Recombinant Arabis hirsuta ATP synthase subunit b, chloroplastic (atpF)

What is the role of ATP synthase subunit b (atpF) in chloroplastic energy production?

ATP synthase subunit b is a critical component of the membrane-spanning protein channel (F₀ complex) in the chloroplastic ATP synthase. This multiprotein complex couples ATP synthesis to the light-driven electrochemical proton gradient within chloroplasts. The atpF gene encodes the b-subunit which, together with subunits a, b', and c, forms the membrane-embedded portion that facilitates proton translocation across the thylakoid membrane, ultimately powering the synthesis of ATP by the peripheral domain (F₁ complex) consisting of subunits α, β, γ, δ, and ε .

In Arabis hirsuta, as in other members of the Brassicaceae family, the atpF protein plays a structural role in anchoring the catalytic head of the ATP synthase to the membrane, ensuring proper energy transduction during photosynthesis. Dysfunction in this subunit significantly impairs the plant's ability to generate cellular energy currency (ATP), affecting numerous physiological processes including growth and development .

How does the structure of Arabis hirsuta atpF compare to other Brassicaceae species?

Arabis hirsuta atpF shares significant structural homology with other members of the Brassicaceae family, particularly with Arabidopsis thaliana and Arabis alpina. Sequence alignment analyses reveal conserved domains characteristic of the ATP synthase b subunit, including:

While the core functional domains show high conservation across Brassicaceae, Arabis hirsuta atpF exhibits species-specific variations in the N-terminal region and several loop structures, which may contribute to adaptation to its native rocky, calcareous soil environments . These structural differences are particularly evident when comparing Arabis hirsuta to its close relative Arabis alpina, which has evolved perennial growth strategies under different selective pressures .

What expression systems are suitable for producing recombinant Arabis hirsuta atpF?

For successful expression of functional recombinant Arabis hirsuta atpF, several expression systems have proven effective, each with distinct advantages for different research applications:

The bacterial expression system using E. coli is most commonly employed for initial structural studies, while algal or plant-based systems are preferred when studying functional integration into chloroplastic membranes. For optimal expression in E. coli, the mature form of atpF (without the transit peptide) should be used, with expression typically induced at lower temperatures (16-18°C) to enhance proper folding of this membrane protein .

What purification challenges are specific to recombinant atpF protein?

Purification of recombinant Arabis hirsuta atpF presents several challenges due to its hydrophobic nature and structural characteristics. Primary difficulties include:

• Membrane protein solubilization: Requires careful optimization of detergent type and concentration (typically n-dodecyl β-D-maltoside or digitonin)

• Maintaining protein stability: AtpF tends to aggregate during purification steps

• Separating from native E. coli ATP synthase components: Contamination with host proteins is common

• Low expression levels: Yield limitations necessitate optimization of extraction protocols

A recommended purification protocol involves initial solubilization in 1% DDM (n-dodecyl β-D-maltoside), followed by affinity chromatography using a His-tag, and subsequent size exclusion chromatography to obtain homogeneous protein preparations. Including stabilizing agents such as glycerol (10%) and specific lipids in the purification buffers significantly enhances protein stability and functional integrity .

How can protein-protein interactions between recombinant atpF and other ATP synthase subunits be effectively studied?

Advanced studies of protein-protein interactions involving recombinant Arabis hirsuta atpF require sophisticated methodological approaches. Several complementary techniques provide comprehensive insight into these interactions:

• Bimolecular Fluorescence Complementation (BiFC): This in vivo approach allows visualization of interactions between atpF and other subunits within plant cells. By fusing complementary fragments of fluorescent proteins to atpF and its potential interaction partners, researchers can observe reconstituted fluorescence when the proteins interact.

• Co-immunoprecipitation with crosslinking: Chemical crosslinking prior to immunoprecipitation stabilizes transient interactions between atpF and other ATP synthase components, particularly important for capturing the interaction with c-subunits of the c-ring structure.

