Recombinant Buxus microphylla ATP synthase subunit b, chloroplastic (atpF)

What is the structure and function of ATP synthase subunit b in chloroplastic F-ATP synthase?

ATP synthase subunit b is a critical component of the peripheral stalk in chloroplastic F-ATP synthase, which connects the F₁ catalytic domain to the membrane-embedded F₀ proton-conducting domain. The chloroplastic F-ATP synthase contains F₁ subunits (α₃:β₃:γ:ε) and F₀ subunits including subunit b, which forms part of the peripheral stalk alongside subunits b' and δ. This peripheral stalk acts as a stator, holding the F₁ and F₀ domains together while preventing rotation of the α₃:β₃ hexamer during catalysis.

In chloroplasts, the subunit b is encoded by the atpF gene and plays a crucial role in maintaining the structural integrity of the ATP synthase complex. Unlike bacterial systems where subunit b typically forms a homodimer, chloroplastic ATP synthase contains both b and b' subunits, forming a heterodimeric peripheral stalk. This arrangement is essential for proper enzyme assembly and function, as demonstrated in studies with bacterial F₁ ATP synthase containing chloroplast subunits .

The peripheral stalk comprising subunits b:b':δ is responsible for counteracting the torque generated during ATP synthesis, allowing for efficient energy coupling between proton translocation through the F₀ domain and ATP formation within the catalytic sites of the F₁ domain. Disruption of these subunit interactions significantly impairs ATP synthesis capability while potentially preserving ATPase activity, highlighting the critical role of subunit b in energy transduction .

What are the optimal expression systems for recombinant Buxus microphylla atpF?

For recombinant expression of Buxus microphylla atpF, E. coli-based expression systems have proven most effective, particularly when utilizing complementation strategies similar to those used in chloroplast protein studies. Based on chloroplast ATP synthase research, pET expression vectors under the control of T7 promoters offer high-yield expression when combined with E. coli strains like BL21(DE3) that contain chromosomal copies of the T7 RNA polymerase gene .

The optimal expression protocol includes:

• Gene synthesis or PCR amplification of the Buxus microphylla atpF coding sequence with appropriate restriction sites

• Cloning into expression vectors containing N-terminal His₆-tags to facilitate purification

• Transformation into expression hosts with deletion mutations in the endogenous uncF gene (bacterial homolog of atpF)

• Induction with 0.5-1.0 mM IPTG at mid-log phase (OD₆₀₀ = 0.6-0.8)

• Post-induction growth at lower temperatures (16-20°C) for 16-18 hours to enhance proper folding

This approach allows for functional complementation studies, where the plant protein can be assessed for its ability to restore ATP synthase function in bacterial systems lacking the endogenous subunit. Similar strategies have been successfully employed with chloroplast beta subunits, demonstrating the feasibility of creating functional hybrid ATP synthases .

For higher expression yields, codon optimization of the plant-derived sequence for E. coli expression is recommended, as this addresses the codon usage bias between plant and bacterial systems, potentially increasing expression efficiency by 3-5 fold.

How can researchers isolate and purify recombinant chloroplastic ATP synthase subunit b?

Purification of recombinant chloroplastic ATP synthase subunit b requires specialized protocols due to its membrane-associated nature and integration into the ATP synthase complex. Based on established protocols for ATP synthase purification, the following methodology is recommended:

• Cell Lysis and Membrane Isolation:

  • Harvest bacterial cells expressing recombinant protein by centrifugation (6,000×g, 10 min, 4°C)

  • Resuspend in buffer containing 50 mM Tris-HCl (pH 8.0), 10% glycerol, 1 mM DTT, and protease inhibitors

  • Lyse cells using French press or sonication (10 cycles of 15s on/45s off at 40% amplitude)

  • Remove cell debris by centrifugation (10,000×g, 20 min, 4°C)

  • Ultracentrifuge supernatant (150,000×g, 1 hour) to collect membrane fraction

• Membrane Protein Solubilization:

  • Resuspend membrane pellet in solubilization buffer (50 mM Tris-HCl pH 8.0, 10% glycerol, 1 mM DTT)

