Recombinant Acorus americanus ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, chloroplastic (atpH) in Acorus americanus?

ATP synthase subunit c, chloroplastic (atpH) is a critical component of the F0 sector of ATP synthase in the chloroplasts of Acorus americanus. This protein functions as part of the proton channel within the membrane-embedded F0 portion of the ATP synthase complex. The recombinant form (Q4FGF2) encompasses amino acids 1-81 and is typically expressed with an N-terminal His tag in E. coli expression systems. Alternative names include ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein .

What is the amino acid sequence of Acorus americanus atpH protein?

The full amino acid sequence of Acorus americanus ATP synthase subunit c, chloroplastic (atpH) consists of 81 amino acids with the following sequence: MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFM EALTIYGLVVALALLFANPFV . This sequence represents the complete protein without any fusion tags. For research applications, the recombinant protein is often produced with an N-terminal His tag to facilitate purification.

How should recombinant atpH protein be stored and reconstituted for experimental use?

For optimal stability, lyophilized recombinant atpH protein should be stored at -20°C/-80°C upon receipt. Aliquoting is recommended for multiple use scenarios to avoid repeated freeze-thaw cycles, which can compromise protein integrity. For reconstitution, briefly centrifuge the vial to bring contents to the bottom, then reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Adding glycerol to a final concentration of 5-50% is recommended for long-term storage at -20°C/-80°C (50% glycerol is the default recommendation). Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided .

How can atpH protein be effectively used in membrane protein interaction studies?

To study membrane protein interactions involving atpH, researchers should consider these methodological approaches:

• Co-immunoprecipitation assays: Similar to techniques used for ATP synthase subunit interactions with other proteins like ANT, use antibodies against the His tag or specific epitopes of atpH to pull down protein complexes. This can identify interaction partners in reconstituted membrane systems or when expressed in heterologous systems .

• Crosslinking experiments: Implement chemical crosslinking with membrane-permeable reagents followed by mass spectrometry to identify proteins in proximity to atpH within membrane environments.

• Reconstitution in liposomes: Purify recombinant atpH and potential interaction partners, then reconstitute in liposomes to assess functional interactions through activity assays or biophysical measurements.

• Förster resonance energy transfer (FRET): Tag atpH and potential interaction partners with appropriate fluorophores to monitor protein-protein interactions in reconstituted membrane systems.

For reliable results, maintain the native lipid environment whenever possible, as the protein is a lipid-binding membrane protein whose interactions may be highly dependent on the membrane composition .

What strategies should be employed for optimizing expression and purification of recombinant atpH protein?

For optimal expression and purification of recombinant Acorus americanus atpH protein:

• Expression system optimization:

  • Use E. coli strains specialized for membrane protein expression (C41(DE3), C43(DE3))

  • Test multiple induction temperatures (16°C, 25°C, 30°C) to balance expression yield and proper folding

  • Optimize induction parameters (IPTG concentration 0.1-1.0 mM, induction time 4-16 hours)

  • Consider codon optimization for E. coli expression

• Solubilization and purification:

  • Screen detergents for effective solubilization (DDM, LDAO, Triton X-100)

  • Use immobilized metal affinity chromatography (IMAC) leveraging the His tag

  • Include detergent in all purification buffers at concentrations above CMC

  • Consider a secondary purification step (size exclusion chromatography)

  • Maintain pH 8.0 throughout purification as indicated in the storage buffer information

• Quality control:

  • Verify purity via SDS-PAGE (>90% purity should be achievable)

  • Confirm identity via Western blot and/or mass spectrometry

  • Assess protein stability through thermal shift assays

This approach maximizes both yield and functionality of the purified protein for downstream applications .

How can researchers investigate the role of atpH in proton transport and ATP synthesis?

