Recombinant Cupriavidus taiwanensis ATP synthase subunit delta (atpH)

What are the optimal storage and handling conditions for the recombinant protein?

For maximum stability and activity of recombinant Cupriavidus taiwanensis ATP synthase subunit delta, researchers should follow these handling protocols:

Prior to opening, the vial should be briefly centrifuged to bring contents to the bottom. Repeated freezing and thawing should be strictly avoided as this significantly reduces protein stability and activity .

How does the recombinant form differ from the native protein?

The recombinant Cupriavidus taiwanensis ATP synthase subunit delta is produced in mammalian cells rather than in its native bacterial context . This expression system difference creates several important distinctions:

• Post-translational modifications: The mammalian expression system may introduce modifications not present in the native bacterial protein.

• Protein tagging: The recombinant protein may include a tag (specific type determined during manufacturing), which is absent in the native form .

• Purity level: The recombinant protein is purified to >85% as determined by SDS-PAGE, likely higher than what would be obtained from natural sources .

• Environmental context: The native protein functions within the complete ATP synthase complex in a bacterial membrane environment, whereas the recombinant protein is isolated.

• Functional partners: In its native environment, the protein interacts with other ATP synthase subunits that may affect its conformation and activity.

These differences should be considered when designing experiments, particularly those investigating protein-protein interactions or conformational dynamics.

What experimental techniques are commonly used to characterize this protein?

Researchers investigating Cupriavidus taiwanensis ATP synthase subunit delta typically employ multiple complementary techniques:

• Structural characterization:

  • Circular dichroism spectroscopy for secondary structure assessment

  • X-ray crystallography for high-resolution structural determination

  • Cryo-electron microscopy for visualization within the ATP synthase complex

  • Mass spectrometry for precise molecular weight determination

• Functional analysis:

  • ATP synthesis/hydrolysis assays to measure enzymatic activity

  • Proton translocation assays using pH-sensitive dyes

  • ATPase activity measurements in reconstituted systems

• Interaction studies:

  • Co-immunoprecipitation to identify binding partners

  • Surface plasmon resonance for measuring binding kinetics

  • Crosslinking combined with mass spectrometry to map interaction interfaces

  • Proteomic approaches including 2D-PAGE followed by LC-MS/MS for protein identification

• Expression analysis:

  • Western blotting for protein quantification

  • qRT-PCR for mRNA expression analysis

  • Proteomics for comparative expression studies under different conditions

These techniques collectively provide a comprehensive understanding of the protein's structure, function, interactions, and regulation within the ATP synthase complex.

How does environmental stress affect ATP synthase expression and function in Cupriavidus species?

Cupriavidus species, known for their ability to thrive in challenging environments, exhibit significant adaptations in ATP synthase expression and function under various stressors. Research on related species provides valuable insights:

Studies on Cupriavidus metallidurans under simulated microgravity revealed substantial alterations in stress response pathways. Transcription of chaperones DnaK, DnaJ, and GrpE was induced 2.0-2.6 fold under low-shear modeled microgravity conditions . These chaperones are crucial for proper protein folding and may affect the assembly and stability of complex protein structures like ATP synthase.

Metal stress, particularly relevant for Cupriavidus species known for metal resistance, triggers specific adaptations in energy metabolism. Proteome analysis during stress responses has identified ATP synthase among differentially expressed proteins , indicating that energy production machinery undergoes regulation during stress adaptation.

The mechanistic relationships between stress response and ATP synthase function in Cupriavidus appear to involve:

• Altered gene expression of ATP synthase components

• Enhanced chaperone protection of the complex

• Post-translational modifications affecting activity

• Structural adjustments optimizing function under stress

These adaptations likely represent evolutionary strategies enabling Cupriavidus species to maintain energy homeostasis under challenging environmental conditions, contributing to their remarkable ecological versatility.

What is the role of the delta subunit in ATP synthase assembly and stability?

The delta subunit (atpH) plays critical roles in both the assembly process and long-term stability of the complete ATP synthase complex:

In the assembly pathway, the delta subunit serves as a connector between the catalytic F₁ sector and the proton-translocating F₀ sector. Its structural features enable specific interactions with both the gamma subunit of the central stalk and components of the F₁ catalytic hexamer. These interactions are essential for proper positioning of the central stalk relative to the catalytic sites.

For complex stability, the delta subunit provides critical contact points that maintain the structural integrity of the assembled complex during the rotational catalysis mechanism. Without proper delta subunit function, the mechanical coupling between proton translocation and ATP synthesis becomes inefficient.

Molecular details of these roles include:

• N-terminal region interactions with the F₁ sector, particularly the alpha subunits

• C-terminal region associations with the gamma subunit of the central stalk

• Conformational changes during rotational catalysis that maintain efficient energy transfer

The absence or dysfunction of the delta subunit would likely result in impaired assembly of the complete ATP synthase complex and/or compromised energy coupling efficiency, highlighting its essential role despite its relatively small size compared to other ATP synthase components.

