Recombinant Fagopyrum esculentum subsp. ancestrale ATP synthase subunit b, chloroplastic (atpF)

What is the genomic structure of atpF in Fagopyrum esculentum subsp. ancestrale?

The atpF gene in Fagopyrum esculentum subsp. ancestrale is located in the chloroplast genome. Based on comparative genomic analyses of Fagopyrum species, the gene is situated in the Large Single Copy (LSC) region of the chloroplast genome. F. esculentum subsp. ancestrale shares significant sequence similarity with F. esculentum, with the chloroplast genomes of both showing similar AT/TA dinucleotide distributions (approximately 15.02% and 14.95%, respectively) . The complete chloroplast genome sequencing has revealed that most variations between Fagopyrum species exist in the LSC and SSC regions, which would include the atpF gene encoding ATP synthase subunit b .

What differences exist between Fagopyrum esculentum subsp. ancestrale ATP synthase and other plant ATP synthases?

While specific differences in the ATP synthase of F. esculentum subsp. ancestrale compared to other plants are not extensively documented, comparative genomic analysis of Fagopyrum species reveals evolutionary divergence that may affect protein structure and function. The buckwheat genus shows two distinct evolutionary directions between the cymosum group and the urophyllum group, as evidenced by differences in SSR patterns .

F. esculentum and F. esculentum subsp. ancestrale have similar genetic structures, including nearly identical proportions of AT/TA dinucleotide repeats (14.95% and 15.02%, respectively), indicating evolutionary relatedness . These genetic similarities suggest that their ATP synthase complexes likely share structural and functional characteristics distinct from other plant species.

What are the optimal expression systems for producing recombinant chloroplastic ATP synthase subunit b?

Based on successful strategies for expressing other ATP synthase subunits, the following expression systems are recommended:

• E. coli Expression System: BL21 derivative strains have been successfully used for recombinant expression of the c subunit of chloroplast ATP synthase . For the atpF gene product, similar approaches can be employed using vectors such as pMAL-c2x for fusion protein expression.

• Fusion Protein Approach: Expression of the hydrophobic b subunit as a fusion protein with a solubility tag such as Maltose Binding Protein (MBP) significantly improves solubility and expression levels .

• Co-expression with Chaperones: To increase yields of difficult-to-express proteins, co-transformation with plasmids expressing chaperone proteins like DnaK, DnaJ, and GrpE (e.g., using the pOFXT7KJE3 vector) has proven effective .

What methodological challenges must be overcome when expressing membrane-associated ATP synthase components?

Expressing membrane-associated ATP synthase components like the b subunit presents several challenges:

• Hydrophobicity: The b subunit contains hydrophobic regions that can cause aggregation during expression. Using fusion partners like MBP can improve solubility .

• Protein Toxicity: Membrane proteins can be toxic to the host cell. Co-expression with chaperones can mitigate this issue and increase yields .

• Codon Optimization: Using codon-optimized gene inserts based on the host's codon usage bias significantly improves expression levels. This has been successfully demonstrated with the c subunit and is applicable to the b subunit .

• Purification Complications: After cleavage from fusion partners, hydrophobic membrane proteins may aggregate. Column purification techniques must be carefully optimized, with reversed-phase chromatography being particularly effective for hydrophobic subunits .

How can fusion tags be used to optimize recombinant ATP synthase subunit b expression?

Fusion tags significantly improve the expression of hydrophobic ATP synthase subunits through several mechanisms:

• Enhanced Solubility: The MBP tag has been successfully used to express the hydrophobic c₁ subunit as a soluble MBP-c₁ fusion protein . For the b subunit, a similar MBP fusion strategy would enhance solubility by masking hydrophobic regions.

• Simplified Purification: Fusion tags like MBP facilitate affinity purification using dedicated columns, allowing efficient separation from host cell proteins.

• Protease Cleavage Sites: Incorporating specific protease cleavage sites between the fusion tag and the target protein enables tag removal after purification. For example, a Factor Xa cleavage site can be used to separate MBP from the target protein .

• Expression Monitoring: Fusion with tags enables easier monitoring of expression levels using standard techniques like SDS-PAGE before committing to full-scale purification.

What purification protocol yields the highest purity for recombinant ATP synthase subunit b?

A multi-step purification protocol based on successful approaches with other ATP synthase subunits includes:

• Affinity Chromatography: For MBP-fusion proteins, amylose resin affinity chromatography provides the initial purification step .

• Proteolytic Cleavage: After initial purification, the fusion protein is cleaved using an appropriate protease (e.g., Factor Xa) to separate the target protein from the fusion tag.

• Reversed-Phase Chromatography: The cleaved target protein can be further purified using reversed-phase chromatography, which is particularly effective for hydrophobic membrane proteins .

• Size Exclusion Chromatography: A final polishing step using size exclusion chromatography can remove any remaining contaminants or aggregates.

This protocol has been shown to yield highly purified subunits with confirmed alpha-helical secondary structure, as demonstrated with the c subunit of spinach chloroplast ATP synthase .

How can the structural integrity of recombinant ATP synthase subunit b be verified?

Verification of structural integrity involves multiple complementary techniques:

• Circular Dichroism (CD) Spectroscopy: CD spectroscopy can confirm the correct alpha-helical secondary structure of the purified protein, as has been done for the c subunit .

• Immunological Techniques: Using antibodies specific to the β subunit of ATP synthase, similar approaches can be developed for the b subunit. Available techniques include Western blot (WB), Blue Native-PAGE (BN-PAGE), and Immunofluorescence (IF) .

