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

What is Oenothera biennis ATP synthase subunit b, chloroplastic (atpF)?

Oenothera biennis (German evening primrose) ATP synthase subunit b, chloroplastic (atpF) is a protein component of the F-type ATP synthase complex located in the chloroplast thylakoid membrane. This protein is alternatively known as "ATP synthase F(0) sector subunit b" or "ATPase subunit I," and is encoded by the atpF gene in the chloroplast genome . The protein forms part of the peripheral stalk of the ATP synthase complex, which is critical for energy production in chloroplasts through photophosphorylation.

The atpF protein plays a structural role in linking the F1 catalytic domain (where ATP synthesis occurs) to the F0 membrane domain (which forms the proton channel). This connection is essential for the functional coupling between proton translocation across the membrane and the rotary mechanism that drives ATP synthesis. The protein's importance in maintaining the structural integrity of ATP synthase makes it a valuable target for research on bioenergetic processes in plants.

How should recombinant Oenothera biennis ATP synthase subunit b be stored for optimal stability?

For optimal stability and activity maintenance of recombinant Oenothera biennis ATP synthase subunit b, the following storage conditions are recommended:

It is strongly emphasized that repeated freezing and thawing cycles should be avoided as this can significantly reduce protein stability and activity . For reconstituted proteins, manufacturers recommend adding glycerol to a final concentration of 50% and making small aliquots to minimize freeze-thaw cycles. This approach maintains protein integrity for experiments requiring functional activity and structural studies.

Which expression systems are commonly used for producing recombinant atpF?

Multiple expression systems are utilized for the production of recombinant ATP synthase subunit b, each offering distinct advantages depending on research requirements:

The choice of expression system depends on several factors including the intended application, required post-translational modifications, and experimental design considerations. For functional studies requiring properly folded and active protein, insect cell or yeast expression systems are often preferred due to their ability to perform eukaryotic post-translational modifications . For structural studies or applications requiring high purity, baculovirus expression systems have demonstrated success with ATP synthase components .

What are the key differences between chloroplastic and mitochondrial ATP synthase subunits?

Chloroplastic and mitochondrial ATP synthase subunits exhibit significant differences that reflect their specialized functions in distinct organelles:

These differences are particularly important for researchers designing experiments that target specific organellar ATP synthase complexes. For instance, antibodies against chloroplastic ATP synthase subunit alpha (AtpA) may cross-react with mitochondrial ATP synthase in some cases , while specific antibodies against mitochondrial ATP synthase beta subunit (AtpB) have been developed to distinguish between these complexes . Understanding these differences is essential for proper experimental design and interpretation of results in bioenergetic studies.

How can I optimize immunoprecipitation protocols for Oenothera biennis ATP synthase subunit b?

Immunoprecipitation (IP) of ATP synthase subunit b requires careful optimization to ensure specific isolation of protein complexes. Based on protocols tested with related ATP synthase components, the following comprehensive approach is recommended:

• Antibody Selection and Validation:

  • Use polyclonal antibodies against conserved regions of ATP synthase subunit b

  • Validate antibody specificity via Western blot before IP experiments

  • Consider using recommended dilutions (1:5000 for Western blot, 1:200 for IP)

• Sample Preparation Optimization:

  • Extract proteins under non-denaturing conditions to preserve complex integrity

  • Buffer composition: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate

  • Include protease inhibitor cocktail to prevent degradation

  • Clear lysates via centrifugation (14,000 × g, 10 min, 4°C)

  • Protein concentration: 1-5 mg/ml total protein is optimal

• Pre-clearing Step:

  • Incubate lysate with Protein A/G beads (50 μl bead slurry per 1 ml lysate)

  • Rotate for 1 hour at 4°C

  • Remove beads by centrifugation (1,000 × g, 5 min)

  • This step reduces non-specific binding significantly

• Immunoprecipitation Procedure:

  • Incubate pre-cleared lysate with antibody (2-5 μg per mg of protein)