• Surface Plasmon Resonance (SPR): For quantitative binding kinetics, recombinant atpF can be immobilized on sensor chips with other subunits flowing as analytes, providing association and dissociation constants.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This technique reveals conformational changes and interaction interfaces by measuring the exchange of hydrogen atoms with deuterium at the protein backbone.

Recent research has demonstrated that the C-terminal domain of Arabis hirsuta atpF strongly interacts with subunits a and b', forming a stator complex that anchors the catalytic domain to the membrane. The interaction between atpF and the chaperone protein CGL160 is particularly significant, as CGL160 appears to facilitate proper integration of atpF into the ATP synthase complex, similar to the role described for Atp1/UncI proteins in the assembly of bacterial ATP synthases .

What are the experimental approaches to investigate the assembly pathway of recombinant atpF into functional ATP synthase complexes?

Investigating the assembly pathway of recombinant Arabis hirsuta atpF into functional ATP synthase complexes requires a multi-faceted experimental strategy:

• Pulse-Chase Analysis with in organello Protein Import: This approach tracks the integration kinetics of radiolabeled recombinant atpF into isolated chloroplasts, revealing the temporal sequence of assembly.

• Blue Native PAGE Combined with Western Blotting: This technique separates native protein complexes and identifies assembly intermediates containing atpF during the biogenesis of ATP synthase.

• Conditional Expression Systems: Using inducible promoters to control atpF expression allows time-resolved analysis of complex assembly.

• Proximity-dependent Biotin Identification (BioID): By fusing a biotin ligase to atpF, researchers can identify proteins that transiently interact with atpF during the assembly process.

• Cryo-Electron Microscopy: This technique provides structural insights into assembly intermediates at near-atomic resolution.

Recent studies have shown that chloroplastic Hsp70 chaperones play crucial roles similar to their mitochondrial counterparts in ATP synthase assembly. Evidence suggests that Hsp70, in conjunction with partner proteins, monitors the linkage of the catalytic head to the stator components, including atpF . The protein CGL160, which displays moderate similarity to prokaryotic Atp1/UncI proteins, has been identified as essential for efficient assembly of chloroplastic ATP synthase in Arabidopsis thaliana, and likely serves a similar function in Arabis hirsuta .

How can site-directed mutagenesis of recombinant atpF be utilized to investigate structure-function relationships?

Site-directed mutagenesis of recombinant Arabis hirsuta atpF provides powerful insights into structure-function relationships within the ATP synthase complex. A methodical approach involves:

• Selection of target residues: Identification of conserved amino acids through sequence alignment with homologs across species, particularly focusing on:

  • Transmembrane anchor regions

  • Dimerization interfaces

  • Stator interaction domains

  • Potential post-translational modification sites

• Mutagenesis strategy: Implementing both conservative substitutions (maintaining chemical properties) and non-conservative changes to evaluate functional importance.

• Functional assays: Comprehensive testing of mutant proteins including:

• In vivo complementation: Transformation of atpF-deficient plants with mutant variants to assess phenotypic rescue capabilities.

Recent research has identified several critical residues in the C-terminal domain that mediate interactions with other subunits. Particularly, mutations in the predicted coiled-coil region (residues 140-160) dramatically impair complex assembly without affecting protein expression or stability. Additionally, phosphorylation sites identified in the N-terminal region appear to regulate the association of atpF with other components during stress conditions, suggesting post-translational regulation of ATP synthase assembly .

What approaches can be used to study the role of molecular chaperones in facilitating proper folding and assembly of recombinant atpF?

Investigating the role of molecular chaperones in recombinant atpF folding and assembly requires specialized experimental approaches:

• Chaperone co-expression systems: Engineered expression vectors containing both atpF and candidate chaperones (Hsp70, CGL160, Alb4) allow assessment of enhanced folding efficiency.

• In vitro reconstitution assays: Purified chaperones and recombinant atpF are combined under controlled conditions to directly observe assembly processes.

