Recombinant Buxus microphylla ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, chloroplastic (atpH) from Buxus microphylla and what is its significance?

ATP synthase subunit c, chloroplastic (atpH) from Buxus microphylla (Japanese boxwood) is a critical component of the chloroplast ATP synthase complex. As part of the F0 sector, this protein forms a multimeric ring in the thylakoid membrane that facilitates proton translocation across the membrane. This proton movement drives the rotation of the ring, which is mechanically coupled to ATP synthesis in the F1 sector of the complex . The protein consists of 81 amino acids and is encoded by the atpH gene in the chloroplast genome . The study of this protein is significant for understanding energy conversion mechanisms in photosynthetic organisms and may provide insights into the evolutionary adaptations of energy metabolism in different plant species.

What expression systems are most effective for producing recombinant Buxus microphylla ATP synthase subunit c?

Escherichia coli is the predominant expression system for recombinant production of Buxus microphylla ATP synthase subunit c, chloroplastic (atpH) . The protein can be successfully expressed with an N-terminal His-tag to facilitate purification . When designing your expression system, consider that heterologous expression of membrane proteins like subunit c can be challenging due to potential toxicity to the host cells or formation of inclusion bodies. A strategy similar to that used for spinach chloroplast subunit c might be effective, where optimization of expression conditions (including temperature, induction timing, and media composition) significantly impacts yield . Expression in E. coli BL21(DE3) strains under the control of T7 promoter has proven successful for similar proteins, with expression typically induced using IPTG at lower temperatures (16-25°C) to enhance proper folding.

What purification strategies yield high-purity recombinant Buxus microphylla ATP synthase subunit c?

Purification of recombinant Buxus microphylla ATP synthase subunit c (His-tagged) typically begins with immobilized metal affinity chromatography (IMAC) using Ni-NTA resins . For optimal results:

• Perform cell lysis under denaturing conditions (8M urea) or with detergents appropriate for membrane proteins (such as DDM, LDAO, or OG).

• Include protease inhibitors during lysis to prevent degradation.

• Consider a two-step purification approach: IMAC followed by size exclusion chromatography.

• Verify purity using SDS-PAGE (>90% purity is typically achievable) .

• For functional studies, include a detergent exchange step to transition the protein into a more physiologically relevant detergent or lipid environment.

If reconstituting the multimeric ring is a research goal, methods similar to those developed for spinach chloroplast ATP synthase may be adapted, which involve careful selection of detergents and lipids for proper reassembly of the oligomeric structure .

What are the optimal storage conditions for recombinant Buxus microphylla ATP synthase subunit c?

For maximal stability and activity of recombinant Buxus microphylla ATP synthase subunit c, the following storage conditions are recommended:

• Store lyophilized powder at -20°C/-80°C upon receipt .

• After reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL, add glycerol to a final concentration of 5-50% (50% is standard) .

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles, which significantly reduce stability .

• For short-term usage (up to one week), working aliquots can be stored at 4°C .

• For long-term storage, keep at -20°C/-80°C in buffer containing Tris/PBS with 6% trehalose at pH 8.0 .

It's crucial to briefly centrifuge the vial prior to opening to bring contents to the bottom, particularly after shipping or long-term storage .

How can researchers reliably reconstitute the multimeric c-ring from recombinant Buxus microphylla ATP synthase subunit c monomers?

Reconstitution of the multimeric c-ring from recombinant monomers is a challenging process requiring precise control of experimental conditions. Based on methodologies developed for similar proteins:

• Detergent selection is critical: Screen multiple detergents including DDM, LDAO, and specialized lipid-like detergents such as CHAPSO or digitonin .

