Recombinant Syntrophomonas wolfei subsp. wolfei ATP synthase subunit c (atpE)

What is Recombinant Syntrophomonas wolfei subsp. wolfei ATP synthase subunit c (atpE)?

Recombinant Syntrophomonas wolfei subsp. wolfei ATP synthase subunit c (atpE) is a full-length protein (72 amino acids) that functions as a component of the ATP synthase complex in the anaerobic bacterium Syntrophomonas wolfei. This protein corresponds to UniProt ID Q0AUC8 and is encoded by the atpE gene (also known as Swol_2387). The protein can be recombinantly expressed with various tags (commonly His-tag) in bacterial expression systems like E. coli for research purposes.

The protein has several alternative designations in scientific literature, including ATP synthase F(0) sector subunit c, F-type ATPase subunit c (shortened to F-ATPase subunit c), and Lipid-binding protein. The complete amino acid sequence is MGVALGAGLAVSIAGIGGGIGMGIAGGKAFEAIARQPEVGGDVRTLLFITLAFIETLTIYGLLIAFMLVGKA, representing the full 1-72 amino acid sequence of the native protein.

What is the biological context of Syntrophomonas wolfei as an organism?

Syntrophomonas wolfei is an anaerobic syntrophic microorganism with specialized metabolic capabilities for degrading short-chain fatty acids. The bacterium converts these fatty acids to acetate, hydrogen, and/or formate through a process that is thermodynamically unfavorable in isolation. Morphologically, S. wolfei is characterized as a gram-negative, slightly helical rod with round ends featuring two to eight flagella laterally inserted along the concave side of the cell.

What structural and functional characteristics define ATP synthase subunit c in S. wolfei?

ATP synthase subunit c in Syntrophomonas wolfei functions as a critical component of the F-type ATP synthase complex, which is involved in cellular energy conversion. Structurally, this 72-amino acid protein is characterized by its membrane-embedded nature, consistent with its role in the Fo sector of the ATP synthase. The protein sequence (MGVALGAGLAVSIAGIGGGIGMGIAGGKAFEAIARQPEVGGDVRTLLFITLAFIETLTIYGLLIAFMLVGKA) suggests multiple hydrophobic regions that facilitate membrane integration.

Functionally, ATP synthase subunit c participates in the formation of the c-ring structure within the membrane-embedded Fo portion of ATP synthase. This c-ring plays a crucial role in the rotary mechanism of ATP synthesis, converting the proton motive force into mechanical energy that drives ATP production. In the context of S. wolfei's syntrophic lifestyle, ATP synthase likely plays an important role in energy conservation during the thermodynamically challenging process of fatty acid degradation, potentially interfacing with reverse electron transfer mechanisms that are essential for syntrophic growth.

How does ATP synthase subunit c relate to reverse electron transfer in syntrophic metabolism?

ATP synthase subunit c plays an integral role in the energetic framework supporting reverse electron transfer in Syntrophomonas wolfei. Syntrophic butyrate metabolism involves thermodynamically unfavorable production of hydrogen and/or formate from butyryl-CoA, a process that requires energy input through reverse electron transfer. The ATP synthase complex, which includes the c subunit, is involved in establishing and utilizing the proton gradient necessary for this energetically uphill process.

Research has demonstrated that hydrogen production from butyrate in S. wolfei requires the presence of a proton gradient. ATP synthase functions bidirectionally in this context - it can utilize the proton gradient to generate ATP under favorable conditions, but during reverse electron transfer, it may operate in conjunction with other membrane complexes to support the energetically unfavorable electron flow. Proteomic analyses of S. wolfei membranes have identified not only ATP synthase components but also a membrane-bound hydrogenase (Hyd2) and iron-sulfur oxidoreductases that work together in this process. The expression of these components is upregulated during syntrophic growth conditions, suggesting their coordinated role in facilitating reverse electron transfer necessary for syntrophic metabolism.

What is the relationship between ATP synthase subunit c and protein acylation in S. wolfei?

