Recombinant Oenothera argillicola ATP synthase subunit b, chloroplastic (atpF)

What are the optimal storage conditions for recombinant Oenothera argillicola ATP synthase subunit b?

For optimal stability and activity retention, store recombinant Oenothera argillicola ATP synthase subunit b in Tris-based buffer containing 50% glycerol. Short-term storage (up to one week) can be maintained at 4°C for working aliquots . For long-term preservation, store at -20°C, or preferably at -80°C for extended periods . Importantly, repeated freeze-thaw cycles significantly compromise protein integrity and should be strictly avoided; therefore, preparing single-use aliquots upon initial thawing is strongly recommended . When planning experiments requiring multiple uses, create appropriate volume working aliquots to minimize structural damage from temperature fluctuations.

How does Oenothera argillicola ATP synthase subunit b compare to homologous proteins in other plant species?

Oenothera argillicola ATP synthase subunit b shares structural and functional homology with corresponding proteins across plant species, particularly within the Myrtales order. Comparative genomic analysis places O. argillicola in context with other species as demonstrated in this data comparison:

Data extracted from comparative plastid genome analysis .

This comparison reveals that O. argillicola has a slightly larger protein coding region with similar GC content compared to related species. The conservation of functional domains across these diverse taxa suggests evolutionary pressure to maintain ATP synthase structure, particularly in the membrane-spanning F₀ complex where subunit b resides.

What is the recommended reconstitution protocol for lyophilized recombinant atpF protein?

For optimal reconstitution of lyophilized recombinant Oenothera argillicola ATP synthase subunit b, follow this methodological approach:

• Allow the lyophilized protein to equilibrate to room temperature (18-25°C) before opening to prevent moisture condensation.

• Reconstitute in a Tris-based buffer (pH 7.5-8.0) containing 50% glycerol and appropriate salt concentration (typically 100-150 mM NaCl) .

• Gently resuspend by pipetting rather than vortexing to prevent protein denaturation.

• Allow complete solubilization by incubating at 4°C with gentle rotation for 30-60 minutes.

• Centrifuge briefly (5 minutes at 10,000 × g) to remove any particulate matter.

• Prepare working aliquots in appropriate volumes for single-use applications.

• Flash-freeze aliquots in liquid nitrogen before transferring to -80°C for long-term storage .

This protocol maximizes protein stability while minimizing structural damage during the reconstitution process.

How can researchers distinguish between native and recombinant Oenothera argillicola ATP synthase subunit b in experimental systems?

Distinguishing between native and recombinant Oenothera argillicola ATP synthase subunit b requires a multi-faceted analytical approach:

For quantitative analysis, researchers should develop a calibration curve using purified recombinant protein at known concentrations, enabling accurate determination of experimental samples through densitometric analysis of Western blots or ELISA-based quantification.

What experimental approaches are most effective for studying the integration of recombinant atpF into functional ATP synthase complexes?

Investigating the integration of recombinant Oenothera argillicola ATP synthase subunit b into functional complexes requires sophisticated experimental approaches:

• Blue Native PAGE (BN-PAGE): Use non-denaturing BN-PAGE to preserve protein-protein interactions while separating intact ATP synthase complexes. Follow with Western blotting using anti-atpF antibodies to confirm incorporation.

• Sucrose gradient ultracentrifugation: Fractionate chloroplast membranes on 10-50% sucrose gradients, followed by immunoblotting of collected fractions to track atpF distribution within assembled complexes of different sizes.

• Co-immunoprecipitation with interaction partners: Use antibodies against other ATP synthase subunits (particularly atpA or atpH) to co-precipitate interacting proteins, then detect recombinant atpF through tag-specific antibodies or mass spectrometry.

• Förster resonance energy transfer (FRET): Generate fluorescently labeled atpF and potential interaction partners to measure energy transfer as an indicator of physical proximity within the complex.

• Enzymatic activity coupling assays: Measure ATP synthesis rates in reconstituted systems containing recombinant atpF versus native protein to assess functional integration.

• Cryo-electron microscopy: Visualize the structural incorporation of tagged recombinant atpF within the assembled ATP synthase complex at near-atomic resolution.

What evolutionary insights can be gained from studying Oenothera argillicola atpF in comparison to other Myrtales species?

Evolutionary analysis of Oenothera argillicola atpF yields significant phylogenetic insights when compared across Myrtales taxa:

• Sequence conservation patterns: The atpF gene in O. argillicola maintains core functional domains despite exhibiting species-specific variations in non-critical regions. Comparative analysis reveals that O. argillicola has distinctive plastome characteristics, including a large ~56kb inversion in the LSC region between the accD/rbcL and rps16/trnQ UUG regions, which affects the genomic context of atpF .