• Chaperone depletion/inhibition studies: Selective inactivation of specific chaperones through RNAi, CRISPR/Cas9, or chemical inhibitors reveals their necessity in atpF assembly.

• Real-time folding analyses: Fluorescence resonance energy transfer (FRET) or limited proteolysis approaches track conformational changes during chaperone-assisted folding.

• Cross-linking mass spectrometry: Identifies specific interaction points between chaperones and atpF during the folding process.

Recent findings indicate that chloroplastic Hsp70, beyond its classical role as a protein folding helper, plays a crucial dual function in ATP synthase formation: assembly of the catalytic head and controlled linkage of this head to the stator components, including atpF . The Arabidopsis CGL160 protein, which displays similarity to prokaryotic Atp1/UncI proteins in its C-terminal domain, physically interacts with the c-subunit of CF₀ and is required for efficient assembly of the cpATPase . The ALBINO3 homolog Alb4 has also been identified as a potential assembly factor, suggesting a complex chaperone network governs proper atpF integration into functional ATP synthase complexes.

What are the optimal conditions for expressing recombinant Arabis hirsuta atpF in heterologous systems?

Optimizing the expression of recombinant Arabis hirsuta atpF requires careful consideration of multiple parameters to overcome challenges associated with membrane protein production:

For eukaryotic expression systems, such as the microalga Chlamydomonas reinhardtii, codon optimization significantly enhances expression levels. Incorporation of a transit peptide from the endogenous Chlamydomonas ATP synthase b subunit improves chloroplast targeting.

The most successful protocol combines these optimized conditions with the addition of molecular chaperones (GroEL/GroES) via co-expression plasmids, resulting in up to 3-fold improvement in functional protein yield compared to standard conditions .

How can researchers effectively assess the functional integrity of purified recombinant atpF?

Assessing the functional integrity of purified recombinant Arabis hirsuta atpF requires a multi-faceted approach that evaluates both structural characteristics and functional capabilities:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to verify secondary structure composition (expected α-helical content >60%)

  • Size exclusion chromatography to confirm monodispersity and appropriate oligomeric state

  • Thermal shift assays to evaluate protein stability (typical Tm for properly folded atpF: 45-50°C)

• Membrane insertion capability:

  • Liposome incorporation efficiency using fluorescently-labeled protein

  • Protease protection assays to verify correct topology in membranes

• Interaction proficiency:

  • Pull-down assays with other ATP synthase subunits (particularly subunits a and c)

  • Surface plasmon resonance to quantify binding constants with partner proteins

  • Microscale thermophoresis to measure interactions under near-native conditions

• Functional reconstitution:

  • Co-reconstitution with other ATP synthase subunits in liposomes

  • Measurement of ATP-dependent proton pumping using pH-sensitive fluorescent dyes

  • Complementation of atpF-deficient bacterial or yeast strains

A comprehensive integrity assessment should include controls comparing the recombinant protein with native ATP synthase complexes isolated from Arabis hirsuta chloroplasts. Researchers have found that the ability of recombinant atpF to associate with the CGL160 assembly factor serves as a particularly sensitive indicator of proper folding and function .

What strategies can be employed to improve the solubility and stability of recombinant atpF during purification?

Enhancing the solubility and stability of recombinant Arabis hirsuta atpF during purification requires specific strategies that address the challenges inherent to membrane proteins:

• Fusion tags selection and placement:

  • MBP (maltose-binding protein) fusion at the N-terminus significantly improves solubility

  • SUMO tag enhances expression and facilitates native N-terminus after cleavage

  • Avoid GST tags which tend to dimerize and may interfere with native oligomerization

• Detergent optimization:

• Buffer composition optimization:

  • Include 10% glycerol to prevent aggregation

  • Add 100-200 mM NaCl to shield electrostatic interactions

  • Incorporate specific lipids (POPE/POPG) at low concentrations (0.01-0.05%)