• Lipid incorporation strategies:

  • Utilize a mixed micelle approach with synthetic lipids (DOPC, DOPG) at specific lipid:protein ratios

  • Gradually remove detergent using Bio-Beads or dialysis to promote controlled assembly

  • Consider incorporating native lipids extracted from chloroplasts to enhance assembly efficiency

• Environmental factors affecting assembly efficiency:

  Factor	Optimal Range	Effect on Assembly
  pH	6.5-8.0	Higher pH favors deprotonated state of key acidic residues
  Temperature	20-30°C	Lower temperatures reduce aggregation but slow assembly
  Ionic strength	50-150 mM	Moderate salt concentrations stabilize interactions
  Divalent cations	2-5 mM Mg2+	Enhance stability of the assembled complex

• Verification methods: Assess assembly using analytical ultracentrifugation, blue native PAGE, negative-stain electron microscopy, or cross-linking mass spectrometry to confirm appropriate stoichiometry and structural integrity .

The reconstituted c-ring can be further employed for functional studies or structural analyses, providing insights into the proton translocation mechanism and rotational dynamics of this critical component of ATP synthase.

What site-directed mutagenesis approaches are most informative for studying the function of Buxus microphylla ATP synthase subunit c?

Site-directed mutagenesis of recombinant Buxus microphylla ATP synthase subunit c can provide valuable insights into structure-function relationships. Key mutagenesis targets and approaches include:

• Essential proton-binding site residues:

  • The conserved acidic residue (glutamate) in the transmembrane domain is critical for proton translocation

  • Substitution with glutamine (E→Q) to prevent protonation while maintaining size

  • Substitution with aspartate (E→D) to alter pKa while preserving charge

  • Adjacent residues that modulate the pKa of the acidic residue

• Interface residues affecting c-ring assembly:

  • Residues at monomer-monomer interfaces that determine stoichiometry and stability

  • Conservative substitutions to alter hydrophobicity or hydrogen bonding potential

  • Introduction of disulfide bridges to trap specific conformational states

• Experimental readouts for functional analysis:

  • In vitro proton translocation assays using reconstituted proteoliposomes

  • Thermostability assays to assess structural integrity

  • Binding studies with other ATP synthase components

  • ATP synthesis measurements when incorporated into complete ATP synthase complexes

• Control mutagenesis:

  • Introduction of mutations in non-essential regions as controls

  • Systematic alanine scanning to identify previously unrecognized functional regions

When expressing mutant variants, maintain identical expression and purification conditions to enable direct comparisons. Combining mutagenesis with structural studies (such as cryo-EM) can provide mechanistic insights into how specific residues contribute to proton translocation and rotary catalysis.

How do environmental conditions affect the stability and function of recombinant Buxus microphylla ATP synthase subunit c?

The stability and function of recombinant Buxus microphylla ATP synthase subunit c are significantly influenced by environmental conditions, which must be carefully controlled in experimental settings:

• pH sensitivity:

  • The protonation state of the key acidic residue is pH-dependent

  • Functional studies should examine pH ranges from 5.5 (luminal pH during photosynthesis) to 8.0 (stromal pH)

  • Stability typically decreases at extreme pH values (<5.0 or >9.0)

• Temperature effects:

  • Thermal stability typically ranges from 4-40°C, with diminished stability at higher temperatures

  • Temperature-dependent unfolding studies (using CD spectroscopy or differential scanning calorimetry) can reveal thermodynamic parameters

  • Native temperature adaptation may differ between Buxus microphylla and other species, warranting comparative studies

• Ionic strength and specific ion effects:

  • Monovalent ions (Na+, K+) at physiological concentrations (100-150 mM) typically stabilize the protein

  • Divalent cations (Mg2+, Ca2+) at 1-5 mM can enhance stability but may compete with proton binding at high concentrations

  • Anion effects should not be overlooked, particularly for chloride and phosphate

• Detergent and lipid environment:

  • Functional reconstitution requires appropriate detergent selection

  • The lipid composition significantly affects both stability and activity

  • Native-like lipid environments (with MGDG, DGDG, SQDG, and PG) most closely mimic physiological conditions but may be challenging to reproduce experimentally

Understanding these environmental factors is essential for designing robust experimental protocols and interpreting results in a physiologically relevant context. Researchers should systematically evaluate these parameters when establishing new assays or comparing results across different experimental systems.

What comparative insights can be gained by studying ATP synthase subunit c across different plant species, including Buxus microphylla?

