Recombinant Desulfotalea psychrophila ATP synthase subunit b (atpF)

What is Desulfotalea psychrophila and why is its ATP synthase of interest?

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium capable of growth at temperatures below 0°C. As an abundant member of microbial communities in permanently cold marine sediments, these bacteria play significant roles in global carbon and sulfur cycles . The ATP synthase of this psychrophilic organism is of particular interest because it must function efficiently at low temperatures, suggesting potential structural and functional adaptations compared to mesophilic homologs. These adaptations may provide insights into cold-temperature enzyme functionality and evolution.

What is the molecular structure of D. psychrophila ATP synthase subunit b?

The ATP synthase subunit b (atpF) from D. psychrophila is a membrane protein component of the F0 sector of ATP synthase. According to available sequence data, the full-length protein consists of 242 amino acids . The amino acid sequence is: MKENIKRVLPFLLVLLFAFAPLALASAPVDAPAPADAPADALPSAKVISAPADAGVIEAV ELEHATVTGAHDVAVAHVADSLSHEKLMDLFWRVLNFAVLMAILIKFGAKPIANALSGRQ QRVKSEVEDLEARRIVAEKEFRQFEAKLANVEKDIDSIVDKAVAQAEIEKAKILERAEQA AADIQKSAEQAIQNEIANAKRSLKNDAADQAAVMAEELIVKHLTADDQVKIVEDYLAKVG AV . This sequence likely contains regions responsible for membrane anchoring, interactions with other subunits, and contributions to the functional architecture of the ATP synthase complex.

What is the genomic context of the atpF gene in D. psychrophila?

The atpF gene in D. psychrophila has the ordered locus name DP0830 . It is part of the 3,523,383 bp circular chromosome that contains 3,118 predicted genes . While specific details about the ATP synthase operon organization in D. psychrophila are not provided in the search results, bacterial ATP synthase genes are typically organized in operons. The genome of D. psychrophila also contains two plasmids of 121,586 bp and 14,663 bp, though it's unclear if any ATP synthase-related genes are present on these plasmids .

What are the optimal expression and purification conditions for recombinant D. psychrophila ATP synthase subunit b?

Based on protocols for similar proteins, recombinant D. psychrophila ATP synthase subunit b can be successfully expressed in E. coli expression systems, similar to the approach used for the related ATP synthase subunit a (atpB) . For optimal expression, researchers should consider:

• Expression vector selection: Vectors with strong inducible promoters (T7, tac) are recommended

• E. coli strain selection: BL21(DE3) or derivatives optimized for membrane protein expression

• Induction conditions: Lower temperatures (16-20°C) may improve folding of psychrophilic proteins

• Purification approach: Affinity chromatography using appropriate tags determined during production

Purification typically achieves >85% purity as determined by SDS-PAGE . The recombinant protein should be stored in a Tris-based buffer with 50% glycerol for stability . For extended storage, maintain at -20°C or -80°C, with working aliquots kept at 4°C for up to one week to avoid degradation from repeated freeze-thaw cycles .

How can researchers functionally characterize the D. psychrophila ATP synthase b subunit?

Functional characterization of the ATP synthase subunit b requires multiple complementary approaches:

• Protein-protein interaction studies:

  • Co-immunoprecipitation with other ATP synthase subunits

  • Cross-linking experiments to identify interacting partners

  • Yeast two-hybrid or bacterial two-hybrid assays

• Structural studies:

  • Circular dichroism (CD) spectroscopy to assess secondary structure

  • Nuclear magnetic resonance (NMR) for structural characterization

  • X-ray crystallography if crystals can be obtained

• Temperature-dependent activity assays:

  • ATP synthesis/hydrolysis measurements at different temperatures (focus on 0-15°C range)

  • Compare kinetic parameters (Km, Vmax) with mesophilic homologs

• Reconstitution experiments:

  • Incorporation into liposomes to assess membrane association

  • Reconstitution with other ATP synthase subunits to assess complex formation

What are the challenges in studying cold-adapted ATP synthases like that of D. psychrophila?

Research on cold-adapted ATP synthases presents several unique challenges:

• Protein stability issues: Cold-adapted proteins are often more flexible and less stable at moderate temperatures compared to mesophilic counterparts, making handling during purification and analysis difficult.

• Expression difficulties: Expression in mesophilic hosts like E. coli may result in improper folding or aggregation if the recombinant protein retains cold-adapted properties.

• Functional assessment complexity: Standard enzymatic assays may not reflect natural activity if not performed at appropriate low temperatures with suitable buffers that mimic the native environment.

• Structural determination challenges: The inherent flexibility of cold-adapted proteins can complicate structural studies using traditional methods like X-ray crystallography.

• Reconstitution barriers: Reconstituting functional complexes may require specific lipid compositions that mirror the cold environment membrane adaptations.

How does the structure of ATP synthase subunit b contribute to cold adaptation in D. psychrophila?