To investigate the role of atpH in proton transport and ATP synthesis, researchers should implement these methodological approaches:

• Reconstitution in proteoliposomes:

  • Purify recombinant atpH and other ATP synthase subunits

  • Reconstitute in liposomes with defined lipid composition

  • Establish a proton gradient across the membrane using pH shifts or ionophores

  • Measure ATP synthesis rates under varying conditions

• Patch-clamp electrophysiology:

  • Similar to techniques used to study ATP synthase channels in mitoplasts, adapt patch-clamp methodology to proteoliposomes containing reconstituted atpH

  • Measure ion conductance under various conditions to assess channel formation and activity

  • Test effects of known ATP synthase inhibitors and modulators

• pH-sensitive fluorescent probes:

  • Incorporate pH-sensitive fluorescent dyes into proteoliposomes

  • Monitor proton transport in real-time during ATP synthesis/hydrolysis

  • Correlate proton movement with ATP synthesis rates

• Site-directed mutagenesis:

  • Create specific mutations in the atpH sequence to identify key residues involved in proton transport

  • Assess how these mutations affect ATP synthesis efficiency

These approaches provide complementary data on the functional role of atpH in the context of the complete ATP synthase complex .

What is the significance of atpH in chloroplast genetics and evolution studies?

The atpH gene holds significant value in chloroplast genetics and evolutionary studies for several reasons:

• Phylogenetic marker: The atpF-atpH intergenic region serves as an effective chloroplast DNA marker for phylogenetic studies in plants, including species differentiation within the Araceae family, to which Acorus belongs. This region shows appropriate levels of sequence variation for resolving relationships at various taxonomic levels .

• Evolutionary conservation: The conserved nature of the atpH coding sequence across plant lineages makes it valuable for deep evolutionary studies, while its intergenic regions show sufficient variability for resolving closer relationships.

• Methodological approach for evolutionary studies:

  • Extract total genomic DNA from plant tissues

  • Amplify the atpF-atpH region using validated primers

  • Sequence the amplicons using standard methods

  • Analyze sequence divergence and construct phylogenetic trees

  • Compare evolutionary rates with other chloroplast markers

• Organellar genome evolution: Comparing atpH sequences and organization across plant lineages provides insights into chloroplast genome evolution, gene transfer events, and adaptation to different photosynthetic requirements.

The atpH region's utility as a genetic marker has been demonstrated in various plant groups, making it valuable for biodiversity and evolutionary research .

How does the structure-function relationship of atpH contribute to ATP synthase activity under different environmental conditions?

The structure-function relationship of atpH in ATP synthase under varying environmental conditions reveals sophisticated adaptive mechanisms:

• pH-dependent structural adaptations:

  • At pH 6.5, the protonation state of key residues in atpH changes, affecting its interaction with other subunits like the γ-subunit

  • These interactions influence channel formation properties and can be studied using pH-controlled reconstitution systems

  • Research methodology should include circular dichroism spectroscopy at varying pH to monitor secondary structure changes

• Membrane lipid composition effects:

  • atpH functions as a lipid-binding protein, with its activity modulated by membrane composition

  • Experimental approach: Reconstitute atpH in liposomes with systematically varied lipid compositions and measure functional parameters

  • Quantify protein-lipid interactions using native mass spectrometry or hydrogen-deuterium exchange

• Stress response mechanisms:

  • Under oxidative or heavy metal stress (like antimony), photosynthetic efficiency decreases in Acorus species

  • This affects ATP synthase function, potentially through modifications of subunits like atpH

  • Investigate by measuring ATP synthesis rates in chloroplasts isolated from plants exposed to different stressors

• Oligomeric state variations:

  • atpH may form different oligomeric assemblies under varying conditions

  • These can be visualized using cryo-electron microscopy and native gel electrophoresis

  • Correlate oligomeric states with functional parameters such as proton conductance and ATP synthesis rates

This multi-faceted approach reveals how atpH structure dynamically adapts to environmental conditions, maintaining ATP synthase function across various physiological states .

What are the challenges and solutions for studying interactions between atpH and other ATP synthase subunits?