• Selection pressure analysis: Calculate the ratio of non-synonymous to synonymous substitutions (Ka/Ks) to identify regions under positive or purifying selection. Previous studies in Oenothera have identified 215 pairwise combinations where Ka/Ks could be determined, revealing evolutionary constraints on critical functional domains .

• Genomic rearrangement impact: O. argillicola possesses unique genomic structures including two copies of the initiator tRNA trnfM CAU which differ by a single nucleotide polymorphism . This genomic context provides insights into the evolutionary forces shaping chloroplast gene organization.

• Codon usage bias: Analysis of the GC content in protein-coding regions (43% in O. argillicola) compared with other Myrtales species provides evidence of selection pressure on codon optimization .

Researchers investigating evolutionary aspects should employ phylogenetic reconstruction methods incorporating both sequence data and structural gene arrangements to fully capture the evolutionary trajectory of atpF across the Myrtales order.

What methodological considerations are important when using recombinant atpF for structure-function relationship studies?

Structure-function studies of recombinant Oenothera argillicola ATP synthase subunit b require careful methodological considerations:

• Protein folding verification: Employ circular dichroism spectroscopy to confirm that recombinant atpF adopts its native secondary structure, focusing particularly on alpha-helical content expected in transmembrane regions.

• Membrane integration assays: Use liposome reconstitution experiments to assess the ability of recombinant atpF to integrate into lipid bilayers, as proper membrane insertion is critical for function.

• Site-directed mutagenesis strategy: Design a systematic mutagenesis approach targeting:

  • Conserved residues in transmembrane domains

  • Potential interaction surfaces with other ATP synthase subunits

  • Regions unique to O. argillicola compared to homologs

• Expression system selection: Consider chloroplast-derived expression systems that provide the appropriate cellular environment for correct folding and post-translational modifications of this chloroplastic protein.

• Biophysical interaction measurements: Employ isothermal titration calorimetry or surface plasmon resonance to quantify binding affinities between atpF and interacting partners, particularly subunit c (atpH) .

• Functional reconstitution controls: Always include wild-type protein controls alongside mutant variants in functional assays to establish clear structure-function correlations.

When interpreting results, researchers should consider that the functional activity of isolated subunit b may differ significantly from its behavior within the assembled ATP synthase complex.

How can researchers optimize expression systems for producing high-quality recombinant Oenothera argillicola atpF?

Optimizing expression of recombinant Oenothera argillicola ATP synthase subunit b requires strategic consideration of multiple parameters:

• Expression system selection: Compare prokaryotic (E. coli) and eukaryotic (yeast, insect, plant) expression systems, noting that chloroplast proteins often express well in bacterial systems but may lack critical post-translational modifications.

• Codon optimization strategy: Analyze the GC content (43% in native gene) and design a codon-optimized sequence that preserves critical structural elements while maximizing expression efficiency in the chosen host.

• Vector design considerations:

  • Incorporate a cleavable N-terminal signal sequence for membrane targeting

  • Select affinity tags that minimally interfere with protein folding

  • Include TEV or PreScission protease sites for tag removal

• Expression conditions optimization matrix:

• Purification strategy optimization:

  • Employ detergent screening to identify optimal membrane protein solubilization conditions

  • Implement two-step purification combining affinity chromatography with size exclusion or ion exchange

  • Validate final protein quality through mass spectrometry and activity assays

• Storage buffer optimization: Test various buffer compositions containing 50% glycerol with different pH values (7.0-8.5) and salt concentrations (50-200 mM) to maximize stability .

Researchers should perform small-scale expression trials before scaling up, analyzing both soluble and membrane fractions to track protein localization and folding efficiency.

How can recombinant Oenothera argillicola atpF be utilized in photosynthesis research?

Recombinant Oenothera argillicola ATP synthase subunit b offers valuable applications in photosynthesis research through several methodological approaches:

• Reconstitution studies: Incorporate purified recombinant atpF into liposomes containing other ATP synthase components to create minimal functional systems for studying energy transduction mechanisms.

• Inhibitor binding studies: Use recombinant atpF to screen for novel ATP synthase inhibitors that specifically target the F₀ sector, potentially leading to new herbicide development.

• Protein-protein interaction mapping: Employ recombinant atpF as bait in pull-down assays to identify previously unknown interaction partners within the thylakoid membrane proteome.

• Comparative energetics analysis: Compare ATP synthesis efficiency between systems containing O. argillicola atpF versus homologous proteins from other species to investigate evolutionary adaptations in energy conversion.

