Recombinant Barbarea verna ATP synthase subunit b, chloroplastic (atpF)

What is ATP synthase subunit b (atpF) and what is its role in chloroplastic energy production?

ATP synthase subunit b (atpF) is a critical peripheral stalk component of the chloroplastic ATP synthase complex. This complex plays an essential role in the final stage of photosynthesis by harnessing the proton gradient established during light-dependent reactions to synthesize ATP. The peripheral stalk, composed of subunits b and b' (encoded by atpF and ATPG respectively), functions as a critical structural element that prevents rotation of specific portions of the ATP synthase during catalysis .

The peripheral stalk connects the membrane-embedded Fo portion with the catalytic F1 portion, providing the stationary framework necessary for rotational catalysis. Without properly functioning atpF, the entire ATP synthase assembly process is compromised, preventing efficient energy production in the chloroplast .

How does Barbarea verna atpF compare to homologous genes in other plant species?

The atpF gene in Barbarea verna shares significant structural and functional similarities with homologous genes in related Brassicaceae species. Comparative analyses with other chloroplast genomes reveal that atpF belongs to the highly conserved group of chloroplast-encoded genes essential for photosynthesis .

While the specific sequence of B. verna atpF may contain species-specific variations, its core functional domains remain conserved. For context, in related chloroplast genomes such as those in Boswellia sacra, ATP synthase-related genes maintain key structural elements despite evolutionary divergence . The conservation of atpF across plant species underscores its essential role in chloroplast function and plant survival.

What is known about the genomic organization of atpF in Barbarea verna chloroplasts?

The atpF gene is located in the chloroplast genome of Barbarea verna. Like most chloroplast-encoded ATP synthase components, it is part of a conserved gene cluster within the chloroplast DNA. While the specific organization in B. verna has not been extensively characterized in the provided search results, insights can be drawn from related species.

In most plants, the atpF gene contains an intron that must be properly spliced for functional expression. The presence of this intron is evolutionarily significant, as some species have lost introns in certain chloroplast genes, potentially affecting their stress response capabilities. For instance, the maintenance of introns in genes like clpP in some species has been associated with enhanced ability to combat heat and drought stress .

What expression systems are most suitable for producing recombinant B. verna atpF protein?

For successful production of recombinant B. verna atpF protein, Escherichia coli-based expression systems have proven effective for chloroplast proteins as evidenced by successful expression of the related atpH subunit . When selecting an expression system, researchers should consider:

• Codon optimization: Adapting the plant chloroplast codon usage to match E. coli preferences for improved expression efficiency

• Vector selection: Vectors containing strong inducible promoters (T7, tac)

• Fusion tags: N-terminal His-tagging appears effective for chloroplast ATP synthase subunits, facilitating purification while maintaining function

• Expression conditions: Lower temperatures (16-20°C) often improve folding of membrane-associated proteins

• Host strains: C41(DE3) or C43(DE3) strains, specifically designed for membrane protein expression

Expression trials should include small-scale optimization before scaling up, with protein expression verified by SDS-PAGE and Western blotting.

What are effective purification strategies for isolating recombinant atpF protein?

Purification of recombinant atpF presents challenges due to its membrane-associated nature. Based on successful approaches with related ATP synthase subunits, the following strategy is recommended:

• Cell lysis: Sonication or pressure-based disruption in buffer containing mild detergents (0.5-1% DDM or LDAO)

• Initial purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin, leveraging the N-terminal His-tag

• Secondary purification: Size exclusion chromatography to separate aggregates and contaminants

• Detergent exchange: If necessary, replace extraction detergent with a milder option (0.03-0.05% DDM) during final purification steps

• Quality control: Assess purity by SDS-PAGE (>90% purity standard) and confirm identity by mass spectrometry

For structural studies, additional steps including ion exchange chromatography may improve homogeneity. Final product should be stored in glycerol-containing buffer at -80°C to maintain stability, with freeze-thaw cycles minimized .

How can researchers verify the proper folding and functionality of recombinant atpF?

Verifying proper folding and functionality of recombinant atpF requires multiple complementary approaches:

• Circular dichroism (CD) spectroscopy: To assess secondary structure content and proper folding

• Limited proteolysis: Correctly folded proteins show distinct protection patterns against proteolytic digestion

• Interaction assays: Co-immunoprecipitation or pull-down assays to verify binding to other ATP synthase subunits

• Reconstitution assays: Incorporation of purified atpF into liposomes or nanodiscs to assess membrane integration

• Functional complementation: Transformation of atpF-deficient mutants to assess functional rescue

Particular attention should be paid to the interaction between atpF and other peripheral stalk components, especially the ATPG-encoded b' subunit, as their coordinated assembly is critical for ATP synthase function. Mass spectrometry can be used to verify the presence of expected post-translational modifications that might be essential for function .