Studying interactions between atpH and other ATP synthase subunits presents several challenges with corresponding methodological solutions:

• Challenge: Maintaining native membrane environment
  Solution:

  • Use nanodiscs or styrene-maleic acid lipid particles (SMALPs) to extract ATP synthase complexes with surrounding native lipids

  • Implement on-membrane crosslinking prior to solubilization

  • Validate findings using multiple detergent systems to identify detergent-specific artifacts

• Challenge: Distinguishing direct vs. indirect interactions
  Solution:

  • Combine proximity labeling techniques (BioID, APEX) with mass spectrometry

  • Perform in vitro binding assays with purified components

  • Use computational molecular docking validated by targeted mutagenesis of predicted interface residues

• Challenge: Dynamic and transient interactions
  Solution:

  • Implement time-resolved crosslinking during different functional states

  • Use hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Apply single-molecule FRET to capture transient interactions

• Challenge: Functional relevance of observed interactions
  Solution:

  • Design mutations that specifically disrupt predicted interactions without affecting protein folding

  • Measure functional consequences (proton transport, ATP synthesis) of disrupted interactions

  • Correlate interaction strength with functional parameters under varying conditions

Researchers have demonstrated the interaction between ATP synthase subunits (particularly subunit c) and other proteins like ANT through co-immunoprecipitation, suggesting similar approaches would be effective for studying atpH interactions .

How can recombinant atpH be utilized in developing novel antimicrobial compounds targeting bacterial ATP synthase?

Recombinant Acorus americanus atpH can serve as a valuable tool in developing antimicrobial compounds through these research approaches:

• Comparative structural analysis:

  • Perform detailed structural comparisons between plant atpH and bacterial homologs

  • Identify unique structural features that could be exploited for selective targeting

  • Use computational approaches to identify potential binding pockets present in bacterial but not plant ATP synthases

• High-throughput screening platform:

  • Develop parallel screening systems using both plant atpH and bacterial homologs

  • Screen compound libraries for molecules that selectively inhibit bacterial ATP synthase

  • Methodology: Reconstitute proteins in liposomes with pH-sensitive dyes to monitor proton transport inhibition

• Structure-based drug design:

  • Use structural data to design compounds that selectively bind bacterial ATP synthase

  • Validate binding and selectivity through biophysical techniques (ITC, SPR, MST)

  • Iteratively optimize compounds based on structure-activity relationships

• Resistance mechanism studies:

  • Generate resistant bacterial strains through directed evolution

  • Sequence ATP synthase genes to identify resistance mutations

  • Use this information to design second-generation compounds that overcome resistance

• In vitro to in vivo transition:

  • Test promising compounds against whole bacteria

  • Confirm ATP synthase as the target using metabolomics and cellular ATP measurements

  • Validate selectivity by testing effects on plant chloroplast function

This systematic approach leverages structural and functional knowledge of ATP synthase to develop selective antimicrobial compounds with lower probability of cross-reactivity with plant or human homologs .

What are the optimal buffer conditions for maintaining atpH stability and functionality in vitro?

Optimal buffer conditions for atpH stability and functionality require careful consideration of multiple parameters:

For functional studies, additional considerations include:

• Incorporation of 2-5 mM MgCl₂ for studies involving ATP binding or hydrolysis

• Addition of appropriate lipids (phosphatidylcholine/phosphatidylethanolamine) if reconstituting into membranes

• Use of high-purity water and analytical grade reagents to prevent contamination

Monitor protein stability regularly using analytical size exclusion chromatography or dynamic light scattering to detect aggregation .

How can researchers effectively incorporate recombinant atpH into liposomes for functional studies?

Effective incorporation of recombinant atpH into liposomes for functional studies requires a systematic approach:

• Liposome preparation:

  • Select lipid composition mimicking chloroplast membranes (include MGDG, DGDG, SQDG, and phosphatidylglycerol)

  • Prepare multilamellar vesicles through hydration of dried lipid films

  • Form unilamellar vesicles via extrusion through polycarbonate membranes (100-200 nm pore size)

  • Control liposome size distribution using dynamic light scattering

• Protein incorporation strategies:

  • Method A - Direct incorporation: Add detergent-solubilized atpH during liposome formation

  • Method B - Detergent-mediated insertion: Partially solubilize preformed liposomes with mild detergents (e.g., Triton X-100 at sub-solubilizing concentrations) before adding protein

  • Method C - Fusion with proteoliposomes: Create protein-containing small liposomes that can fuse with larger target liposomes

• Detergent removal techniques:

  • Bio-Beads SM-2 adsorption (controlled addition over 2-4 hours)