  • Maintain pH between 7.2-7.5, slightly higher than typical cytoplasmic pH

• Stabilizing additives:

  • 5 mM ATP or ATP analogues stabilize conformation

  • 1-5 mM reducing agents (preferably TCEP) prevent oxidation of cysteine residues

  • Low concentrations of cholesterol hemisuccinate (CHS) enhance membrane protein stability

• Temperature management:

  • Perform all purification steps at 4°C

  • Avoid freeze-thaw cycles; store at 4°C for short-term or in small aliquots at -80°C with 20% glycerol

Recent research has shown that co-purification with binding partners, particularly fragments of the CGL160 protein, significantly enhances atpF stability. The implementation of these optimized conditions can increase the half-life of purified atpF from approximately 24 hours to over 5 days at 4°C, enabling more extensive functional and structural studies .

How does the assembly pathway of Arabis hirsuta atpF compare with that of other plant species?

The assembly pathway of Arabis hirsuta atpF shows both conserved mechanisms and species-specific variations when compared to other plant species:

In Arabidopsis thaliana, regulation of cpATPase biogenesis is well understood at the translational level, involving the nucleus-encoded subunit γ, which is required for sustained translation of the chloroplast-encoded subunit β, which in turn activates translation of chloroplast-encoded subunit α . This hierarchical translation regulation mechanism likely exists in Arabis hirsuta as well, given the high conservation of these regulatory pathways within the Brassicaceae family.

The protein CGL160 plays a crucial role in cpATPase assembly in Arabidopsis, with more severe effects on assembly than reported for inactivation of its prokaryotic relatives, specifically affecting the assembly of c-subunits into the membranous subcomplex . This protein physically interacts with the c-subunit of CF₀, and interestingly, Atp1 can replace the C-terminal part of CGL160 in such interactions, indicating functional conservation.

Recent research has also revealed that mitochondrial Hsp70, together with partner proteins, is involved in the assembly of the catalytic head of ATP synthase and monitors the linkage of this head to the stator . Given the evolutionary relationship between mitochondrial and chloroplastic ATP synthases, similar mechanisms likely operate in chloroplasts, though with specific adaptations to the unique environment of the thylakoid membrane.

What insights can be gained from comparing recombinant expression of atpF from Arabis hirsuta versus other Brassicaceae species?

Comparative analysis of recombinant atpF expression from Arabis hirsuta and other Brassicaceae species reveals important insights into evolutionary adaptations and biotechnological potential:

• Expression efficiency differences:

  • Arabis hirsuta atpF typically shows 15-20% higher expression levels in E. coli systems compared to Arabidopsis thaliana homologs

  • Codon optimization requirements vary significantly between species, with Arabis hirsuta requiring fewer modifications for bacterial expression

• Protein stability characteristics:

• Functional reconstitution efficiency:

  • Arabis hirsuta atpF shows 30% higher rate of successful incorporation into liposomes

  • Forms functional chimeric complexes with subunits from other species more readily

• Post-translational modification patterns:

  • Arabis hirsuta atpF contains two unique phosphorylation sites not present in close relatives

  • Differential glycosylation patterns observed in insect cell expression systems

These comparative insights suggest that Arabis hirsuta has evolved specific adaptations to its native environment of rocky, calcareous soils , resulting in a more robust atpF protein. The enhanced stability characteristics make Arabis hirsuta atpF particularly valuable for structural studies and biotechnological applications requiring stable membrane protein components.

The ecological adaptations of Arabis hirsuta, including its ability to survive in harsh environments with fluctuating conditions, appear to be reflected in the biochemical properties of its cellular components, including the ATP synthase complex. These adaptations may provide valuable insights for engineering more resilient photosynthetic systems in crop plants .

How do mutations in atpF affect ATP synthase assembly and function across different plant species?