How can researchers effectively study interactions between the delta subunit and other ATP synthase components?

Investigating interactions between the ATP synthase delta subunit and other components requires sophisticated approaches that capture both static and dynamic aspects of these relationships:

In vitro interaction analysis approaches:

• Surface Plasmon Resonance (SPR): This label-free technique enables real-time measurement of binding kinetics between purified delta subunit and other ATP synthase components. By immobilizing either the delta subunit or its potential binding partners on a sensor chip, researchers can determine association and dissociation rates, as well as binding affinities.

• Isothermal Titration Calorimetry (ITC): This technique measures the heat released or absorbed during binding interactions, providing thermodynamic parameters (ΔH, ΔS, ΔG) that characterize the interaction between the delta subunit and other components.

• Cross-linking Mass Spectrometry (XL-MS): By using chemical cross-linkers of defined length, researchers can capture transient interactions and identify specific residues involved in subunit interfaces through subsequent mass spectrometry analysis.

Structural visualization approaches:

• Cryo-Electron Microscopy: Recent advances allow visualization of the entire ATP synthase complex, revealing the structural arrangement of the delta subunit relative to other components. This is particularly valuable for understanding the architectural role of this connector subunit.

• FRET (Förster Resonance Energy Transfer): By labeling the delta subunit and potential interaction partners with appropriate fluorophores, researchers can monitor dynamic interactions and conformational changes during ATP synthase function.

In vivo approaches:

• Bacterial Two-Hybrid Assays: These genetic systems allow detection of protein-protein interactions in living cells by linking interaction to reporter gene expression.

• Co-immunoprecipitation with specific antibodies: Using antibodies against the delta subunit to pull down the entire complex, followed by proteomic analysis, can identify associated proteins under physiological conditions .

When designing such experiments, researchers should consider the potential impact of recombinant tags on protein interactions and include appropriate controls to validate the physiological relevance of observed interactions.

What comparative insights emerge from studying ATP synthase across different Cupriavidus species?

Comparative analysis of ATP synthase across Cupriavidus species reveals evolutionary adaptations related to their diverse ecological niches:

The genus Cupriavidus encompasses species adapted to various challenging environments, including metal-contaminated soils, plant nodules, and clinical settings. Comparing ATP synthase components, including the delta subunit, across these species provides insights into both conserved features essential for core function and specialized adaptations.

Research on Cupriavidus metallidurans, known for its exceptional metal resistance, has revealed sophisticated stress response mechanisms that may protect essential energy-generating machinery like ATP synthase. While Cupriavidus taiwanensis, originally isolated from root nodules and capable of nitrogen fixation, may show adaptations related to the high-energy demands of symbiotic relationships.

Comparative genomic and proteomic analyses suggest that while the core machinery of ATP synthesis is highly conserved, regulatory elements and subtle structural modifications exist that likely optimize ATP synthase function in different ecological contexts. For instance, metal-adapted species may show modifications that maintain function despite potential metal interference with proton gradients.

This comparative approach helps distinguish fundamental mechanisms of ATP synthesis from species-specific adaptations, contributing to our understanding of how this essential energy-generating machine has been fine-tuned through evolution to function effectively across diverse environmental conditions.

What are the implications of studying bacterial ATP synthase for antimicrobial development?

Bacterial ATP synthase, including the delta subunit from organisms like Cupriavidus taiwanensis, represents a promising target for novel antimicrobial development for several reasons:

• Essential function: ATP synthase is critical for bacterial energy metabolism, making it an attractive target where inhibition leads to bacterial death or significant growth impairment.

• Structural distinctions: Despite functional conservation, significant structural differences exist between bacterial and mammalian ATP synthases. The delta subunit and other components show sufficient divergence to potentially allow selective targeting of bacterial enzymes.

• Novel mechanism of action: As antibiotic resistance increases, new targets with different mechanisms of action from traditional antibiotics are urgently needed. ATP synthase inhibitors would represent a mechanistically distinct class of antimicrobials.

• Broad-spectrum potential: The conservation of ATP synthase across bacterial species suggests that effective inhibitors might show broad-spectrum activity.

• Resistance insights: Studying adaptations in ATP synthase from environmentally resilient bacteria like Cupriavidus species may reveal natural resistance mechanisms that could be anticipated and countered in drug development.

Research approaches investigating ATP synthase as an antimicrobial target include:

• Structure-based drug design targeting unique features of bacterial subunits

• High-throughput screening for selective inhibitors of bacterial ATP synthesis

• Combination approaches targeting both ATP synthase and other cellular processes

• Development of prodrugs activated specifically in bacterial environments

The detailed characterization of components like the delta subunit provides crucial structural information to guide these antimicrobial development efforts.