  • Dialysis against detergent-free buffer (48-72 hours with multiple buffer changes)

  • Gel filtration to separate detergent monomers

• Validation of incorporation:

  • Confirm protein orientation using protease protection assays

  • Quantify incorporation efficiency through protein:lipid ratio analysis

  • Verify functionality by measuring proton translocation using pH-sensitive fluorescent dyes

• Functional characterization:

  • Establish proton gradients using acid-base transitions or light-driven proton pumps

  • Measure ATP synthesis capacity under varying conditions

  • Monitor effects of known inhibitors to confirm specific activity

This methodical approach ensures reliable reconstitution of functionally active atpH in liposome systems suitable for detailed biophysical and biochemical characterization .

What are the current limitations in understanding atpH function in non-photosynthetic plastids?

Current limitations in understanding atpH function in non-photosynthetic plastids present significant research challenges:

• Knowledge gaps in non-photosynthetic ATP synthesis:

  • ATP synthesis in non-photosynthetic plastids remains poorly characterized compared to chloroplasts

  • While research has shown that chromoplasts contain ATP synthase with an atypical γ-subunit that functions in ATP synthesis, the role of subunit c (atpH) in these specialized plastids is less understood

  • The interaction between atpH and the atypical γ-subunit in non-photosynthetic plastids requires investigation

• Methodological challenges:

  • Difficulty isolating pure, intact non-photosynthetic plastids (e.g., amyloplasts, chromoplasts)

  • Limited availability of tissue-specific antibodies for plastid ATP synthase components

  • Challenges in measuring ATP synthesis in organello for non-photosynthetic plastids

  • Need for improved protocols to study membrane protein complexes in specialized plastids

• Research approaches to address limitations:

  • Develop improved isolation protocols for non-photosynthetic plastids

  • Implement comparative proteomics between different plastid types

  • Utilize plastid-targeted fluorescent ATP sensors to monitor ATP dynamics in vivo

  • Apply single-particle cryo-EM to resolve structural differences in ATP synthase from different plastid types

• Future research directions:

  • Investigate post-translational modifications of atpH in different plastid types

  • Examine stoichiometry of ATP synthase components in non-photosynthetic plastids

  • Study regulatory mechanisms controlling ATP synthase assembly and activity during plastid differentiation

  • Explore potential moonlighting functions of atpH beyond ATP synthesis

Understanding atpH function in non-photosynthetic plastids would significantly enhance our knowledge of energy metabolism across diverse plant tissues and developmental stages .

How might environmental stressors affect atpH expression and function in Acorus species?

Environmental stressors significantly impact atpH expression and function in Acorus species through multiple mechanisms:

• Heavy metal stress effects:

  • Antimony (Sb) exposure in Acorus calamus significantly reduces chlorophyll content and photosynthetic parameters (Pn, Gs, E)

  • These photosynthetic impairments likely affect chloroplast ATP synthase function, including atpH

  • Research approach: Combine chlorophyll fluorescence measurements with quantification of ATP synthase subunit expression under controlled Sb exposure

• Oxidative stress implications:

  • Environmental stressors generate reactive oxygen species that can damage chloroplast proteins

  • ATP synthase subunits, including atpH, may undergo oxidative modifications affecting assembly and function

  • Methodology: Use redox proteomics to identify specific modifications to atpH under stress conditions

• pH stress and atpH function:

  • Environmental stressors often alter cellular and organellar pH

  • At pH 6.5, ATP synthase function changes significantly, as observed in mitochondrial studies

  • Research approach: Measure ATP synthesis rates in chloroplasts isolated from stressed plants at various pH values

• Adaptation mechanisms:

  • Acorus species show varying tolerance to environmental stresses

  • This may involve adaptations in ATP synthase composition or regulation

  • Comparative studies between stress-tolerant and sensitive Acorus species could reveal key adaptations

• Quantifiable impacts:

  • Plant height decreases by up to 35% under severe Sb stress

  • Photosynthetic parameters (Pn) decrease by 38.2% under high Sb concentrations

  • These physiological changes correlate with energy production capacity and likely reflect changes in ATP synthase function

Understanding these stress responses provides insight into plant adaptation mechanisms and potential applications in phytoremediation or stress-tolerant crop development .