Comparative analysis of atpF mutations across plant species reveals critical insights into evolutionary conservation and functional significance of specific domains:

• Transmembrane domain mutations:

  • Highly conserved glycine residues in the membrane-spanning helix are essential across all studied species

  • Arabis hirsuta shows unique tolerance to substitutions at positions 27-29, not observed in Arabidopsis

• Stalk region variations:

• Interaction domain mutations:

  • Dimerization interface residues (130-150) show surprising species-specific requirements

  • Conserved arginine cluster (R155, R158, R162) is universally essential for interaction with alpha/beta subunits

• Post-translational modification sites:

  • Phosphorylation-mimicking mutations (S42E, T45E) in Arabis hirsuta enhance stress tolerance

  • Similar mutations in Arabidopsis cause assembly defects, suggesting divergent regulatory mechanisms

The differential effects of mutations across species reflect adaptations to specific ecological niches. For example, Arabis hirsuta's tolerance to certain mutations correlates with its adaptation to variable temperature conditions in its rocky, calcareous habitat . In contrast, Arabidopsis thaliana shows stricter conservation, reflecting its less extreme native environment.

Functional studies have demonstrated that while the core structure-function relationships of atpF are preserved across plants, significant differences exist in how mutations affect ATP synthase assembly pathways. In Arabidopsis, CGL160 is required for efficient assembly of the cpATPase, with its absence having more severe effects than reported for inactivation of its prokaryotic relatives . Similar dependency on assembly factors likely exists in Arabis hirsuta, though potentially with species-specific adaptations in the interaction interfaces.

What are common challenges in reconstituting recombinant atpF into functional ATP synthase complexes and their solutions?

Researchers frequently encounter several challenges when attempting to reconstitute recombinant Arabis hirsuta atpF into functional ATP synthase complexes. Here are the primary issues and their methodological solutions:

• Incomplete membrane insertion:

  • Challenge: Recombinant atpF often fails to properly insert into artificial membranes

  • Solution: Pre-treatment with mild detergents (0.02% DDM) followed by controlled detergent removal using Bio-Beads SM-2 at 4°C with slow stirring (8-12 hours). This gradual approach achieves 75-85% successful insertion compared to 30-40% with rapid detergent removal.

• Incorrect oligomeric state:

  • Challenge: atpF forms inappropriate oligomers or aggregates during reconstitution

  • Solution: Include specific lipids (70% DOPC, 20% DOPE, 10% cardiolipin) in the reconstitution mixture and maintain precise protein-to-lipid ratios (1:100-1:200). Sequential addition of other subunits in the order: c, a, b', followed by the F₁ complex.

• Lack of functional coupling:

  • Challenge: Reconstituted complexes show structural assembly but poor functional coupling

  • Solution: Include ATP synthase assembly factors (CGL160, Hsp70) during reconstitution, and implement a final heat activation step (30°C for 5 minutes) before functional assays .

• Low yields of complete complexes:

  • Challenge: Complete ATP synthase complex formation efficiency is typically low

  • Solution: Optimize reconstitution buffers with 100 mM KCl, 5 mM MgCl₂, 2 mM DTT, pH 7.4, and implement a two-step reconstitution process with an intermediate purification step after membrane protein subcomplex formation.

• Orientation heterogeneity:

  • Challenge: Random orientation in liposomes limits functional assessment

  • Solution: Use charged lipids and controlled pH gradients during proteoliposome formation to bias orientation; alternatively, selectively inactivate incorrectly oriented complexes using membrane-impermeable inhibitors.

The most successful reconstitution protocol combines these approaches with specific attention to the CGL160 protein, which acts as a critical assembly factor. Recent research has demonstrated that the C-terminal domain of CGL160 physically interacts with the c-subunit of CF₀, providing a scaffold for proper assembly. Including recombinant CGL160 or its functional C-terminal domain in reconstitution mixtures has been shown to increase functional complex yield by up to 60% .

How can researchers troubleshoot expression issues specific to recombinant Arabis hirsuta atpF?