How does atpF contribute to chloroplast ATP synthase assembly and stability?

ATP synthase subunit b (atpF) plays a crucial role in the assembly and stability of the chloroplast ATP synthase complex through several mechanisms:

• Scaffold function: atpF forms part of the peripheral stalk that connects the membrane-embedded Fo portion to the catalytic F1 portion, providing structural support for the entire complex

• Assembly coordination: Research on ATP synthase biogenesis indicates that peripheral stalk subunits like atpF are required early in the assembly process, serving as nucleation points for complex formation

• Stability maintenance: Without proper atpF expression, ATP synthase complexes show impaired accumulation, as demonstrated in knock-out mutants of the related peripheral stalk component ATPG

• Proteolytic regulation: Studies with FTSH protease mutants suggest that peripheral stalk components help protect other ATP synthase subunits from proteolytic degradation

In Chlamydomonas reinhardtii, frame-shift mutations in atpF completely prevent ATP synthase function and accumulation, highlighting its essential role in complex integrity . Comparative studies suggest this mechanism is likely conserved in Barbarea verna and other higher plants.

What specific amino acid residues in atpF are critical for its function?

While the search results don't provide specific information about critical amino acid residues in B. verna atpF, general principles regarding functionally important residues in ATP synthase peripheral stalk subunits can be inferred:

• Transmembrane anchor residues: Hydrophobic amino acids in the membrane-spanning domain that correctly position the peripheral stalk

• Interaction interface residues: Amino acids mediating interactions with other ATP synthase subunits, particularly those forming the stator complex

• Structural motifs: Residues forming coiled-coil structures common in peripheral stalks that provide mechanical rigidity

• Evolutionary conservation: Amino acids conserved across species are likely functionally critical

The highly conserved nature of ATP synthase components across species suggests that sequence alignment of B. verna atpF with well-characterized homologs would identify these critical residues. Site-directed mutagenesis studies targeting these conserved regions would then confirm their functional significance.

How do environmental stresses affect atpF expression and function in Barbarea verna?

Environmental stresses significantly impact chloroplast gene expression, including atpF, though specific data for B. verna is limited. Based on research in related plants:

• Drought stress: Plants exposed to drought conditions often show altered regulation of photosynthetic genes. The presence of psbA in B. sacra, which protects photosystem II from oxidative stress during drought, suggests similar protective mechanisms might exist for ATP synthase components

• Herbivore pressure: In related species like Barbarea vulgaris, herbivore attack (e.g., diamondback moth infestation) triggers complex transcriptional responses in chloroplast-related genes

• Temperature extremes: Heat stress may require specific chaperones for proper folding of membrane proteins like atpF

• Light intensity: High light conditions generally increase demand for ATP synthase function and may upregulate expression of its components

Research on the protective gene clpP suggests that plants adapted to arid environments have evolved specific mechanisms to maintain chloroplast function under stress. Similar adaptive mechanisms likely influence atpF expression and protein stability in B. verna .

What strategies can overcome solubility issues when working with recombinant atpF?

As a peripheral membrane protein, atpF presents solubility challenges that can be addressed through multiple strategies:

• Fusion partners: Addition of solubility-enhancing fusion partners (MBP, SUMO, or Trx) at the N-terminus

• Detergent screening: Systematic testing of detergents including:

  • Mild non-ionic detergents (DDM, LMNG)

  • Zwitterionic detergents (LDAO, Fos-choline)

  • Novel amphipathic polymers (SMA, amphipols)

• Truncation constructs: Expressing soluble domains separately from transmembrane regions

• Co-expression approaches: Simultaneously expressing interaction partners that might stabilize atpF

• Cell-free expression systems: Direct synthesis into detergent micelles or nanodiscs

Optimization of buffer conditions is also critical, exploring various pH values, salt concentrations, and stabilizing additives (glycerol, specific lipids, osmolytes). The successful expression of the related chloroplastic ATP synthase subunit c (atpH) as a His-tagged protein in E. coli suggests that similar approaches might work for atpF.

How can researchers differentiate between nuclear and chloroplast-encoded versions of ATP synthase components?

Differentiating between nuclear and chloroplast-encoded ATP synthase components requires multiple analytical approaches:

• Genetic origin analysis:

  • Chloroplast DNA extraction and sequencing to confirm gene location

  • Analysis of characteristic features of chloroplast genes (promoter elements, ribosome binding sites)

  • Examination of codon usage patterns typical of chloroplast genomes

• Protein analysis:

  • N-terminal sequencing to identify chloroplast transit peptides on nuclear-encoded proteins

  • Mass spectrometry to detect post-translational modifications specific to each genetic origin

  • Immunolocalization with compartment-specific markers

• Expression dynamics:

  • Analysis of transcriptional responses under conditions affecting specifically chloroplast or nuclear gene expression

  • Evaluation of polycistronic transcripts (common in chloroplasts) versus monocistronic mRNAs

• Molecular biology approaches:

  • Use of specific inhibitors of chloroplast or cytoplasmic translation

  • Transformation experiments targeting either the chloroplast or nuclear genome

This distinction is important as chloroplast-encoded components like atpF are subject to different regulatory mechanisms than nuclear-encoded subunits like ATPG .