• Mass Spectrometry: Mass spectrometry can verify the molecular weight and post-translational modifications of the purified protein.

• Functional Assays: Reconstitution experiments can assess the ability of the recombinant subunit to integrate into functional ATP synthase complexes.

What analytical methods are most effective for characterizing protein-protein interactions in ATP synthase complexes?

Several analytical methods are particularly effective for studying interactions within the ATP synthase complex:

• Blue Native-PAGE (BN-PAGE): This technique is effective for analyzing intact protein complexes and has been successfully used with ATP synthase components at dilutions of 1:5000 .

• Crosslinking Studies: Chemical crosslinking followed by mass spectrometry can identify interaction surfaces between subunits.

• Fluorescence Resonance Energy Transfer (FRET): FRET experiments can measure distances between specific residues in different subunits, as demonstrated in studies of the DELSEED-loop of the β subunit .

• Molecular Modeling: Computational approaches based on crystal structures can predict interaction interfaces between subunits.

How can recombinant ATP synthase subunits be used to study the mechanism of proton translocation?

Recombinant ATP synthase subunits provide valuable tools for mechanistic studies:

• Site-Directed Mutagenesis: Recombinant expression allows for systematic mutation of specific residues in the b subunit to identify amino acids critical for interaction with other subunits or for maintaining structural integrity .

• Reconstitution Experiments: Purified recombinant subunits can be used in reconstitution experiments to study their integration into functional complexes. This has been a goal for the recombinant c subunit of spinach chloroplast ATP synthase .

• Chimeric Proteins: Creating chimeric proteins with sections from different species can help identify regions responsible for species-specific characteristics.

• Proton Translocation Assays: Reconstituted complexes containing recombinant subunits can be used in liposome-based assays to measure proton translocation efficiency.

What experimental approaches can determine the stoichiometry of ATP synthase complexes in Fagopyrum esculentum?

The stoichiometry of ATP synthase complexes can be investigated using:

• Mass Spectrometry: Quantitative mass spectrometry can determine the relative abundance of different subunits in purified complexes.

• Cryo-Electron Microscopy: Cryo-EM analysis of purified complexes can reveal the structural arrangement and stoichiometry of subunits.

• Biochemical Crosslinking: Chemical crosslinking followed by SDS-PAGE can identify adjacent subunits and their stoichiometry.

• Reconstitution Studies: Using defined ratios of recombinant subunits in reconstitution experiments can help determine the optimal stoichiometry for functional complexes.

The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring (cₙ) in the enzyme, which is organism-dependent . Understanding this stoichiometry is crucial for unraveling the energetics of ATP synthesis in Fagopyrum esculentum.

How do mutations in the DELSEED-loop region of ATP synthase affect coupling efficiency?

Studies on the DELSEED-loop of the β subunit provide insights into coupling mechanisms that could be applied to subunit b research:

• Deletion Mutants: Analysis of deletion mutants with 7-14 amino acids removed from the DELSEED-loop revealed that a 10-residue deletion lost ATP synthesis ability while retaining ATPase activity, and a 14-residue deletion abolished all enzymatic activity .

• Charge-Altering Mutations: An AALSAAA mutant, in which all negative charges of the DELSEED motif were removed, showed normal MgATP binding patterns with a high-affinity site still present, indicating that the negative charges are not essential for this function .

• FRET Experiments: Fluorescence resonance energy transfer experiments confirmed that certain deletions shortened the DELSEED-loop by approximately 10 Å, defining the minimum length required for coupling catalysis and rotation .

How does the evolutionary divergence of Fagopyrum species affect ATP synthase structure and function?

Comparative genomics of Fagopyrum species reveals evolutionary patterns with potential implications for ATP synthase:

• Divergent Groups: Fagopyrum species have been divided into two divergent evolutionary groups (cymosum and urophyllum) based on nucleotide repeat patterns . These genetic differences may translate to structural and functional variations in their ATP synthases.

• SSR Patterns: The proportion of CA/TG repeats in the cymosum group (~0.96%) is much higher than in the urophyllum group (~0.44%), while the AAT/TTA type is absent in the cymosum group but present in F. longistylum (~0.87%), F. leptopodum (~0.89%), F. luojishanense (~0.89%), and F. urophyllum (~1.27%) .

• Genetic Similarity: F. esculentum and F. esculentum subsp. ancestrale have similar AT/TA dinucleotide distributions (14.95% and 15.02% respectively), supporting their close genetic relationship .

• Implications for ATP Synthase: These genetic differences may affect the structure and function of chloroplast-encoded proteins, including ATP synthase subunits, potentially leading to variations in energy conversion efficiency among different Fagopyrum species.

What biotechnological applications could benefit from recombinant Fagopyrum ATP synthase components?

Recombinant ATP synthase components from Fagopyrum species offer several biotechnological applications:

• Bioenergetic Systems: Recombinant ATP synthase components could be used to develop artificial bioenergetic systems for ATP production or as molecular motors.

• Protein Engineering: The unique properties of Fagopyrum ATP synthase components could inform the design of engineered proteins with enhanced stability or activity.

• Therapeutic Applications: Components like recombinant buckwheat trypsin inhibitor (rBTI) have shown beneficial effects in age-related protein aggregation and mitochondrial dysfunction models . Similar approaches could be explored with ATP synthase components.

• Research Tools: Recombinant ATP synthase subunits serve as valuable tools for studying energy conversion mechanisms and could aid in developing assays for measuring ATP synthesis efficiency.

• Structural Biology: Providing purified components for structural studies using techniques like X-ray crystallography or cryo-electron microscopy to elucidate the detailed structure of plant ATP synthases.