While specific structural features of D. psychrophila ATP synthase subunit b related to cold adaptation are not directly addressed in the search results, several hypotheses can be proposed based on known principles of cold adaptation:

• Amino acid composition analysis: The protein sequence (MKENIKRVLPFLLVLLFAFAPLALASAPVDAPAPADAPADALPSAKVISAPADAGVIEAV ELEHATVTGAHDVAVAHVADSLSHEKLMDLFWRVLNFAVLMAILIKFGAKPIANALSGRQ QRVKSEVEDLEARRIVAEKEFRQFEAKLANVEKDIDSIVDKAVAQAEIEKAKILERAEQA AADIQKSAEQAIQNEIANAKRSLKNDAADQAAVMAEELIVKHLTADDQVKIVEDYLAKVG AV) likely contains:

  • Reduced proline and arginine content in flexible regions

  • Increased glycine content for enhanced flexibility

  • Reduced hydrophobic core packing

  • Strategic placement of charged residues to destabilize rigid structures

• Membrane interaction adaptations: The membrane-spanning regions may contain modifications that maintain appropriate flexibility and function within the cold, rigid membrane environment of D. psychrophila.

• Interaction interfaces: The surfaces that interact with other ATP synthase subunits may feature adaptations that maintain functional assembly at low temperatures while allowing for efficient energy transduction.

What expression systems are most suitable for recombinant D. psychrophila ATP synthase subunit b?

For optimal expression of D. psychrophila ATP synthase subunit b, researchers should consider:

E. coli appears to be a viable expression system based on successful expression of related proteins such as ATP synthase subunit a (atpB) from D. psychrophila and ATP synthase subunit b from Bacillus pumilus .

What are the recommended storage and handling procedures for D. psychrophila ATP synthase proteins?

To maintain stability and functionality of recombinant D. psychrophila ATP synthase subunit b:

• Storage recommendations:

  • Short-term (up to 1 week): Store at 4°C in appropriate buffer

  • Medium-term: Store at -20°C in 50% glycerol

  • Long-term: Store at -80°C in 50% glycerol

• Buffer composition:

  • Tris-based buffer with 50% glycerol, optimized for protein stability

  • Consider including protease inhibitors to prevent degradation

• Handling precautions:

  • Avoid repeated freeze-thaw cycles which can reduce activity

  • Briefly centrifuge vials before opening to collect contents at the bottom

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

• Shelf life considerations:

  • Liquid form: approximately 6 months at -20°C/-80°C

  • Lyophilized form: approximately 12 months at -20°C/-80°C

How can researchers perform functional reconstitution of ATP synthase complexes incorporating D. psychrophila subunits?

Functional reconstitution of ATP synthase complexes requires careful consideration of multiple factors:

• Purification of individual subunits:

  • Each subunit should be purified to >85% purity using appropriate chromatography techniques

  • Consider co-expression of multiple subunits for stable complex formation

• Liposome preparation:

  • Use lipid compositions that mimic D. psychrophila membranes (if known) or consider E. coli polar lipid extract as a starting point

  • Prepare liposomes by detergent removal methods (e.g., dialysis, Bio-Beads)

• Reconstitution protocol:

  • Mix purified subunits in appropriate stoichiometric ratios

  • Add to detergent-destabilized liposomes

  • Remove detergent slowly to allow complex assembly and membrane incorporation

  • Assess functional reconstitution through ATP synthesis/hydrolysis assays

• Temperature considerations:

  • Perform reconstitution at temperatures relevant to D. psychrophila (0-15°C)

  • Test function across a temperature range to assess cold adaptation

How does the ATP synthase machinery in D. psychrophila compare with those in other extremophiles?

D. psychrophila represents an interesting point of comparison for extremophile ATP synthases:

• Comparison with thermophiles:
  Unlike hyperthermophilic organisms like Archaeoglobus fulgidus (another sulfate reducer), D. psychrophila has evolved adaptations for cold environments rather than heat stability . While A. fulgidus ATP synthases require structural rigidity to function at high temperatures, D. psychrophila likely features increased flexibility for function at low temperatures. Comparative genomics between these organisms reveals "many striking differences, but only a few shared features" .

• Comparison with other psychrophiles:
  D. psychrophila carries "nine putative cold shock proteins and nine potentially cold shock-inducible proteins" which may interact with or regulate ATP synthase function at low temperatures. This abundance of cold-adaptation proteins suggests sophisticated mechanisms for bioenergetic function in cold environments.

• Evolutionary considerations:
  The ATP synthase complex represents a conserved molecular machine across diverse organisms, but extremophile versions show specialized adaptations. D. psychrophila's ATP synthase likely represents an important example of cold adaptation in this essential energy-generating complex.

What genomic insights inform our understanding of ATP synthase function in D. psychrophila?

Genomic analysis of D. psychrophila provides several insights relevant to ATP synthase function:

• Regulatory systems: D. psychrophila "encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases" . These may be involved in regulating ATP synthase expression in response to environmental conditions.

• Metabolic context: The genome reveals "the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system" . These systems work in concert with ATP synthase to maintain cellular energetics.

• Absence of certain features: The genome shows "the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc" , suggesting potentially unique electron transport pathways feeding into the ATP synthase system.