Recombinant Chloranthus spicatus 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 component of the peripheral stalk of the F0F1-ATP synthase complex in chloroplasts. It functions as part of the "stator" along with subunits a, d, F6, and OSCP, helping to prevent rotation of the α3β3 hexamer relative to subunit a during catalysis . The peripheral stalk is essential for maintaining structural integrity of the complex while allowing the central rotor components to turn. In chloroplasts, atpF plays a critical role in coupling proton movement across the thylakoid membrane to ATP synthesis, utilizing the proton gradient established by photosynthetic electron transport .

The ATP synthase complex consists of two major functional domains: F1, located in the chloroplast stroma (equivalent to the mitochondrial matrix), and F0, embedded in the thylakoid membrane. The F1 domain contains the catalytic sites for ATP synthesis, while the F0 domain forms the proton channel. Subunit b serves as a critical connector between these domains, ensuring proper transmission of energy from proton movement to ATP synthesis .

How does Chloranthus spicatus atpF differ from other plant species' homologous proteins?

Comparative sequence analysis reveals that while core functional domains remain highly conserved, variations occur primarily in regions involved in peripheral interactions and regulation. These differences may reflect adaptations to specific environmental conditions or metabolic requirements of Chloranthus spicatus. A detailed structural comparison would require analysis beyond the available search results.

What expression systems are most effective for producing recombinant Chloranthus spicatus atpF protein?

Multiple expression systems have been employed for recombinant production of ATP synthase subunits, each with distinct advantages. Based on available data for Chloranthus spicatus proteins, the following systems have proven effective:

• Yeast expression system: Suitable for producing properly folded eukaryotic proteins with post-translational modifications .

• E. coli expression system: Offers high yields and is frequently used for chloroplast proteins due to the prokaryotic origin of chloroplasts .

• Baculovirus expression system: Provides a eukaryotic environment with high expression levels .

• Mammalian cell expression system: Offers the most sophisticated post-translational processing but typically with lower yields .

For chloroplast proteins specifically, E. coli systems have shown success as demonstrated in research where spinach chloroplast ATP synthase subunits were functionally expressed in bacterial systems . When expressing Chloranthus spicatus atpF, the E. coli system often provides the optimal balance between yield, proper folding, and experimental simplicity, particularly for structural and functional studies.

What are the optimal conditions for purifying recombinant atpF protein while maintaining structural integrity?

Purification of recombinant atpF requires careful attention to maintain the protein's native structure. The recommended protocol includes:

• Cell lysis: Gentle lysis methods using enzymatic approaches (lysozyme for E. coli) or mild detergents are preferred over harsh mechanical disruption.

• Initial purification: If the recombinant protein includes an affinity tag (commonly used with Chloranthus spicatus proteins), affinity chromatography provides a powerful first step . Immobilized metal affinity chromatography (IMAC) with Ni-NTA resin is effective for His-tagged proteins.

• Buffer optimization: Maintaining a pH between 7.0-8.0 and including 10-15% glycerol helps stabilize the protein structure. For membrane-associated proteins like atpF, inclusion of mild detergents (0.05-0.1% n-dodecyl β-D-maltoside) may be necessary.

• Secondary purification: Size exclusion chromatography or ion exchange chromatography can improve purity beyond 85% (the typical threshold for initial characterization) .

• Quality control: SDS-PAGE analysis confirms protein purity (target >85%), while circular dichroism spectroscopy can verify proper secondary structure .

Purified proteins should be stored at -80°C in small aliquots to avoid freeze-thaw cycles that can compromise structural integrity.

How can researchers verify the functionality of purified recombinant atpF protein?

Verifying functionality of isolated atpF is challenging since it normally functions as part of the larger ATP synthase complex. Several approaches can be used:

• Binding assays: Measuring interaction with other ATP synthase subunits using techniques such as surface plasmon resonance, pull-down assays, or co-immunoprecipitation. Particularly, interaction with the F1 domain components should be detectable.

• Reconstitution experiments: Incorporating purified atpF into liposomes along with other ATP synthase components to restore partial function. Complete reconstitution has been achieved with bacterial ATP synthase and can be adapted for chloroplast proteins .

• Structural integrity assessment: Circular dichroism spectroscopy and thermal shift assays can confirm proper folding and stability.