What potential applications exist for using atpH structure for directed evolution of ATP synthase with novel properties?

Directed evolution of ATP synthase using atpH structure offers promising applications for creating novel bioenergetic systems:

• Enhanced environmental tolerance:

  • Create ATP synthase variants with improved thermal stability through directed evolution of atpH

  • Develop screening systems using pH-sensitive fluorescent proteins to identify variants with broader pH optima

  • Apply error-prone PCR and DNA shuffling to generate diverse atpH libraries

  • Methodology: Reconstitute variant proteins in liposomes and screen for ATP synthesis under extreme conditions

• Modified ion selectivity:

  • Engineer atpH to transport alternative ions (Na⁺, K⁺) instead of H⁺

  • Identify key residues for ion selectivity through computational modeling and site-directed mutagenesis

  • Create hybrid proteins incorporating features from bacterial Na⁺-dependent ATP synthases

  • Application: Develop bio-inspired ion pumps for novel energy harvesting systems

• Altered regulatory properties:

  • Modify atpH to alter its interaction with regulatory subunits like the atypical γ-subunit found in chromoplasts

  • Design variants with altered response to regulatory metabolites

  • Screening approach: Develop high-throughput assays measuring ATP synthesis under various regulatory conditions

• Bio-hybrid technologies:

  • Create atpH variants capable of incorporating non-natural amino acids with novel properties

  • Develop chimeric proteins combining atpH with other membrane proteins for new functionalities

  • Application: Bio-electronic interfaces where modified ATP synthase responds to electrical stimuli

• Therapeutic applications:

  • Compare plant atpH with human mitochondrial ATP synthase to identify structural differences

  • Develop selective inhibitors of bacterial ATP synthase for antimicrobial applications

  • Design modified atpH-based peptides that can modulate mitochondrial permeability transition for potential disease therapies

These applications leverage the fundamental role of atpH in bioenergetics while exploring its potential for engineering novel functional properties through directed evolution approaches .

What are common challenges in expressing recombinant atpH, and how can they be addressed?

Common challenges in recombinant atpH expression and their solutions include:

Quality control methods to ensure properly expressed atpH:

• Verify size and purity via SDS-PAGE (should show >90% purity)

• Confirm identity via Western blot using anti-His antibodies

• Assess secondary structure via circular dichroism to verify proper folding

• Test functionality through reconstitution in liposomes and proton transport assays

Following these troubleshooting strategies ensures production of high-quality recombinant atpH suitable for downstream research applications .

How can researchers validate the functional integrity of purified recombinant atpH protein?

Validating the functional integrity of purified recombinant atpH requires a multi-faceted approach:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy: Compare the spectrum with known profiles of properly folded c-subunits to verify secondary structure elements

  • Size exclusion chromatography: Confirm monodispersity and appropriate oligomeric state

  • Thermal shift assays: Measure protein stability and appropriate melting temperature

  • Limited proteolysis: Properly folded proteins show characteristic digestion patterns

• Membrane incorporation assays:

  • Liposome flotation assays: Verify the protein's ability to associate with lipid membranes

  • Freeze-fracture electron microscopy: Visualize proper membrane integration

  • Sucrose density gradient ultracentrifugation: Confirm association with membrane fractions

• Functional assays:

  • Proton transport measurements:

    • Reconstitute atpH in liposomes containing pH-sensitive fluorescent dyes

    • Establish pH gradients and measure fluorescence changes

    • Compare transport rates with published values for ATP synthase c-subunits

  • Assembly competence:

    • Test ability to associate with other ATP synthase subunits in vitro

    • Use pull-down assays to verify specific interactions with partner subunits

• Biophysical characterization:

  • Surface plasmon resonance (SPR): Measure binding kinetics with known interaction partners

  • Isothermal titration calorimetry (ITC): Quantify thermodynamic parameters of binding events

  • Hydrogen-deuterium exchange mass spectrometry: Map properly folded regions and flexible domains

• Reference controls:

  • Run parallel analyses with well-characterized c-subunits from model organisms

  • Include negative controls using denatured protein samples

  • Quantify the percentage of functionally active protein in preparations

This comprehensive validation approach ensures that purified recombinant atpH maintains its native structural and functional properties, which is critical for accurate interpretation of subsequent experimental results .