Comparative analysis of ATP synthase subunit c across plant species, including Buxus microphylla, provides valuable evolutionary and functional insights:

• Evolutionary conservation and divergence:

  • Core functional regions (proton-binding site, transmembrane domains) show high conservation

  • Species-specific variations may correlate with environmental adaptations

  • Phylogenetic analysis can reveal evolutionary relationships and selective pressures

• Structural variations affecting c-ring stoichiometry:

  • The number of c subunits in the ring (typically 8-15) varies across species

  • This variation directly impacts the H+/ATP ratio and thus the bioenergetic efficiency

  • Comparative analysis of interface residues may explain stoichiometric differences

• Methodological approach for comparative studies:

  • Multiple sequence alignment of subunit c from diverse plant species

  • Homology modeling based on available high-resolution structures

  • Heterologous expression of subunit c from multiple species under identical conditions

  • Functional reconstitution to compare activity parameters

• Experimental data table comparing key properties across species:

  Species	Sequence Identity to Buxus (%)	Key Sequence Variations	Predicted c-ring Stoichiometry	Notable Functional Differences
  Buxus microphylla	100	Reference sequence	Unknown	Reference properties
  Spinacia oleracea	~70-80 (estimated)	Variations in loop regions	14	Well-characterized in literature
  Arabidopsis thaliana	~75-85 (estimated)	Conserved core, variable termini	14	Model system with genetic tools
  Cyanobacteria	~40-50 (estimated)	More divergent sequence	13-15	Evolutionary predecessor

This comparative approach can reveal how natural variation in subunit c contributes to functional adaptation of ATP synthase across different photosynthetic organisms and environmental niches.

What are the most effective approaches for studying the interaction between Buxus microphylla ATP synthase subunit c and other components of the ATP synthase complex?

Investigating interactions between Buxus microphylla ATP synthase subunit c and other components of the ATP synthase complex requires sophisticated biochemical and biophysical approaches:

• Co-purification and pull-down assays:

  • Express recombinant subunit c with affinity tags (His, FLAG, etc.)

  • Use differentially tagged components to verify direct interactions

  • Carefully optimize detergent conditions to maintain native-like interactions

  • Mass spectrometry analysis of pull-down fractions to identify interaction partners

• Cross-linking mass spectrometry (XL-MS):

  • Apply chemical cross-linkers of varying lengths to capture transient interactions

  • MS/MS analysis to identify cross-linked peptides and map interaction interfaces

  • Data interpretation using molecular modeling to generate structural constraints

  • Compare cross-linking patterns under different functional states (e.g., with/without nucleotides)

• Surface plasmon resonance (SPR) and microscale thermophoresis (MST):

  • Quantitative measurement of binding affinities between subunit c and other ATP synthase components

  • Determination of binding kinetics (kon, koff) under various conditions

  • Competition assays to identify binding sites

• Cryo-electron microscopy:

  • Single-particle analysis of the entire ATP synthase complex

  • Focused classification to resolve conformational states of the c-ring

  • Local refinement to improve resolution at interaction interfaces

  • Fitting of atomic models into EM density to interpret molecular details

• Functional coupling assays:

  • Measure ATP synthesis or hydrolysis with reconstituted complexes

  • Analyze how mutations in subunit c affect interactions with other components

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Assessment of rotational dynamics using single-molecule techniques

These methodologies can provide complementary information about the structural and functional integration of subunit c within the larger ATP synthase complex, offering insights into how energy conversion is achieved through coordinated protein-protein interactions.

What experimental design strategies are most effective for studying recombinant Buxus microphylla ATP synthase subunit c?

Effective experimental design for studying recombinant Buxus microphylla ATP synthase subunit c requires careful consideration of multiple factors:

• Systematic parameter optimization:

  • Apply systems thinking approach to link experimental variables and outcomes

  • Design factorial experiments to identify critical parameters and interaction effects

  • Develop a standardized workflow that maintains consistency across experiments

• Control selection:

  • Include positive controls (well-characterized ATP synthase subunit c from other species)

  • Implement negative controls (non-functional mutants, irrelevant proteins of similar size/hydrophobicity)

  • Use internal controls to normalize for experimental variation

• Validation approaches:

  • Employ multiple independent methods to confirm key findings

  • Verify protein identity using mass spectrometry

  • Confirm proper folding using circular dichroism or limited proteolysis

  • Assess functional competence through reconstitution assays

• Statistical considerations:

  • Perform power analysis to determine appropriate sample sizes

  • Use appropriate statistical tests for data analysis (t-tests, ANOVA, non-parametric methods)

  • Report effect sizes alongside p-values to indicate biological significance

These experimental design strategies enhance reproducibility and enable systematic investigation of the structural and functional properties of recombinant Buxus microphylla ATP synthase subunit c.