• Structural biology applications: Use purified recombinant atpF in crystallization trials or as a component for cryo-EM studies of the complete chloroplast ATP synthase.

When designing these experiments, researchers should consider the unique genomic context of O. argillicola, which contains a large ~56kb inversion in the LSC region that may influence gene expression patterns compared to other model plants .

What analytical techniques are most effective for detecting conformational changes in recombinant atpF during ATP synthesis?

Detecting conformational changes in recombinant Oenothera argillicola ATP synthase subunit b during catalytic cycles requires sophisticated biophysical approaches:

• Time-resolved fluorescence spectroscopy: Introduce single cysteine residues at strategic positions through site-directed mutagenesis, label with environment-sensitive fluorophores, and monitor spectral shifts during ATP synthesis.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare deuterium incorporation patterns between active and inactive states to identify regions with altered solvent accessibility during the catalytic cycle.

• Single-molecule FRET: Engineer FRET donor-acceptor pairs at key positions to monitor distance changes between specified residues during proton translocation.

• Electron paramagnetic resonance (EPR) spectroscopy: Introduce spin labels at specific sites to detect changes in local environment during ATP synthase operation.

• Limited proteolysis coupled with MS: Compare proteolytic susceptibility patterns between different functional states to identify regions that become exposed or protected during conformational transitions.

• Cross-linking mass spectrometry: Use bifunctional cross-linkers with varying spacer lengths to capture dynamic interactions between atpF and neighboring subunits during different stages of ATP synthesis.

When interpreting data, researchers should correlate observed conformational changes with specific steps in the ATP synthesis mechanism, particularly focusing on how proton movement through the F₀ sector couples to conformational changes in F₁.

How can researchers develop effective antibodies against Oenothera argillicola atpF for immunological studies?

Developing high-specificity antibodies against Oenothera argillicola ATP synthase subunit b requires a methodical approach:

• Epitope selection strategy:

  • Analyze the amino acid sequence (MKNVTDSFVSLVHWPSAGSFGFNTDILATNPINLSVVLGVLIFFGKGVLSDLLDNRKQRILNTIRNSEELREGAIEQLEKARARLQDVQIEAEGYRAYGYFGIDEQRHESINSTYKTLEQLENNKNESIHFEQQRAINQVRQQIFQQALQGALGTLNSCLNNELHLRTISANIVLFGSMKELTD) using epitope prediction algorithms.

  • Prioritize hydrophilic, surface-exposed regions unique to O. argillicola atpF.

  • Avoid transmembrane segments (approximately residues 32-52) which are poorly immunogenic.

• Immunization protocol optimization:

  • Use either full-length recombinant protein or selected peptide conjugates.

  • Employ multiple immunization routes and adjuvant combinations.

  • Implement a prolonged immunization schedule with at least 4-5 boosts for maximum affinity maturation.

• Antibody screening methodology:

  • Develop a multi-tiered screening approach using ELISA, Western blot, and immunoprecipitation.

  • Test cross-reactivity against homologous proteins from related species.

  • Validate antibody specificity using knockout/knockdown systems or competing peptides.

• Affinity purification strategy:

  • Immobilize recombinant atpF on solid support for affinity purification.

  • Perform sequential elution with increasing stringency to isolate high-affinity antibodies.

  • Characterize purified antibodies for titer, specificity, and applications compatibility.

For researchers requiring monoclonal antibodies, implement hybridoma screening with native and denatured protein to select clones recognizing different epitope conformations.

What challenges exist in studying protein-protein interactions between atpF and atpH in Oenothera argillicola?

Investigating protein-protein interactions between ATP synthase subunits b (atpF) and c (atpH) in Oenothera argillicola presents several methodological challenges:

• Membrane protein solubilization: Both atpF and atpH are integral membrane proteins with hydrophobic domains . Researchers must carefully optimize detergent conditions that maintain native-like interactions while providing sufficient solubilization for analysis.

• Stoichiometry considerations: The ATP synthase F₀ complex contains one atpF subunit but multiple atpH subunits (typically 8-15 depending on species). This asymmetric stoichiometry complicates interpretation of binding data.

• Dynamic nature of interactions: The atpF-atpH interaction changes during the catalytic cycle as protons move through the complex. Static analytical methods may miss these transient conformational states.

• Reconstitution system requirements: Creating artificial membrane systems that properly orient both proteins requires careful lipid composition optimization to match the native thylakoid environment.

• Detection system limitations: Traditional yeast two-hybrid systems are poorly suited for membrane proteins; modified split-ubiquitin or BACTH (Bacterial Adenylate Cyclase Two-Hybrid) systems are more appropriate but require extensive optimization.