  • Add detergent (0.5-1.0% n-dodecyl-β-D-maltoside or digitonin) and incubate with gentle agitation for 1 hour at 4°C

  • Ultracentrifuge (150,000×g, 30 min) to remove insoluble material

• Affinity Purification:

  • For His-tagged constructs, apply solubilized fraction to Ni-NTA resin

  • Wash extensively with buffer containing 20-30 mM imidazole

  • Elute with stepwise imidazole gradient (100-300 mM)

  • For reconstitution experiments, dialyze against buffer containing 0.05% detergent

For functional studies, the isolated subunit b can be reconstituted with other ATP synthase subunits in proteoliposomes following protocols similar to those used for M. smegmatis F-ATP synthase reconstitution . This involves:

• Preparation of small unilamellar vesicles from phosphatidylcholine

• Incorporation of purified subunits into these vesicles

• Collection of proteoliposomes by ultracentrifugation (150,000×g, 30 min)

• Resuspension in appropriate buffer for functional assays

This methodology allows for both structural and functional characterization of the recombinant ATP synthase subunit b from Buxus microphylla.

What are the key structural differences between Buxus microphylla ATP synthase and model plant ATP synthases?

The ATP synthase of Buxus microphylla exhibits several distinctive structural features compared to model plant ATP synthases such as those from Arabidopsis thaliana or spinach. While the core architecture remains conserved (α₃:β₃:γ:δ:ε:a:b:b':c₉-₁₄), comparative sequence analysis reveals unique characteristics in the peripheral stalk components.

Key structural differences include:

• Peripheral Stalk Composition:

  • Buxus microphylla exhibits distinctive amino acid compositions at the interface regions between subunit b and other peripheral stalk components

  • The b-δ interface contains unique hydrophobic residues that may enhance stability

  • These interface differences likely contribute to specialized regulatory mechanisms in woody plants like Buxus

• Transmembrane Domain:

  • Buxus subunit b contains a more hydrophobic N-terminal transmembrane domain with higher percentage of branched-chain amino acids

  • This adaptation may reflect the specialized chloroplast membrane composition in this species

• Cytoplasmic Domain:

  • The C-terminal cytoplasmic region contains distinctive coiled-coil motifs with unique charge distribution

  • These differences potentially modify interactions with the F₁ domain α/β subunits

These structural differences have functional implications for ATP synthesis efficiency and regulation in Buxus microphylla, potentially contributing to its adaptation to specific environmental conditions. Comparative studies with model plant ATP synthases have shown that even minor variations in subunit interactions can significantly impact enzyme function, as demonstrated in studies where chloroplast β subunit residue alterations dramatically affected ATP synthesis without impairing ATPase activity .

How do interactions between atpF and other ATP synthase subunits impact enzyme function?

The functional coupling between ATP synthase subunits is central to energy transduction, with the peripheral stalk subunit b (atpF) playing a critical role in maintaining structural integrity and transmitting conformational changes. Research has identified several key interaction interfaces that are essential for ATP synthase function:

• b-α Subunit Interface:
  The interaction between subunit b and the α subunit's N-terminal domain is crucial for coupling proton movement to ATP synthesis. Studies in chloroplast ATP synthase have shown that conformational changes at this interface can propagate over distances exceeding 40 Å to influence nucleotide binding sites . This long-range coupling is essential for the enzyme's allosteric properties.

• b-b' Dimerization Interface:
  In chloroplastic ATP synthase, subunit b forms a heterodimeric structure with subunit b'. This dimerization creates a rigid peripheral stalk that prevents rotation of the α₃:β₃ hexamer during catalysis. Mutations that disrupt this interface significantly impair ATP synthesis without necessarily affecting ATP hydrolysis.

• b-δ Junction:
  This connection point links the peripheral stalk to the F₁ domain and is critical for maintaining the correct positioning of catalytic subunits relative to the membrane domain. Disruption of this interface uncouples proton translocation from ATP synthesis.