Recent research has uncovered a potential connection between ATP synthase subunit c and metabolite-driven protein acylation in Syntrophomonas wolfei. During fatty acid degradation, S. wolfei produces various reactive acyl-Coenzyme A species (RACS) as intermediates. These RACS have been correlated with acyl-lysine modifications in other prokaryotic and eukaryotic systems, where they serve as important regulatory mechanisms for metabolic processes.

Mass spectrometry-based proteomic studies have characterized acylome profiles of S. wolfei subspecies grown on different carbon substrates. While the specific acylation status of ATP synthase subunit c has not been fully elucidated in the available research, the prevalence of acylation modifications in S. wolfei suggests that membrane proteins involved in energy transduction, including ATP synthase components, may be subject to regulatory acylation. These modifications could potentially serve as a feedback mechanism linking the metabolic state of the cell (specifically the levels of various acyl-CoA intermediates) to the activity of energy-conserving complexes like ATP synthase, which would be particularly important during syntrophic growth conditions.

How does recombinant ATP synthase subunit c compare to native protein in structure and function?

Table 1: Comparison of Native and Recombinant ATP synthase subunit c(atpE)

What are the optimal storage and handling protocols for recombinant S. wolfei ATP synthase subunit c?

Proper storage and handling of recombinant Syntrophomonas wolfei ATP synthase subunit c is critical for maintaining protein integrity and activity. Based on established protocols, the following comprehensive approach is recommended:

For long-term storage, maintain the protein at -20°C or preferably -80°C in an appropriate buffer system. The recombinant protein is typically supplied in Tris/PBS-based buffer containing 6% trehalose at pH 8.0 or in Tris-based buffer with 50% glycerol. The addition of glycerol or trehalose serves as a cryoprotectant to prevent damage during freeze-thaw cycles.

For working solutions, it is recommended to thaw the protein on ice and prepare working aliquots to avoid repeated freeze-thaw cycles, which can significantly degrade protein quality. Working aliquots can be stored at 4°C for up to one week. Before opening any vial, briefly centrifuge to bring the contents to the bottom, particularly if the protein is supplied as a lyophilized powder.

For reconstitution of lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% is recommended for aliquots intended for long-term storage, with 50% being the standard recommendation. Protein purity should be greater than 90% as determined by SDS-PAGE to ensure experimental reliability.

What expression systems are most effective for producing recombinant S. wolfei ATP synthase subunit c?

E. coli represents the most widely used and validated expression system for recombinant Syntrophomonas wolfei ATP synthase subunit c production. This bacterial expression system offers several advantages for the production of this membrane protein:

The relatively small size (72 amino acids) of ATP synthase subunit c makes it amenable to expression in E. coli without the toxicity issues often encountered with larger membrane proteins. His-tagging at the N-terminus has been successfully implemented, facilitating purification via affinity chromatography without apparent interference with protein folding. The resulting recombinant protein typically achieves greater than 90% purity following optimized purification protocols.

When designing expression constructs, researchers should consider:

• Codon optimization for E. coli if working with the native S. wolfei sequence

• Signal sequence considerations for membrane protein targeting

• Tag placement (N-terminal His-tagging has been validated)

• Induction conditions (temperature, inducer concentration, and duration)

• Lysis and solubilization methods appropriate for membrane proteins

While E. coli remains the standard, alternative expression systems might be considered for specific research applications. For instance, cell-free protein synthesis systems could potentially offer advantages for producing membrane proteins like ATP synthase subunit c with fewer complications related to toxicity or inclusion body formation.

What methodological approaches can be used to study protein-protein interactions involving ATP synthase subunit c?

Investigating protein-protein interactions involving ATP synthase subunit c from Syntrophomonas wolfei requires specialized approaches that accommodate its membrane-embedded nature. Several complementary methodologies can be employed:

Blue Native Gel Electrophoresis (BN-PAGE): This technique has proven effective for studying membrane protein complexes in S. wolfei. BN-PAGE allows separation of native protein complexes and can be coupled with activity assays performed directly in the gel. Research has successfully used this approach to identify membrane-bound complexes including ATP synthase components from S. wolfei grown under various conditions.