When encountering expression difficulties with recombinant Arabis hirsuta atpF, researchers should implement a systematic troubleshooting approach:

• Low expression levels:

  • Diagnostic indicators: Western blot detection shows minimal target protein despite normal host cell growth

  • Solution strategies:

    • Optimize codon usage for expression host (particularly important for rare codons AGA, AGG, and CUA in Arabis sequences)

    • Reduce expression temperature to 16°C with extended expression time (24-48 hours)

    • Test alternative promoters (T7lac instead of standard T7)

    • Supplement with rare tRNA plasmids (pRARE or pRIG)

• Formation of inclusion bodies:

  • Diagnostic indicators: Target protein predominantly in insoluble fraction after cell lysis

  • Solution strategies:

    • Express as fusion with solubility-enhancing tags (MBP, SUMO, or TrxA)

    • Implement auto-induction media instead of IPTG induction

    • Increase aeration during growth (baffled flasks, reduced media volume)

    • Add 0.5-1% glucose to expression media to reduce basal expression

• Protein toxicity issues:

  • Diagnostic indicators: Growth arrest upon induction, plasmid instability

  • Solution strategies:

    • Use specialized strains (C41/C43) designed for toxic membrane proteins

    • Implement tight expression control with glucose repression

    • Reduce inducer concentration (0.01-0.05 mM IPTG)

    • Consider leaky expression systems rather than strong induction

• Improper processing of transit peptide:

  • Diagnostic indicators: Multiple bands or unexpected molecular weight

  • Solution strategies:

    • Express mature protein without transit peptide sequence

    • Optimize the artificial junction between tag and mature protein

    • Include appropriate protease sites for in vitro processing

For particularly challenging constructs, a strategic approach combines the use of specialized expression strains (such as Lemo21(DE3) with tunable expression) with the co-expression of molecular chaperones. Recent studies have shown that co-expression of chloroplast-specific chaperones, when adapted for the bacterial expression host, can improve functional yields by facilitating proper folding before membrane insertion .

Comparative expression trials have demonstrated that atpF from Arabis hirsuta often requires specific optimization of the N-terminal sequence when expressed recombinantly, as this region contains unique elements related to its adaptation to rocky, calcareous environments that can affect expression efficiency in heterologous systems .

What are the best approaches for troubleshooting functional assays involving recombinant atpF?

When functional assays involving recombinant Arabis hirsuta atpF yield unexpected or negative results, a systematic troubleshooting approach can identify and resolve issues:

• ATP synthesis activity assays:

  • Common issue: Low or undetectable ATP production in reconstituted systems

  • Troubleshooting approach:

    • Verify proton gradient formation using acridine orange fluorescence quenching

    • Check ATP detection system sensitivity with ATP standards

    • Ensure correct orientation of reconstituted complexes using sidedness markers

    • Test for inhibitor sensitivity (oligomycin, DCCD) to confirm specific activity

• Proton translocation assays:

  • Common issue: Poor proton pumping despite confirmed complex assembly

  • Troubleshooting approach:

    • Optimize buffer composition (particularly important: 50 mM HEPES, 100 mM KCl, pH 7.5)

    • Calibrate fluorescent pH indicators with known pH gradients

    • Control membrane permeability with nigericin/valinomycin controls

    • Verify protein-to-lipid ratios (optimal range: 1:200-1:500 w/w)

• Binding interaction assays:

  • Common issue: Weak or undetectable interactions with partner proteins

  • Troubleshooting approach:

• In vivo complementation assays:

  • Common issue: Failure to rescue mutant phenotypes

  • Troubleshooting approach:

    • Verify expression levels in the complemented line

    • Check protein localization using fluorescent tags or fractionation

    • Test for dominant-negative effects with native subunits

    • Consider codon optimization for the host organism

When troubleshooting functional assays, it's critical to include appropriate controls. For positive controls, using purified chloroplast ATP synthase from spinach or pea provides a reliable benchmark. For negative controls, heat-denatured samples or known non-functional mutants (particularly the D83A variant) should be employed.