• Complementation studies: Similar to work performed with spinach chloroplast subunits, testing whether the recombinant atpF can complement deficiencies in model organisms with mutations or deletions in the corresponding gene .

These approaches provide complementary information about protein functionality and should be used in combination for comprehensive characterization.

What experimental designs can effectively study protein-protein interactions between atpF and other ATP synthase subunits?

Several experimental approaches can elucidate the interactions between atpF and other ATP synthase components:

• Cross-linking coupled with mass spectrometry: Chemical cross-linkers can capture transient interactions, followed by mass spectrometry identification of interaction partners. This approach has successfully mapped subunit interfaces in ATP synthase complexes .

• FRET (Förster Resonance Energy Transfer): By labeling atpF and potential interaction partners with appropriate fluorophores, researchers can detect proximity and conformational changes during function.

• Cryo-electron microscopy: This technique allows visualization of the entire ATP synthase complex at near-atomic resolution, revealing the position and interactions of atpF within the native complex structure .

• Yeast two-hybrid or bacterial two-hybrid systems: These genetic approaches can screen for interactions between atpF and other subunits, though they may miss interactions that require the membrane environment.

• Co-expression and co-purification: Engineering systems to co-express atpF with other subunits and analyzing the resulting complexes can provide valuable information about assembly interactions .

The most robust approach combines multiple techniques to overcome the limitations of individual methods.

How do mutations in atpF affect ATP synthase assembly and function?

Mutations in atpF can have profound effects on ATP synthase assembly and function, disrupting energy production. Research approaches to study these effects include:

• Site-directed mutagenesis: Introducing specific mutations into recombinant atpF based on conserved residues or disease-associated variants. Similar to the approach used for the beta subunit where changing a cysteine residue to tryptophan blocked ATP synthesis without significantly affecting ATPase activity .

• Expression in model systems: Expressing mutant atpF in suitable model organisms and assessing phenotypic effects on growth, energy production, and ATP synthase assembly.

• In vitro reconstitution: Incorporating mutant atpF into reconstituted ATP synthase complexes and measuring functional parameters like ATP synthesis rates and proton pumping.

• Structural analysis: Using techniques like hydrogen-deuterium exchange mass spectrometry to determine how mutations affect protein dynamics and conformation.

These studies can reveal critical residues and domains within atpF that are essential for proper assembly and function of the ATP synthase complex. For example, studies with chloroplast beta subunits showed that residue 63, located at the interface between alpha and beta subunits, is critical for coupling nucleotide binding to proton movement .

What strategies can address poor expression yields of recombinant atpF protein?

Improving expression yields of recombinant atpF protein can be achieved through several strategies:

• Codon optimization: Adjusting the coding sequence to match the codon usage bias of the expression host often dramatically improves yields.

• Expression vector selection: Testing different promoters (T7, tac, AOX1 for yeast) can optimize transcription levels.

• Host strain selection: For E. coli expression, specialized strains like BL21(DE3) pLysS, Rosetta, or Origami can improve yields for challenging proteins .

• Fusion tags: Incorporation of solubility-enhancing fusion partners such as MBP (maltose-binding protein), SUMO, or Thioredoxin can improve expression and solubility.

• Expression conditions optimization: Systematically varying temperature (typically lowering to 18-25°C), induction timing, and inducer concentration can significantly impact yields.

• Adding specific chaperones: Co-expression with molecular chaperones like GroEL/ES can facilitate proper folding and increase yields of functional protein.

For membrane-associated proteins like atpF, lower induction temperatures (16-20°C) and extended expression times often yield better results than standard conditions.

How can researchers overcome issues related to protein instability during purification and storage?

Maintaining stability of atpF protein throughout purification and storage requires specific strategies:

• Buffer optimization: Screening different buffer compositions, including:

  • pH ranges (typically 7.0-8.0)

  • Salt concentrations (150-300 mM NaCl)

  • Addition of stabilizing agents (10-15% glycerol, 1-5 mM DTT or TCEP)

  • Testing various detergents for membrane-associated regions

• Protease inhibition: Including a complete protease inhibitor cocktail during initial extraction and purification steps.

• Temperature control: Performing all purification steps at 4°C and minimizing time between steps.

• Storage optimization: Lyophilization has proven effective for long-term storage of Chloranthus spicatus proteins . Alternatively, flash-freezing small aliquots in liquid nitrogen and storing at -80°C prevents degradation from repeated freeze-thaw cycles.