  • Rotation overnight at 4°C ensures maximal antigen capture

  • Add 50 μl Protein A/G beads and incubate 2-4 hours at 4°C

  • Perform 4-5 washes with buffer containing reduced detergent (0.1%)

  • Final wash with detergent-free buffer

• Elution and Analysis:

  • Elute bound proteins with 50 μl SDS-PAGE sample buffer (2% SDS, 10% glycerol, 50 mM Tris-HCl pH 6.8)

  • Heat at 80°C for 10 minutes to ensure complete dissociation

  • Analyze by Western blot or mass spectrometry

• Critical Controls:

  • Input sample (5-10% of pre-IP lysate)

  • IgG control (non-relevant antibody of same species)

  • Beads-only control

  • Known interaction partner as positive control

• Troubleshooting Guide:

By implementing these optimizations, researchers can successfully isolate ATP synthase subunit b complexes for studying protein-protein interactions, post-translational modifications, and assembly states.

What structural differences exist between chloroplastic and mitochondrial ATP synthase subunit b?

The structural differences between chloroplastic and mitochondrial ATP synthase subunit b reflect their specialized functions and evolutionary history:

• Sequence and Domain Organization:

  • Both contain N-terminal transmembrane anchors but with different hydrophobicity patterns

  • The chloroplastic atpF (Oenothera biennis) contains a single transmembrane segment followed by an extended stalk

  • Mitochondrial ATP5PB (human homolog) has a more complex membrane interaction domain

  • C-terminal domains form extended α-helical structures but with organelle-specific interaction surfaces

• Dimerization Interface:

  • Chloroplastic atpF forms obligate homodimers through coiled-coil interactions

  • The dimerization interface contains characteristic leucine-zipper-like motifs

  • Mitochondrial subunit b has more polar residues at the dimerization interface

  • These differences affect the stability and flexibility of the peripheral stalk

• Interaction with Catalytic Components:

  • Chloroplastic atpF interacts with specific F₁ subunits adapted for photophosphorylation

  • Mitochondrial ATP5PB interacts with additional mitochondria-specific subunits

  • These adaptations reflect the different regulatory mechanisms in each organelle

• Experimental Methods for Structural Comparison:

• Functional Implications:

  • The structural differences contribute to organelle-specific ATP synthase functions

  • Chloroplastic features adapted to light-dependent regulation

  • Mitochondrial features evolved for response to respiratory substrates

  • Understanding these differences is crucial for designing organelle-specific interventions

These structural distinctions provide valuable insights for researchers studying organelle-specific bioenergetics and designing experiments to target specific ATP synthase complexes or subunits.

How do post-translational modifications affect the function of ATP synthase subunit b?

Post-translational modifications (PTMs) of ATP synthase subunit b play crucial roles in regulating complex assembly, stability, and activity. While specific PTM data for Oenothera biennis atpF is limited, research on homologous proteins provides significant insights:

• Types of PTMs Affecting ATP Synthase Components:

  a) Phosphorylation:

  • Affects protein-protein interactions within the ATP synthase complex

  • Key sites: Serine and threonine residues in the C-terminal domain

  • Detection methods: Phospho-specific antibodies, Phos-tag gels, mass spectrometry

  • Functional impact: Can alter complex stability and catalytic efficiency

  b) Oxidative Modifications:

  • Nitration of tyrosine residues has been shown to elicit FoF1-ATPase activity loss

  • Oxidative stress can lead to carbonylation of vulnerable residues

  • Detection methods: Anti-nitrotyrosine antibodies, OxyBlot, redox proteomics

  • Functional impact: Generally inhibitory to ATP synthase function

  c) Acetylation:

  • Occurs primarily on lysine residues in the soluble domain

  • Affects interaction with other subunits and complex stability

  • Detection: Anti-acetyl-lysine antibodies, mass spectrometry

  • Functional impact: Can alter regulatory properties

• Methodological Approaches for Studying PTMs:

  a) Sample Preparation:

  • Extract proteins with phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄)

  • Include protease inhibitors to prevent degradation

  • Rapid extraction on ice to preserve labile modifications

  b) Analytical Techniques:

  • 2D-PAGE followed by Western blotting with modification-specific antibodies

  • Immunoprecipitation (using protocol in section 2.1) combined with mass spectrometry

  • Enrichment strategies for specific modifications (IMAC for phosphopeptides)

  c) Targeted Proteomics Workflow:

  • Protein extraction and digestion with trypsin

  • Enrichment of modified peptides

  • LC-MS/MS analysis with multiple reaction monitoring

  • Data analysis using specialized software (MaxQuant, Skyline)

• PTM Distribution and Functional Impact:

• Experimental Design for PTM Studies:

  a) Comparative Analysis:

  • Control vs. stress conditions (high light, oxidative, heat stress)

  • Developmental stages (young vs. mature leaves)

  • Different tissues (mesophyll vs. bundle sheath cells)

  b) Mutagenesis Approach:

  • Generate phospho-mimetic (S/T to D/E) or phospho-deficient (S/T to A) mutants

  • Express in heterologous systems or plant models

  • Assess functional consequences using activity assays

Understanding these modifications provides valuable insights into how plants regulate ATP synthase activity in response to changing environmental conditions and developmental stages, offering potential targets for enhancing photosynthetic efficiency.

What approaches can be used to study the role of ATP synthase subunit b in bioenergetics?

Investigating the role of ATP synthase subunit b (atpF) in bioenergetics requires integrating structural, biochemical, and physiological approaches:

By systematically applying these complementary approaches, researchers can develop a comprehensive understanding of how ATP synthase subunit b contributes to bioenergetic processes in chloroplasts, providing insights into fundamental aspects of photosynthetic energy conversion and potential targets for optimization.

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

Mutations in the atpF gene encoding ATP synthase subunit b can significantly impact the assembly and function of the ATP synthase complex. A systematic analysis of mutation effects reveals:

• Categories of atpF Mutations and Their Functional Impacts:

  a) Transmembrane Domain Mutations:

  • Affect membrane anchoring and complex stability

  • May disrupt proton translocation pathways

  • Often result in complete loss of ATP synthase assembly

  b) Dimerization Interface Mutations:

  • Compromise formation of the b-b dimer essential for peripheral stalk

  • Destabilize connections between F₁ and F₀ sectors

  • Result in uncoupled complexes that maintain ATP hydrolysis but lack synthesis

  c) F₁ Interaction Domain Mutations:

  • Disrupt connections to the catalytic F₁ sector

  • Affect energy coupling between proton transport and ATP synthesis

  • May allow F₁ sector to dissociate from the membrane

• Methodological Approaches for Studying atpF Mutations:

  a) Generation of Mutations:

  • Site-directed mutagenesis of the Oenothera biennis atpF sequence

  • Expression of mutant proteins in heterologous systems

  • Introduction of mutations into model organisms via CRISPR/Cas9

  b) Functional Characterization Framework:

  • ATP synthesis/hydrolysis assays with isolated complexes

  • Proton pumping measurements using pH-sensitive dyes

  • Growth phenotype analysis under different light conditions

  c) Structural Assessment Techniques:

  • Blue Native PAGE to analyze complex assembly states

  • Co-immunoprecipitation to test subunit interactions

  • Cryo-EM analysis of complex integrity

• Comprehensive Analysis of Mutation Effects:

• Systematic Mutation Analysis Strategy:

  a) Alanine Scanning:

  • Replace consecutive residues with alanine

  • Identify regions critical for function

  • Target identified regions for further analysis

  b) Conservation-Based Approach:

  • Focus on residues conserved across species

  • Compare the sequence from Oenothera biennis with other plants

  • Prioritize residues with high evolutionary conservation

  c) Structure-Guided Mutagenesis:

  • Based on homology models or available structures

  • Target predicted interaction surfaces

  • Introduce charge reversal mutations at key interfaces

This systematic approach to studying atpF mutations provides valuable insights into the structure-function relationships of ATP synthase and can inform strategies for engineering improved photosynthetic efficiency in crop plants.