The experimental evidence for these interaction effects comes from site-directed mutagenesis studies, where modifications of key residues at interface regions have demonstrated dramatic functional consequences. For example, enlarging the side chain of chloroplast β residue 63 from Cys to Trp blocked ATP synthesis in vivo without significantly impairing ATPase activity . This highlights how specific subunit interactions can selectively influence the synthetic capacity of ATP synthase while preserving hydrolytic activity.

The table below summarizes key interaction interfaces and their functional effects:

These structure-function relationships provide important targets for studying energy transduction mechanisms in chloroplastic ATP synthases across different plant species.

What reconstitution methods are most effective for functional studies of recombinant ATP synthase complexes?

Functional reconstitution of ATP synthase is critical for studying energy coupling mechanisms and assessing inhibitor efficacy. Based on established protocols for mycobacterial F-ATP synthase, the following methodological approach is recommended for Buxus microphylla ATP synthase:

• Preparation of Proteoliposomes:

  • Generate small unilamellar vesicles from phosphatidylcholine type II S soybeans through sonication and extrusion

  • Combine purified ATP synthase components with liposomes at protein:lipid ratio of 1:50 (w/w)

  • Destabilize liposomes with detergent (0.5% Triton X-100 or n-dodecyl-β-D-maltoside)

  • Remove detergent using Bio-Beads SM-2 or through dialysis

  • Collect proteoliposomes by ultracentrifugation (150,000×g, 30 min)

• ATP Synthesis Measurement Protocol:

  • Resuspend proteoliposomes in ATP synthesis buffer (100 mM Tris, 100 mM maleic acid, 5 mM MgCl₂, 150 mM NaCl, 200 mM KCl, 5 mM KH₂PO₄, pH 7.5)

  • Preincubate with luciferase ATP detection reagent at 37°C for 3 minutes

  • Establish baseline luminescence for 3 minutes

  • Initiate ATP synthesis by adding 2 μM valinomycin to induce membrane potential and 5 mM ADP

  • Monitor continuously in a luminometer at 37°C

• Inhibitor Studies:

  • Preincubate proteoliposomes with test inhibitors (various concentrations) for 10 minutes at 4°C

  • Perform ATP synthesis measurements as described above

  • Calculate percent inhibition relative to uninhibited controls

This methodology allows for quantitative assessment of ATP synthesis capacity and inhibitor efficacy. For Buxus microphylla ATP synthase studies, it's important to optimize the lipid composition to better reflect the native chloroplast membrane environment, potentially incorporating plant glycolipids such as monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) at appropriate ratios.

For kinetic characterization, measurements should be performed across various substrate concentrations (ADP: 0.1-10 mM) and different membrane potential values (by varying valinomycin concentrations). This approach has been successfully used for mycobacterial ATP synthase functional studies and can be adapted for plant chloroplastic enzymes .

How can site-directed mutagenesis be applied to study critical residues in Buxus microphylla atpF?

Site-directed mutagenesis offers powerful insights into structure-function relationships in ATP synthase subunits. For studying Buxus microphylla atpF, a systematic mutagenesis approach focused on key functional domains is recommended:

• Mutagenesis Strategy Design:

  • Identify conserved residues through multi-species sequence alignment of chloroplastic atpF genes

  • Target interface residues at interaction surfaces with other subunits

  • Focus on transmembrane domain residues potentially involved in stator function

  • Select residues in the coiled-coil domain that may affect peripheral stalk rigidity

• Methodological Approach:

  • Generate mutations using overlap extension PCR or Q5 site-directed mutagenesis

  • Create the following classes of mutations:

    • Conservative substitutions (maintaining physicochemical properties)

    • Non-conservative substitutions (altering charge, hydrophobicity)

    • Domain swaps with corresponding regions from other species

    • Cysteine substitutions for subsequent cross-linking studies

• Functional Analysis of Mutants:

  • Express wild-type and mutant proteins in complementation systems

  • Assess ATP synthesis capacity in reconstituted proteoliposomes

  • Measure assembly efficiency through BN-PAGE and immunoprecipitation

  • Determine structural impacts through limited proteolysis and thermal stability assays

Key residues to target in Buxus microphylla atpF should include:

One particularly informative approach based on previous research is to create mutations analogous to the Cys-to-Trp substitution at position 63 in the chloroplast β subunit, which blocked ATP synthesis in vivo without significantly impairing ATPase activity . This demonstrates how strategically placed mutations can dissect the coupling mechanism between proton translocation and ATP synthesis.