Co-immunoprecipitation (Co-IP): Using antibodies against the His-tag of recombinant ATP synthase subunit c or against native epitopes, researchers can pull down protein complexes and identify interaction partners through mass spectrometry. This approach is particularly valuable for discovering novel interaction partners.

Cross-linking Mass Spectrometry: Chemical cross-linking followed by mass spectrometry analysis can provide detailed information about spatial proximity of proteins within complexes. This is especially useful for membrane protein assemblies like ATP synthase where traditional structural biology approaches may be challenging.

Proteomic Differential Expression Analysis: Comparing membrane proteomes under different growth conditions (e.g., syntrophic vs. non-syntrophic growth) can identify proteins that co-express with ATP synthase components, suggesting functional relationships. This approach has already revealed differential expression of hydrogenase and iron-sulfur oxidoreductase components during syntrophic growth of S. wolfei.

Bacterial Two-Hybrid Systems: Modified for membrane proteins, these systems can detect direct interactions between ATP synthase subunit c and candidate partners in a cellular context.

How can researchers interpret experimental results in the context of syntrophic metabolism?

Interpreting experimental results involving ATP synthase subunit c from Syntrophomonas wolfei requires contextualizing findings within the unique energetic constraints of syntrophic metabolism. When analyzing data, researchers should consider the following framework:

Energy Balance Perspective: Syntrophic butyrate metabolism operates near thermodynamic equilibrium with very small free energy yields. Therefore, small changes in protein expression, modification, or activity may have magnified impacts on metabolic function. Data should be interpreted with awareness that ATP synthase is part of a tightly regulated energy conservation system where subtle effects may be physiologically significant.

Comparative Analysis Approach: Results should be compared across multiple growth conditions, particularly:

• Syntrophic growth (e.g., with Methanospirillum hungatei or Dehalococcoides mccartyi)

• Non-syntrophic growth (e.g., on crotonate)

• Growth under varying hydrogen partial pressures

This comparative approach has successfully revealed differential expression of energy metabolism components under syntrophic conditions. For example, hydrogenase gene expression increases during syntrophic butyrate growth compared to crotonate growth, indicating specific adaptation to syntrophic conditions.

Integration with Metabolite Data: Correlate protein findings with metabolomic data when available, particularly focusing on acyl-CoA intermediates and their relationship to protein acylation patterns. This integrative approach can reveal how ATP synthase regulation might be linked to metabolic state through post-translational modifications.

Functional Redundancy Assessment: Consider that S. wolfei may possess multiple mechanisms for energy conservation with potentially overlapping functions. Results suggesting minimal impact from manipulating a single component may reflect redundant systems rather than lack of importance.

What are the key considerations when comparing ATP synthase subunit c across different bacterial species?

Comparative analysis of ATP synthase subunit c across bacterial species provides valuable evolutionary and functional insights but requires careful consideration of several factors:

Sequence Homology Analysis: While ATP synthase is highly conserved across domains of life, specific adaptations in the c subunit sequence may reflect specialization for different environments or metabolic strategies. Sequence alignment tools should be employed to identify conserved motifs versus variable regions that might indicate functional specialization.

Structural Comparison: Despite sequence variations, secondary and tertiary structural features of subunit c are often conserved. The c-ring stoichiometry (number of c subunits per ring) can vary between species and correlates with the thermodynamic efficiency of the ATP synthase complex. This consideration is especially relevant when comparing organisms adapted to different energy regimes.

Physiological Context: S. wolfei's ATP synthase operates in a uniquely constrained energetic environment requiring reverse electron transfer for syntrophic growth. When comparing to non-syntrophic organisms, consider how these fundamentally different energetic constraints might influence ATP synthase function and regulation.

Table 2: Comparative Analysis Framework for ATP Synthase Subunit c

Evolutionary Perspective: Consider horizontal gene transfer events versus vertical inheritance when interpreting similarities and differences. ATP synthase genes generally show vertical inheritance patterns, but exceptions exist, particularly in specialists like S. wolfei that have adapted to ecological niches with unique energetic constraints.