Researchers should consider complementary approaches including in situ crosslinking, biolayer interferometry with nanodiscs, and native mass spectrometry to build a comprehensive interaction model.

What are common pitfalls in working with recombinant Oenothera argillicola atpF and how can they be addressed?

Working with recombinant Oenothera argillicola ATP synthase subunit b presents several technical challenges that researchers should anticipate and address methodically:

• Protein aggregation during purification:

  • Problem: Hydrophobic transmembrane domains promote self-association.

  • Solution: Optimize detergent type and concentration; consider mixed micelle systems (e.g., DDM/CHS); maintain samples below 4°C throughout purification.

• Loss of function after tag removal:

  • Problem: Protease treatment or harsh cleavage conditions disrupt protein structure.

  • Solution: Design constructs with longer flexible linkers between tag and protein; use gentler proteases like TEV instead of thrombin; optimize buffer conditions during cleavage.

• Poor solubility in aqueous buffers:

  • Problem: The hydrophobic nature of atpF limits solubility in standard buffers.

  • Solution: Maintain 50% glycerol in storage buffer ; include low concentrations of non-ionic detergents; avoid freezing concentrated protein solutions.

• Inconsistent results in functional assays:

  • Problem: Variable incorporation into multi-subunit complexes affects activity measurements.

  • Solution: Standardize complex reconstitution protocols; measure incorporation efficiency before functional testing; use internal controls in each experiment.

• Degradation during storage:

  • Problem: Proteolytic degradation despite protease inhibitor use.

  • Solution: Store at -80°C rather than -20°C for long-term ; add multiple protease inhibitor classes; avoid repeated freeze-thaw cycles.

When troubleshooting, implement systematic changes to one parameter at a time while maintaining detailed records of protein behavior under each condition.

How can researchers optimize ELISA protocols specifically for Oenothera argillicola atpF quantification?

Optimizing ELISA protocols for Oenothera argillicola ATP synthase subunit b quantification requires systematic method development:

• Antibody selection and optimization:

  • Test both monoclonal and polyclonal antibodies recognizing different epitopes.

  • Determine optimal primary antibody concentration through checkerboard titration.

  • For sandwich ELISA, identify complementary capture/detection antibody pairs recognizing non-overlapping epitopes.

• Surface chemistry optimization:

  • Compare high-binding polystyrene plates with streptavidin-coated surfaces for biotinylated capture antibodies.

  • Test oriented antibody immobilization using Protein A/G or anti-Fc antibodies to improve sensitivity.

  • Optimize blocking conditions (BSA vs. casein vs. commercial blockers) to minimize background.

• Sample preparation protocol development:

  • Validate extraction efficiency from different experimental matrices.

  • Determine detergent compatibility with antibody binding.

  • Develop sample dilution protocols to ensure measurements within the linear range.

• Calibration strategy:

  • Use the same recombinant Oenothera argillicola atpF preparation for standard curves.

  • Prepare standards in matrix-matched solutions mimicking experimental samples.

  • Implement 4PL or 5PL curve fitting rather than linear regression for improved accuracy.

• Quality control implementation:

  • Include positive and negative controls on each plate.

  • Monitor inter-assay and intra-assay variability through control samples.

  • Validate with alternative quantification methods (e.g., Western blot, mass spectrometry).

Through methodical optimization of these parameters, researchers can develop ELISA protocols with detection limits in the low ng/mL range for accurate atpF quantification.

How does the genomic context of atpF in Oenothera argillicola compare with other species, and what are the implications for research?

The genomic context of atpF in Oenothera argillicola reveals important evolutionary adaptations with significant research implications:

• Unique genomic features: Oenothera plastid chromosomes contain two copies of the initiator tRNA trnfM CAU which differ by a single nucleotide polymorphism in plastomes I, II, III and IV and are part of a tandem repeat structure . This unique arrangement may influence initiation of translation for plastid-encoded proteins.

• Intron conservation: While some genes like rpl2 have lost introns in certain Lythraceae species , the intron pattern in atpF appears conserved in Oenothera, suggesting different evolutionary pressures on structural genes versus ribosomal protein genes.

These genomic context differences necessitate careful consideration when designing experiments involving plastid transformation or complementation studies, as promoter activity and RNA processing may differ between Oenothera and model plant species.

What can substitution rate analysis of atpF across Oenothera species reveal about evolutionary conservation?

Substitution rate analysis of atpF across Oenothera species provides valuable insights into evolutionary conservation patterns:

Researchers conducting evolutionary studies should implement both site-specific and sliding-window analyses of substitution rates to identify regions under different selection pressures, providing insights into the functional constraints on this important chloroplastic protein.