What are the key considerations for recombinant expression and purification of ATP synthase subunits?

Successful recombinant expression and purification of ATP synthase subunits like delta (atpH) require careful optimization of multiple parameters:

Expression system selection:

While the commercial recombinant Cupriavidus taiwanensis atpH is produced in mammalian cells , researchers may choose from several expression systems based on their specific requirements:

Construct design considerations:

• Affinity tag selection (His₆, GST, MBP) and placement (N- or C-terminal)

• Inclusion of protease cleavage sites for tag removal

• Codon optimization for the chosen expression host

• Signal peptide inclusion for targeted expression

• Fusion partners to enhance solubility if needed

Purification strategy optimization:

• Initial capture: Affinity chromatography

• Intermediate purification: Ion exchange chromatography

• Polishing: Size exclusion chromatography

• Quality control: SDS-PAGE, mass spectrometry, activity assays

• Typical target: >85% purity as verified by SDS-PAGE

Stability considerations:

• Buffer composition: pH, ionic strength, specific ions

• Addition of stabilizing agents: Glycerol (5-50%)

• Storage conditions: -20°C to -80°C for extended storage

• Avoidance of repeated freeze-thaw cycles

Careful attention to these factors increases the likelihood of obtaining functional protein suitable for downstream structural and functional studies.

How can researchers effectively reconstitute ATP synthase activity in vitro?

Reconstituting ATP synthase activity in vitro is challenging but essential for detailed functional studies. The process involves several critical steps:

1. Component preparation:

• Purify individual subunits, including delta (atpH), under conditions that maintain their native structure

• Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL with appropriate stabilizers

• For membrane components, proper detergent selection is crucial

2. Reconstitution strategies:

For functional studies, three main approaches exist:

3. Activity verification methods:

• ATP synthesis measurements:

  • Luciferase-based ATP detection systems

  • Coupled enzyme assays (e.g., hexokinase/glucose-6-phosphate dehydrogenase)

  • 32P-labeled ADP incorporation assays

• ATP hydrolysis measurements:

  • Colorimetric phosphate release assays

  • Coupled enzyme systems (e.g., pyruvate kinase/lactate dehydrogenase)

  • pH-sensitive indicators to monitor proton release

• Proton translocation assessment:

  • pH-sensitive fluorescent dyes (ACMA, pyranine)

  • Proton flux measurements with pH electrodes

  • Membrane potential indicators (oxonol dyes)

4. Assay optimization considerations:

• Temperature and pH optimization for the specific bacterial enzyme

• Substrate concentrations (ATP, ADP, Pi)

• Divalent cation requirements (typically Mg2+)

• Inclusion of coupling factors if needed

• Control experiments with specific inhibitors

What techniques can accurately measure the impact of environmental stressors on ATP synthase?

Investigating how environmental stressors affect ATP synthase structure and function requires a multi-faceted approach combining molecular, biochemical, and biophysical techniques:

Gene and protein expression analysis:

• Transcriptional changes:

  • qRT-PCR for targeted analysis of ATP synthase genes

  • RNA-Seq for genome-wide transcriptional responses, as demonstrated in studies on stress responses in Cupriavidus species

  • Reporter gene constructs to monitor promoter activity in real-time

• Protein level assessment:

  • Western blotting with specific antibodies against ATP synthase subunits

  • Proteomics approaches using 2D-PAGE and LC-MS/MS to identify differential expression

  • Pulse-chase labeling to determine protein turnover rates under stress conditions

Functional assessments:

• ATP synthesis capacity:

  • Measurement of ATP production rates in membrane vesicles or reconstituted systems

  • Determination of P/O ratios (ATP produced per oxygen consumed)

  • Analysis of ATP synthase efficiency under varying stress conditions

• Proton translocation:

  • Fluorescent probe-based assays to measure proton gradient formation

  • Membrane potential measurements under stress conditions

  • Assessment of proton leak across membranes during stress

Structural integrity evaluations:

• Complex stability:

  • Blue Native PAGE to analyze ATP synthase complex integrity

  • Size exclusion chromatography to detect stress-induced complex dissociation

  • Hydrogen-deuterium exchange mass spectrometry to identify regions with altered stability

• Conformation changes:

  • Circular dichroism to monitor secondary structure alterations

  • Intrinsic fluorescence to detect tertiary structure modifications

  • Cross-linking studies to identify changes in subunit interactions

Environmental stress simulation approaches:

These complementary approaches enable researchers to develop a comprehensive understanding of how environmental stressors impact ATP synthase structure, assembly, and function in bacteria like Cupriavidus taiwanensis.

How can bacterial ATP synthase research contribute to bioenergetics understanding in extremophiles?