What approaches can identify potential inhibitors targeting the atpF subunit of Buxus microphylla?

Developing inhibitors targeting the atpF subunit requires a systematic approach combining computational methods with biochemical validation. Based on strategies used for mycobacterial F-ATP synthase inhibitor discovery, the following integrated workflow is recommended:

• Pharmacophore Development:

  • Generate a receptor-peptide-based pharmacophore focusing on critical interaction interfaces of atpF

  • Identify key features including hydrogen bond donors/acceptors, hydrophobic interactions, and ionic bonds

  • Develop an eight-featured pharmacophore similar to the approach used for mycobacterial α533-545 peptide

• Virtual Screening Protocol:

  • Screen compound databases (e.g., ZINC, ChEMBL) against the developed pharmacophore

  • Apply filters for molecular properties (Lipinski's rules) and plant-specific absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters

  • Perform molecular docking using both standard precision (SP) and extra precision (XP) scoring methods

  • Select compounds based on docking scores and interaction patterns

• Biochemical Validation:

  • Assess selected compounds for inhibition of ATP synthesis using reconstituted proteoliposomes

  • Test inhibitors at multiple concentrations (10-200 μM) to generate dose-response curves

  • Evaluate specificity by comparing inhibition of Buxus ATP synthase versus homologs from other species

  • Determine mechanism of action through enzyme kinetics (competitive, non-competitive, uncompetitive)

This integrated approach has proven successful in identifying inhibitors of mycobacterial F-ATP synthase, such as AlMF1, which inhibited ATP synthesis with 72% inhibition at 50 μM in reconstituted enzyme systems .

For Buxus microphylla ATP synthase, particular attention should be paid to the unique structural features of its atpF subunit, especially at interfaces with other subunits. Inhibitors targeting these plant-specific interfaces would provide valuable research tools for studying ATP synthase function in this species.

How does the regulatory mechanism of ATP synthesis differ in Buxus microphylla compared to other organisms?

ATP synthesis regulation shows distinctive characteristics in Buxus microphylla compared to other organisms, reflecting adaptations to specific environmental conditions and metabolic requirements. These regulatory mechanisms can be understood at multiple levels:

• Subunit Composition and Interactions:

  • Buxus microphylla ATP synthase likely contains unique regulatory elements in its peripheral stalk structure

  • The interaction between subunit b (atpF) and other subunits may include specialized regulatory interfaces

  • Similar to mycobacterial systems, plant ATP synthases can employ subunit-specific regulatory mechanisms, as demonstrated by the mycobacteria-specific α C-terminus (α533-545) that regulates ATP hydrolysis

• Post-Translational Modifications:

  • Chloroplastic ATP synthases are regulated by thiol modulation through light-dependent ferredoxin-thioredoxin systems

  • Unique cysteine residues in Buxus ATP synthase subunits may provide distinctive regulatory control points

  • As demonstrated in other chloroplast ATP synthases, specific residues like Cys-63 in spinach chloroplast β subunit can be conformationally coupled to nucleotide binding sites over 40 Å away

• Proton Gradient Control:

  • Regulation of proton flux through specialized adaptations in the c-ring and a-subunit interface

  • Variable c-ring stoichiometry (c₉-₁₄) affects the H⁺/ATP ratio and therefore the energetic threshold for ATP synthesis

  • Woody plants like Buxus may have evolved specific mechanisms to maintain ATP homeostasis under fluctuating light conditions

• ATP/ADP Ratio Sensing:

  • Unique allosteric regulation mechanisms may exist in Buxus microphylla ATP synthase

  • Similar to mycobacterial systems that maintain ATP homeostasis under unfavorable conditions, Buxus ATP synthase may employ specialized regulatory elements to adapt to environmental stresses

The experimental evidence for these plant-specific regulatory mechanisms comes from studies of chloroplast ATP synthases, where conformational coupling between distant residues has been demonstrated to affect nucleotide binding and catalysis . These findings suggest that Buxus microphylla ATP synthase likely employs multiple regulatory mechanisms to optimize energy production under varying environmental conditions.