What methods are most effective for studying atpF-protein interactions?

Several complementary methods are effective for studying atpF protein interactions:

• Co-immunoprecipitation: Using antibodies against atpF or its interaction partners to pull down protein complexes

• Pull-down assays: Leveraging affinity tags (like the His-tag used in recombinant systems) to isolate atpF along with binding partners

• Crosslinking mass spectrometry: Chemical crosslinking followed by mass spectrometry to identify interaction interfaces at amino acid resolution

• Förster Resonance Energy Transfer (FRET): For detecting interactions in native-like environments

• Blue Native PAGE: For analyzing intact membrane protein complexes and subcomplexes

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics and affinities

• Hydrogen-deuterium exchange mass spectrometry: To identify protein regions protected during complex formation

When studying peripheral stalk assembly specifically, examining the interaction between atpF and the nuclear-encoded ATPG component is particularly important, as both proteins form the peripheral stalk and their coordinated expression is critical for ATP synthase biogenesis .

How can structural studies of atpF contribute to understanding energy transduction in chloroplasts?

Structural studies of atpF can provide crucial insights into energy transduction through several research avenues:

• Conformational dynamics: High-resolution structures of atpF in different states would reveal how the peripheral stalk maintains stability during rotational catalysis

• Energy transmission: Identifying how mechanical forces are transmitted through the peripheral stalk during proton translocation

• Assembly interfaces: Mapping the interaction surfaces between atpF and other ATP synthase components

• Species-specific adaptations: Comparing B. verna atpF structure to homologs from plants adapted to different environments could reveal evolutionary adaptations in energy production

Technical approaches should include:

• Cryo-electron microscopy of intact ATP synthase complexes

• X-ray crystallography of isolated peripheral stalk components

• NMR studies of dynamic regions

• Molecular dynamics simulations to model conformational changes

These structural insights would complement functional studies and potentially identify novel targets for enhancing photosynthetic efficiency in agricultural applications.

What role does atpF play in coordinating nuclear and chloroplast gene expression?

ATP synthase assembly requires precise coordination between nuclear and chloroplast genomes, with atpF potentially playing a key regulatory role:

• Signaling pathways: Evidence suggests that the assembly state of ATP synthase components may trigger retrograde signaling from chloroplast to nucleus

• Expression coordination: In Chlamydomonas, peripheral stalk subunits like atpF are required for stable accumulation of other ATP synthase components, suggesting a regulatory role in complex assembly

• Co-evolution of genetic systems: The interaction between chloroplast-encoded atpF and nuclear-encoded ATPG requires synchronized expression and co-evolution of both genetic systems

• RNA stability factors: Nuclear-encoded RNA-binding proteins like the OPR protein MDE1 specifically stabilize chloroplast mRNAs encoding ATP synthase components, demonstrating nuclear control over chloroplast gene expression

This coordination represents a fascinating example of the endosymbiotic relationship between the chloroplast and nuclear genomes, with factors like MDE1 being recruited relatively recently in evolutionary history to regulate chloroplast gene expression .

How might comparative studies of atpF across Brassicaceae species inform evolutionary adaptation of energy systems?

Comparative studies of atpF across Brassicaceae could reveal important evolutionary insights:

• Adaptive evolution: Sequence variations in atpF across species adapted to different environments may reflect selective pressures on energy production

• Functional conservation: Despite sequence divergence, the core function of ATP synthase is likely conserved, with variations potentially reflecting fine-tuning to ecological niches

• Co-evolutionary patterns: Coordinated changes between atpF and interacting partners would indicate co-evolution of the ATP synthase complex

• Stress adaptation signatures: Species from extreme environments may show specific modifications in atpF that enhance ATP synthase stability under stress conditions

In this context, B. verna (native to Eurasia but naturalized elsewhere) provides an interesting comparison point to related species like B. vulgaris, which has evolved specific defense mechanisms against herbivores . Chloroplast genome comparisons similar to those performed between Boswellia sacra and other plants could identify signature adaptations in photosynthetic machinery that reflect ecological adaptation.

Table 1: Key Molecular Characteristics of B. verna atpF and Related ATP Synthase Components

*Estimated range based on typical peripheral stalk components; exact B. verna sequence lengths not specified in search results

Table 2: Experimental Approaches for Studying Recombinant atpF