How might the study of atpH contribute to our understanding of chloroplast evolution and adaptation?

The study of atpH offers significant insights into chloroplast evolution and adaptation through several research avenues:

• Comparative genomics approach:

  • The atpF-atpH intergenic region serves as an effective DNA marker for phylogenetic studies in plants

  • Comparing atpH sequences across diverse plant lineages reveals evolutionary rates and selective pressures

  • Research methodology: Sequence atpH from phylogenetically diverse plant species, particularly focusing on early-diverging lineages like Acorus

  • The resulting data can help reconstruct chloroplast genome evolution and endosymbiotic events

• Structural adaptation mechanisms:

  • atpH structure and function may differ between plants adapted to various environmental conditions

  • Comparative analysis of atpH from plants in extreme environments (high temperature, drought, high salinity) can reveal adaptive modifications

  • Experimental approach: Express and characterize atpH from plants with different photosynthetic adaptations (C3, C4, CAM)

• Co-evolution with interacting partners:

  • atpH interacts with multiple ATP synthase subunits, which constrain its evolutionary trajectory

  • Analyzing co-evolutionary patterns between atpH and other ATP synthase components provides insight into functional constraints

  • Research methodology: Apply co-evolutionary analysis algorithms to ATP synthase subunit sequences across plant lineages

• Horizontal gene transfer assessment:

  • Unusual evolutionary patterns in atpH might indicate horizontal gene transfer events

  • Such events could reveal unexpected evolutionary connections between distant lineages

  • Approach: Conduct phylogenomic analyses to identify discordant evolutionary patterns

• Climate adaptation signatures:

  • Plants in different climates may show adaptations in energy production systems

  • Studying atpH sequences from plants across climate gradients can reveal molecular adaptations to environmental challenges

  • Correlate sequence/structural variations with environmental parameters to identify adaptive traits

These research directions would significantly enhance our understanding of how chloroplast energy production systems evolved and adapted across plant diversification .

What potential exists for engineering modified atpH variants for biotechnological applications?

Engineering modified atpH variants offers promising biotechnological applications across multiple fields:

• Bioenergy production systems:

  • Engineer atpH variants with enhanced coupling efficiency between proton transport and ATP synthesis

  • Create chimeric proteins combining features from bacterial and plant ATP synthases

  • Methodology: Site-directed mutagenesis of key residues followed by functional characterization in reconstituted systems

  • Application: Incorporate into artificial chloroplasts or bacterial systems for improved biofuel production

• Biosensors for environmental monitoring:

  • Develop atpH-based sensors for detecting environmental toxins that affect ATP synthesis

  • Create fusion proteins with fluorescent reporters that respond to functional changes

  • Engineering approach: Introduce environmentally responsive elements at strategic positions

  • Application: Real-time monitoring of water contamination by heavy metals and other toxins that impact photosynthesis

• Nanoscale molecular motors:

  • Utilize the rotary mechanism of ATP synthase for nanomachine development

  • Engineer atpH to function within artificial membrane systems

  • Technique: Bottom-up synthetic biology to create minimal ATP synthase systems

  • Application: Nanoscale pumps, molecular switches, or energy converters

• Pharmacological platforms:

  • Develop screening systems using engineered atpH to identify novel antibiotics targeting bacterial ATP synthase

  • Create variants with binding pockets for specific drug classes

  • Methodology: Structure-based design combined with high-throughput screening

  • Application: Antimicrobial drug discovery platforms

• Bioremediation technologies:

  • Engineer atpH to increase tolerance to heavy metals like antimony

  • Incorporate into plants used for phytoremediation to enhance stress tolerance

  • Approach: Directed evolution to select variants with enhanced stability under heavy metal stress

  • Application: Enhanced phytoremediation systems for contaminated soils

These applications leverage the fundamental properties of atpH while extending its capabilities through protein engineering to address key challenges in biotechnology and environmental management .