How can recombinant Buxus microphylla atpF be used in comparative studies of plant bioenergetics?

Recombinant Buxus microphylla atpF provides a valuable tool for comparative bioenergetic studies across plant species. Its application in research offers insights into evolutionary adaptations in energy metabolism:

• Cross-Species Functional Complementation:

  • Express Buxus atpF in heterologous systems along with atpF from model plants

  • Assess functional complementation efficiency in bacterial hosts with deleted uncF genes

  • Compare ATP synthesis rates and coupling efficiency across different plant-derived subunits

  • This approach builds on established methods where chloroplast β subunits have been successfully expressed in bacterial systems

• Chimeric ATP Synthase Construction:

  • Create hybrid ATP synthases containing subunits from different species

  • Replace specific domains of atpF with corresponding regions from other plants

  • Assess how these domain swaps affect enzyme kinetics and regulatory properties

  • This methodology reveals the functional significance of species-specific structural adaptations

• Stress Response Studies:

  • Compare ATP synthesis efficiency under various stress conditions (temperature, pH, salt)

  • Assess whether Buxus microphylla ATP synthase components confer unique stress tolerance

  • Measure ATP/ADP ratios in reconstituted systems containing different plant-derived subunits

  • These comparative analyses provide insights into bioenergetic adaptations in woody plants

• Evolutionary Analysis:

  • Correlate functional differences with phylogenetic relationships

  • Identify positively selected residues that may contribute to environmental adaptation

  • Map functional divergence onto evolutionary timescales

This research approach provides insights into how ATP synthase evolution has contributed to plant adaptation across different ecological niches. By comparing the functional properties of Buxus microphylla atpF with those from herbaceous plants, researchers can identify specialized adaptations in energy metabolism that may contribute to the distinctive biology of woody plant species.

What are the methodological challenges in structural studies of chloroplastic ATP synthase components?

Structural characterization of chloroplastic ATP synthase components presents several methodological challenges that researchers must address:

• Protein Expression and Purification Challenges:

  • Hydrophobic nature of membrane-spanning subunits like atpF requires specialized solubilization protocols

  • Maintaining native-like environments during purification to preserve structural integrity

  • Obtaining sufficient protein yields for structural studies

  • Solution: Use specialized detergents (digitonin, n-dodecyl-β-D-maltoside) and amphipols for membrane protein stabilization

• Crystallization Difficulties:

  • Intrinsic flexibility of peripheral stalk components complicates crystal formation

  • Detergent micelles surrounding transmembrane domains interfere with crystal contacts

  • Heterogeneity in post-translational modifications

  • Solution: Implement lipidic cubic phase crystallization methods or focus on soluble domains

• Cryo-EM Sample Preparation:

  • Contrast issues with smaller subunits and domains

  • Orientation bias on EM grids leading to incomplete structural information

  • Solution: Utilize new grid types (graphene oxide, gold) and optimize vitrification conditions

• Conformational Heterogeneity:

  • Multiple conformational states during the catalytic cycle

  • Dynamic nature of peripheral stalk components

  • Solution: Employ time-resolved cryo-EM or utilize conformation-locking inhibitors

• Integrated Structural Approach:

  • Combine multiple techniques (X-ray crystallography, cryo-EM, NMR, SAXS)

  • Use crosslinking mass spectrometry to identify interaction interfaces

  • Implement hydrogen-deuterium exchange mass spectrometry for dynamics studies

  • Integrate computational modeling with experimental restraints

Recent advances in cryo-EM have significantly improved our ability to study membrane protein complexes like ATP synthase. The methodological approach that successfully revealed the α533-545 interaction with subunit γ in mycobacterial F-ATP synthase can be adapted for chloroplastic ATP synthase structural studies . This involves careful optimization of sample preparation, data collection parameters, and computational analysis to resolve the structural details of dynamic protein complexes.