• Stabilizing additives: Including specific ligands or binding partners that stabilize the protein's conformation.

A systematic approach to testing these variables using techniques like differential scanning fluorimetry can efficiently identify optimal stability conditions.

How can recombinant Chloranthus spicatus atpF be used to study ATP synthase evolution across plant species?

Recombinant atpF can serve as a valuable tool for evolutionary studies through:

• Comparative structural analysis: Structural comparisons between Chloranthus spicatus atpF and homologs from diverse plant species can reveal evolutionary adaptations. X-ray crystallography or cryo-electron microscopy of recombinant proteins can provide the structural data necessary for these comparisons .

• Chimeric protein studies: Creating chimeric proteins combining domains from Chloranthus spicatus atpF with those from other species can identify functionally critical regions that have been conserved through evolution.

• Cross-species complementation: Testing whether Chloranthus spicatus atpF can functionally replace the equivalent subunit in model organisms provides insight into functional conservation .

• Molecular clock analysis: Detailed sequence comparisons of atpF across plant lineages, particularly focusing on synonymous vs. non-synonymous substitution rates, can reveal evolutionary pressure on different protein regions.

These approaches can uncover how ATP synthase components have evolved to meet the energy requirements of diverse plant species in different environmental niches.

What are the potential applications of studying atpF in understanding bioenergetic disorders?

Although ATP synthase disorders have been primarily studied in mitochondrial contexts, research on chloroplast ATP synthase subunits like atpF has significant translational potential:

• Model system development: Chloroplast ATP synthase can serve as a model system for understanding fundamental aspects of rotary catalysis and energy conversion relevant to mitochondrial disorders .

• Drug development platforms: Recombinant atpF can be used in screening platforms to identify compounds that modulate ATP synthase function, potentially leading to therapeutics for mitochondrial disorders .

• Structural insights: The high degree of conservation in ATP synthase structure means that detailed structural studies of chloroplast subunits can provide insights applicable to understanding human mitochondrial ATP synthase disorders .

• Evolutionary medicine: Comparing ATP synthase across species helps identify critical residues that may be sites of pathogenic mutations in humans. For example, studies have shown that subunit mutations correlate with various pathological forms, including Leigh syndrome .

The bidirectional flow of knowledge between plant and human ATP synthase research offers unique opportunities for addressing bioenergetic disorders.

How do different protein tagging strategies affect the functional studies of recombinant atpF?

Various tagging strategies offer distinct advantages and limitations for atpF functional studies:

For Chloranthus spicatus ATP synthase subunits, in vivo biotinylation using AviTag-BirA technology has been successfully employed . This approach allows for site-specific biotinylation, which is particularly valuable for orientation-controlled immobilization in interaction studies.

The optimal tag choice depends on the specific experimental goals. For structural studies, smaller tags or cleavable tags are preferred, while for interaction studies, tags that facilitate detection or immobilization may be more appropriate.

What reconstitution methods are most effective for studying atpF function in artificial membrane systems?

Reconstitution of atpF into artificial membrane systems enables functional studies in controlled environments. The following methods have proven effective:

• Liposome reconstitution: Incorporation of purified atpF along with other ATP synthase subunits into liposomes created from defined lipid mixtures. This approach allows control over lipid composition and membrane properties.

• Nanodiscs: Using membrane scaffold proteins to create nanometer-scale phospholipid bilayers containing atpF. This system provides a more native-like environment while maintaining solubility and monodispersity.

• Proteoliposomes for activity assays: Creating proteoliposomes with reconstituted ATP synthase components to measure ATP synthesis driven by artificially imposed proton gradients, similar to methods used for bacterial ATP synthase studies .

• Tethered bilayer lipid membranes (tBLMs): These systems allow for electrical measurements across the membrane while incorporating atpF and other ATP synthase components.

The effectiveness of these methods can be assessed by measuring:

• Protein incorporation efficiency using fluorescently labeled proteins

• Orientation of incorporated proteins using protease protection assays

• Functionality through ATP synthesis/hydrolysis assays

• Proton translocation using pH-sensitive dyes

Each method offers distinct advantages for specific experimental questions, with liposome reconstitution being the most widely applicable for functional studies of ATP synthase components.