Studies of ATP synthase from bacteria like Cupriavidus taiwanensis provide critical insights into bioenergetic adaptations in extremophiles and environmentally versatile bacteria:

• Adaptive energy coupling mechanisms: The detailed study of subunits like atpH from Cupriavidus species reveals how energy coupling between proton translocation and ATP synthesis may be optimized for function under challenging conditions. These adaptations may include modified interaction surfaces between connector subunits like delta and other components of the complex.

• Stress response integration with energy metabolism: Research on Cupriavidus metallidurans has demonstrated sophisticated stress response systems, including upregulation of chaperones under conditions like simulated microgravity . Understanding how these responses protect essential energy-generating machinery illuminates survival strategies in extreme environments.

• Metal resistance-energy metabolism connections: Cupriavidus species are renowned for their metal resistance. The relationship between maintaining proton gradients for ATP synthesis and metal detoxification systems represents an important example of how fundamental cellular processes are integrated to enable survival in contaminated environments .

• Comparative insights: By comparing ATP synthase components across extremophiles adapted to different stresses (temperature, pH, salinity, metal exposure), researchers can identify both conserved features essential for function and specific adaptations to particular environmental challenges.

Such research has broader implications beyond understanding individual species, contributing to fundamental questions about:

• The limits of biological energy conversion under extreme conditions

• Evolutionary adaptations in core metabolic machinery

• Design principles for robust energy-generating systems

• Potential biomimetic applications inspired by natural solutions to energetic challenges

What potential biotechnological applications might emerge from recombinant ATP synthase research?

Research on recombinant ATP synthase components, including the delta subunit (atpH) from organisms like Cupriavidus taiwanensis, offers diverse biotechnological opportunities:

Biomedical applications:

• Antimicrobial development: ATP synthase represents a promising target for novel antibiotics with unique mechanisms of action. Structural and functional studies of bacterial-specific features can guide the development of selective inhibitors.

• Drug screening platforms: Reconstituted ATP synthase systems can serve as platforms for screening compounds that modulate its activity, with applications in both antimicrobial discovery and research on mitochondrial disorders.

• Mitochondrial disease models: Bacterial ATP synthase research provides insights relevant to understanding human mitochondrial disorders involving homologous proteins.

Bioenergy applications:

• Biomimetic energy systems: The highly efficient ATP synthase molecular motor (approaching 100% efficiency) serves as inspiration for designing artificial energy conversion systems.

• Biofuel production: Engineered bacteria with optimized ATP synthase could potentially enhance biofuel production by improving energy efficiency.

• Bioelectrochemical systems: ATP synthase principles could inform the development of biological-electronic interfaces for renewable energy applications.

Environmental biotechnology:

• Bioremediation: The metal resistance capabilities of Cupriavidus species, coupled with energy metabolism adaptations, make them promising candidates for bioremediation applications .

• Biosensors: ATP synthase-based biosensors could detect environmental toxins that interfere with energy metabolism.

• Stress-resistant microorganisms: Knowledge of how ATP synthase adapts to stress conditions could guide the engineering of microorganisms with enhanced environmental resilience.

These diverse applications highlight the wide-ranging impact of fundamental research on ATP synthase components like the delta subunit from environmentally versatile bacteria such as Cupriavidus taiwanensis.

What emerging technologies may advance ATP synthase research in the next decade?

Several cutting-edge technologies are poised to transform ATP synthase research, offering unprecedented insights into this molecular machine:

Structural biology advances:

• Cryo-electron microscopy: Continued improvements in resolution and sample preparation will allow visualization of subtle conformational changes during ATP synthesis, potentially capturing the delta subunit's movements during catalysis.

• Integrative structural approaches: Combining techniques like cryo-EM, X-ray crystallography, NMR, and computational modeling to build complete dynamic models of ATP synthase function.

• Time-resolved structural methods: Emerging approaches to capture structural snapshots during the catalytic cycle, revealing the dynamics of energy coupling.

Single-molecule technologies:

• High-speed AFM: Direct visualization of ATP synthase rotary motion in real-time, with potential to observe how the delta subunit participates in this motion.

• Single-molecule FRET: Detailed analysis of conformational dynamics during catalysis with strategic placement of fluorophores on key subunits including delta.

• Optical tweezers and nanomechanical measurements: Direct measurement of forces and mechanical properties during ATP synthase operation.

Systems biology approaches:

Synthetic biology and engineering:

• Designer ATP synthases: Engineering modified ATP synthases with altered properties, potentially including optimized versions of the delta subunit.

• Minimal cells: Incorporation of ATP synthase into synthetic cell systems to study essential requirements for function.

• Biomimetic nanotechnology: Development of artificial molecular motors inspired by ATP synthase principles.

These technological advances will enable researchers to address fundamental questions about ATP synthase function with unprecedented detail, including the specific role of connector subunits like delta in energy coupling and rotary catalysis.