What are common challenges in working with recombinant ATP synthase subunit c and their solutions?

Researchers working with recombinant ATP synthase subunit c from Buxus microphylla and related species frequently encounter several challenges. Below are common issues and evidence-based solutions:

• Poor expression yields:

  • Challenge: Hydrophobic membrane proteins often express poorly in heterologous systems

  • Solutions:

    • Use specialized E. coli strains (C41/C43) designed for membrane protein expression

    • Lower induction temperature (16-18°C) and IPTG concentration (0.1-0.5 mM)

    • Consider fusion partners (MBP, SUMO) to enhance solubility

    • Evaluate cell-free expression systems for toxic proteins

• Protein aggregation during purification:

  • Challenge: Hydrophobic subunit c tends to aggregate when removed from membranes

  • Solutions:

    • Screen multiple detergents (DDM, LDAO, Fos-choline) at various concentrations

    • Add lipids during extraction and purification to stabilize native structure

    • Maintain protein concentration below aggregation threshold (typically <5 mg/mL)

    • Include glycerol (10-20%) to reduce aggregation

• Improper folding and non-functional protein:

  • Challenge: Recombinant protein may not adopt native conformation

  • Solutions:

    • Verify secondary structure using circular dichroism spectroscopy

    • Compare with native protein using limited proteolysis patterns

    • Test functional reconstitution in proteoliposomes

    • Consider refolding protocols if expression in inclusion bodies is unavoidable

• Difficulties in c-ring assembly:

  • Challenge: Monomeric subunit c must assemble into functional c-rings

  • Solutions:

    • Optimize lipid composition for reconstitution (include chloroplast-specific lipids)

    • Use detergent-mediated reconstitution followed by slow detergent removal

    • Monitor assembly using native PAGE or analytical ultracentrifugation

    • Consider co-expression with other ATP synthase components to promote assembly

• Inconsistent functional assays:

  • Challenge: Activity measurements show high variability

  • Solutions:

    • Standardize proteoliposome preparation (size, lipid composition, protein:lipid ratio)

    • Use internal controls for normalization

    • Perform technical and biological replicates (n≥3)

    • Develop robust quantitative assays with clear positive and negative controls

Addressing these challenges requires systematic troubleshooting and careful optimization of each experimental step, from gene design through expression, purification, and functional characterization.

How can researchers effectively investigate the proton translocation mechanism of Buxus microphylla ATP synthase subunit c?

Investigating the proton translocation mechanism of Buxus microphylla ATP synthase subunit c requires sophisticated experimental approaches that probe both structural and functional aspects:

• Reconstitution systems for functional studies:

  • Proteoliposome preparation with defined lipid composition

  • Co-reconstitution with minimal components needed for function

  • Development of asymmetric vesicles with controlled orientation

  • Optimization of protein:lipid ratios for maximal activity

• Proton flux measurement techniques:

  • pH-sensitive fluorescent dyes (ACMA, pyranine) to monitor ΔpH

  • Potentiometric dyes (Oxonol VI, DiSC3) to monitor membrane potential

  • Microelectrode arrays for direct proton current measurements

  • Stopped-flow spectroscopy for rapid kinetic analysis

• Spectroscopic approaches to monitor conformational changes:

  • Site-specific labeling with environmentally sensitive probes

  • FRET pairs to measure distances during the catalytic cycle

  • EPR spectroscopy with spin labels at key positions

  • Time-resolved fluorescence to capture transient states

• Molecular dynamics simulations complementing experimental data:

  • Atomistic simulations of proton movement through the c-ring

  • Free energy calculations for protonation/deprotonation events

  • Modeling of water wire formation in the proton channel

  • Prediction of conformational changes associated with proton binding

• Experimental data integration:

  Technique	Information Provided	Technical Considerations
  Site-directed mutagenesis	Identifies essential residues	Requires robust functional assay
  pH dependence studies	Reveals pKa values and pH optima	Buffer selection is critical
  Inhibitor studies	Identifies binding sites and mechanisms	Specificity must be verified
  Isotope exchange	Measures actual proton movement	Requires specialized equipment
  Cryo-EM	Visualizes conformational states	High protein concentration needed

By combining these approaches, researchers can develop a comprehensive understanding of how the Buxus microphylla ATP synthase subunit c facilitates proton translocation, a process fundamental to bioenergetic coupling in chloroplasts.

What approaches can be used to study the cytotoxic properties of compounds isolated from Buxus microphylla in relation to ATP synthase function?

Recent studies have identified bioactive compounds from Buxus microphylla with cytotoxic activities , raising interesting questions about potential interactions with ATP synthase. Methodological approaches to investigate these relationships include:

• Compound isolation and characterization:

  • Phytochemical extraction from Buxus microphylla twigs and leaves

  • Isolation of triterpenoid alkaloids (like buxmicrophyllines P-R) using chromatographic techniques

  • Structural characterization via NMR and MS analyses

  • Synthesis of derivatives for structure-activity relationship studies

• In vitro binding and inhibition assays:

  • Direct binding studies using purified recombinant ATP synthase subunit c

  • Competition assays with known ATP synthase inhibitors

  • Enzyme activity assays measuring ATP synthesis/hydrolysis rates

  • Proton translocation assays in reconstituted systems

• Cellular studies correlating cytotoxicity with ATP synthase inhibition:

  • ATP level measurements in treated cells

  • Mitochondrial membrane potential assessments

  • Oxygen consumption rate measurements

  • Cell viability assays across multiple cell lines (HL-60, SMMC-7221, A-549, MCF-7, SW480)

• Data analysis framework:

  Compound	IC50 for Cytotoxicity (μM)	ATP Synthase Inhibition (%)	Structure Type	Proposed Mechanism
  Buxmicrophylline P	Not determined	To be determined	9,10-cycloartane	To be investigated
  Buxmicrophylline Q	Not determined	To be determined	9,10-cycloartane	To be investigated
  Buxmicrophylline R	4.51-15.58	To be determined	9,10-cycloartane	To be investigated

• Molecular modeling and docking:

  • In silico prediction of binding sites on ATP synthase

  • Molecular dynamics simulations of compound-protein interactions

  • Structure-based design of analogs with enhanced specificity

  • Comparison with known ATP synthase inhibitors

This integrated approach can provide insights into whether the cytotoxic properties of Buxus microphylla compounds are mediated through ATP synthase inhibition or other cellular targets, potentially leading to the development of new research tools or therapeutic leads.

What emerging technologies show promise for advancing research on Buxus microphylla ATP synthase subunit c?

Several cutting-edge technologies are poised to transform research on Buxus microphylla ATP synthase subunit c and related proteins:

• Cryo-electron tomography (cryo-ET) with subtomogram averaging:

  • Enables visualization of ATP synthase in its native membrane environment

  • Captures conformational heterogeneity during the catalytic cycle

  • Reveals organization and distribution in thylakoid membranes

  • Provides structural context for protein-protein and protein-lipid interactions

• Single-molecule techniques:

  • Fluorescence microscopy to track rotational dynamics in real-time

  • Magnetic tweezers to measure torque generation

  • Single-molecule FRET to monitor conformational changes

  • Nanodiscs as membrane mimetics for single-molecule studies

• Integrative structural biology approaches:

  • Combining cryo-EM with mass spectrometry and computational modeling

  • Hydrogen-deuterium exchange mass spectrometry for dynamics studies

  • Integrative modeling platforms to combine diverse experimental constraints

  • AlphaFold2 and similar AI tools to predict structures and interactions

• Advanced genetic tools:

  • CRISPR-Cas9 editing of chloroplast genomes

  • Site-specific incorporation of non-canonical amino acids

  • Optogenetic control of ATP synthase activity

  • Development of plant systems with simplified ATP synthase variants

• Nanotechnology applications:

  • Artificial membrane systems with controlled composition

  • Nanopore technologies to study proton translocation

  • Surface-enhanced spectroscopies for increased sensitivity

  • Recombinant Buxus microphylla ATP synthase subunit c infused into plant viral nanoparticles for enhanced stability and delivery

These emerging technologies promise to overcome current limitations in studying membrane proteins like ATP synthase subunit c, offering unprecedented insights into their structure, dynamics, and function. Integration of these approaches will likely drive transformative discoveries in understanding the fundamental mechanisms of ATP synthesis in chloroplasts.

What are the potential applications of research on Buxus microphylla ATP synthase subunit c beyond basic science?

Research on Buxus microphylla ATP synthase subunit c has potential applications that extend beyond fundamental understanding of bioenergetics:

• Bioinspired energy conversion technologies:

  • Development of artificial photosynthetic systems

  • Biomimetic rotary nanomachines for energy conversion

  • Hybrid biological-synthetic systems for solar energy capture

  • Novel materials inspired by the proton-conducting properties of the c-ring

• Agricultural applications:

  • Engineering plants with optimized ATP synthase efficiency

  • Development of herbicides targeting species-specific features of ATP synthase

  • Stress resistance improvement through modified energy metabolism

  • Photosynthetic efficiency enhancement in crops

• Biomedical research connections:

  • Comparative studies with human mitochondrial ATP synthase

  • Drug discovery targeting ATP synthase in pathogens

  • Understanding mechanisms of diseases linked to ATP synthase dysfunction

  • Development of diagnostic tools based on ATP synthase activity

• Biotechnological adaptations:

  • Engineered ATP synthase variants for specialized applications

  • Biosensors utilizing ATP synthase components

  • Bioproduction of ATP for cell-free systems

  • Integration into synthetic cells or organelles

• Environmental monitoring and remediation:

  • Biosensors for detecting ATP synthase inhibitors in the environment

  • Understanding the effects of environmental pollutants on photosynthetic efficiency

  • Applications in phytoremediation technologies

  • Climate change impact assessment on photosynthetic energy conversion

These diverse applications underscore the broader impact of fundamental research on Buxus microphylla ATP synthase subunit c, demonstrating how insights from basic science can translate into practical technologies and solutions for global challenges in energy, agriculture, medicine, and environmental science.

What are the most significant unanswered questions regarding Buxus microphylla ATP synthase subunit c?

Despite advances in our understanding of ATP synthase structure and function, several critical questions about Buxus microphylla ATP synthase subunit c remain unanswered:

• Species-specific adaptations:

  • How does the sequence and structure of Buxus microphylla ATP synthase subunit c contribute to the plant's adaptation to its native environment?

  • Do variations in the c-subunit contribute to differences in photosynthetic efficiency across plant species?

  • What is the stoichiometry of the c-ring in Buxus microphylla, and how does it compare to other plant species?

• Structural dynamics:

  • What conformational changes occur in the c-subunit during proton translocation?

  • How do lipid-protein interactions modulate the function of the c-ring?

  • What is the precise mechanism of coupling between proton movement and ring rotation?

• Regulatory mechanisms:

  • How is the assembly of the c-ring regulated in vivo?

  • Are there post-translational modifications that affect c-subunit function?

  • How is ATP synthase activity coordinated with photosynthetic electron transport?

• Evolutionary considerations:

  • What are the evolutionary pressures that have shaped the conservation of the c-subunit?

  • How has the chloroplast ATP synthase c-subunit evolved compared to mitochondrial and bacterial homologs?

  • What can comparative genomics tell us about the co-evolution of c-subunits with other ATP synthase components?

• Biotechnological potential:

  • Can the c-subunit be engineered for enhanced photosynthetic efficiency?

  • What are the structural determinants of inhibitor binding that could be exploited for herbicide development?

  • Could synthetic biology approaches create novel functions for the c-ring architecture?