive, negative, or neutral selection.

  • Use methods like PAML or HyPhy to detect lineage-specific selection patterns.

• Structure-function correlation:

  • Map conserved residues onto predicted 3D structures of atpC2.

  • Identify co-evolving residues that might be functionally linked using methods like Statistical Coupling Analysis (SCA).

This evolutionary approach should be integrated with knowledge about the unique metabolic features of methanotrophs, particularly their electron transfer systems for methane oxidation . The presence of duplicate ATP synthase genes in M. capsulatus suggests potential functional specialization that may be reflected in the evolutionary patterns observed across methanotrophs. Researchers should also consider the ecological niches of different methanotrophs, as these may drive different selective pressures on energy metabolism genes.

How can researchers overcome solubility issues when expressing recombinant M. capsulatus atpC2?

Solubility challenges are common when expressing recombinant membrane-associated proteins like ATP synthase components. Based on experience with similar proteins, including the ATP synthase subunit a 2 from M. capsulatus , the following strategies can help overcome solubility issues:

• Expression condition optimization:

  • Lower the induction temperature to 16-20°C to slow protein production and facilitate proper folding.

  • Reduce IPTG concentration to 0.1-0.5 mM to decrease expression rate.

  • Extend expression time to 18-24 hours to allow for gradual accumulation of soluble protein.

  • Add compatible solutes like glycine betaine (2-10 mM) to the culture medium to enhance protein stability.

• Fusion tag approaches:

  • Use solubility-enhancing fusion partners such as MBP (maltose-binding protein), SUMO, or Thioredoxin.

  • Position the His-tag at the N-terminus as demonstrated successful for atpB2 .

  • Include a cleavable linker between the tag and atpC2 to allow tag removal after purification.

• Buffer optimization:

  • Include 5-10% glycerol in lysis and purification buffers to stabilize the protein.

  • Add 6% trehalose as used successfully with atpB2 .

  • Test different pH conditions (range 7.0-8.5) to find optimal solubility.

  • Include low concentrations (0.1-1%) of mild detergents like n-dodecyl β-D-maltoside (DDM) or digitonin if the protein associates with membranes.

• Co-expression strategies:

  • Co-express atpC2 with its natural binding partners (e.g., γ subunit) to promote proper folding and complex formation.

  • Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) to assist folding.

• Refolding from inclusion bodies:

  • If soluble expression fails, purify inclusion bodies under denaturing conditions.

  • Use a gradual dialysis refolding protocol with decreasing concentrations of urea or guanidine hydrochloride.

  • Add oxidized and reduced glutathione pairs (3:1 ratio) to facilitate proper disulfide bond formation if applicable.

After successful solubilization, the recombinant protein should be stored as recommended for atpB2: aliquoted at -20°C/-80°C with 5-50% glycerol to prevent freeze-thaw damage .

What are the most promising research areas involving M. capsulatus atpC2?

The study of M. capsulatus atpC2 presents several promising research avenues that could significantly advance our understanding of methanotroph biology and energy metabolism. Based on current knowledge of M. capsulatus and its ATP synthase complexes, the following research directions appear most valuable:

• Functional specialization of duplicate ATP synthases: Investigating why M. capsulatus maintains two sets of ATP synthase genes and how atpC2 contributes to this specialization could reveal novel adaptive mechanisms for energy metabolism in methanotrophs.

• Regulatory role in electron transfer modes: Exploring how atpC2 might help coordinate ATP synthase activity with the three distinct electron transfer modes identified in M. capsulatus could provide insights into the integration of methanotrophy with energy production.

• Structural adaptations for thermotolerance: Examining the structural features that enable atpC2 to function optimally at the higher temperatures preferred by M. capsulatus could reveal principles for designing thermostable proteins.

• Evolutionary comparison across methanotroph clades: Conducting comparative analyses of atpC2 across various methanotrophic bacteria could illuminate how energy metabolism has evolved in different lineages that converged on methane utilization.

• Role in ecological adaptation: Investigating how atpC2 function relates to the ecological niche of M. capsulatus could help explain the success of these bacteria in various environments and their important role in the global carbon cycle.