Recombinant Debaryomyces hansenii ATP synthase subunit a (ATP6)
Description
Definition and Production of Recombinant Debaryomyces hansenii ATP Synthase Subunit a (ATP6)
Recombinant Debaryomyces hansenii ATP synthase subunit a (ATP6) is a bioengineered protein corresponding to the mitochondrial ATP synthase subunit 6 in this halotolerant yeast. ATP6 is a core component of the ATP synthase complex, responsible for proton translocation and ATP synthesis in the mitochondrial inner membrane . The recombinant version is produced via heterologous expression in Escherichia coli, typically fused with an N-terminal His tag for purification .
Sequence and Domain Architecture
The amino acid sequence of ATP6 includes hydrophobic regions critical for membrane integration and proton channel formation . Key motifs include:
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Membrane-spanning domains: Required for embedding in the inner mitochondrial membrane.
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Proton-binding residues: Likely involved in proton translocation .
The recombinant ATP6 retains the native tertiary structure, as evidenced by SDS-PAGE and biochemical assays .
Role in ATP Synthase Assembly and Regulation
ATP6 translation is tightly regulated during mitochondrial biogenesis. Studies in yeast models show that mutations in ATP6 or ATP9 assembly pathways enhance translation rates, suggesting feedback mechanisms to maintain subunit stoichiometry .
Halotolerance and Mitochondrial Adaptation
Debaryomyces hansenii thrives in high-salt environments, with ATP synthase activity linked to osmotic stress responses. Under hyperosmotic conditions, the yeast upregulates alternative oxidases (Aox) to sustain mitochondrial membrane potential, indirectly affecting ATP synthase efficiency .
Mutational Studies
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Catalytic Site Mutations: In Saccharomyces cerevisiae, mutations in ATP synthase subunits (e.g., β-E190Q) disrupt ATP hydrolysis, highlighting conserved functional motifs .
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Structural Stability: Mutations in the γ-subunit of bacterial ATP synthases reduce rotational torque, underscoring the interplay between subunits .
Genetic Engineering Tools
Recent advances in D. hansenii genome editing, including CRISPR-Cas9 and in vivo DNA assembly, enable precise modifications of ATP6 for functional studies . These tools facilitate:
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Gene disruption: To study ATP6’s role in mitochondrial biogenesis.
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Heterologous expression: For producing ATP6 variants in high osmolarity environments .
Industrial Applications
The recombinant ATP6 serves as a model for studying:
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Proton translocation mechanisms: Critical for bioenergy research.
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Halotolerant ATP synthases: Potential targets for engineering stress-resistant microbial cell factories .
Challenges and Future Directions
Product Specs
Note: We prioritize shipping the format readily available in our inventory. However, if you have specific format requirements, please indicate them during order placement. We will accommodate your needs to the best of our ability.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. The shelf life of lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: dha:ATP6
Q&A
What is Debaryomyces hansenii ATP synthase subunit a (ATP6)?
Debaryomyces hansenii ATP synthase subunit a (ATP6) is a mitochondrial protein and essential component of the F1F0-ATP synthase complex. It is also known as ATPase subunit 6, ATP synthase subunit 6, or F-ATPase protein 6 . This protein plays a crucial role in the proton channel of the F0 sector of ATP synthase, facilitating proton movement across the inner mitochondrial membrane during ATP synthesis. In D. hansenii, a halotolerant yeast commonly found in marine environments and food fermentations, ATP6 may have adapted specific characteristics that contribute to the organism's ability to thrive in high-salt conditions .
How is recombinant D. hansenii ATP6 typically produced for research use?
Recombinant D. hansenii ATP6 is typically produced using E. coli expression systems. The commercially available versions include an N-terminal His-tag to facilitate purification . The expression construct generally includes residues 4-246 of the mature protein. After expression, the protein is purified using affinity chromatography, typically followed by additional purification steps to achieve >90% purity as verified by SDS-PAGE . The production in E. coli allows for high yield and consistent quality needed for structural and functional studies.
What are the optimal storage conditions for recombinant D. hansenii ATP6?
For long-term storage, recombinant D. hansenii ATP6 should be stored at -20°C or -80°C . When supplied as a lyophilized powder, it should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with 5-50% glycerol added as a cryoprotectant . For working aliquots, storage at 4°C is recommended, but only for up to one week . Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity . When handling the reconstituted protein, brief centrifugation is recommended prior to opening the vial to ensure all content is at the bottom .
How does D. hansenii ATP6 contribute to the organism's halotolerance?
D. hansenii is known for its remarkable ability to grow in environments with high salt concentrations, such as 1M NaCl or KCl . While the direct role of ATP6 in halotolerance has not been fully characterized, integrated multi-omics studies have revealed that high salt conditions trigger specific transcriptomic and proteomic responses in D. hansenii . As a component of the ATP synthase complex, ATP6 likely plays a role in maintaining energy homeostasis under salt stress conditions, possibly through adaptations that allow efficient ATP production despite osmotic challenges.
ATP synthase activity is essential for cellular energy production, and modifications to this complex may be part of D. hansenii's adaptation to high salt environments. The phosphoproteomic analysis of D. hansenii grown in high salt conditions has provided novel insights into protein regulation under stress, though specific data on ATP6 phosphorylation status requires further investigation .
In vitro ATP Synthase Activity Assay
To assess the functionality of recombinant D. hansenii ATP6, researchers can incorporate the protein into liposomes containing other ATP synthase subunits to reconstitute the complex. ATP synthesis activity can then be measured using luciferin/luciferase assays that detect ATP production rates. The experimental setup should include:
| Component | Concentration | Purpose |
|---|---|---|
| Purified ATP6 | 0.5-5 μg/mL | Target protein |
| Other ATP synthase subunits | Equimolar ratios | Complex reconstitution |
| Phospholipids (e.g., POPC) | 10 mg/mL | Liposome formation |
| ADP | 1 mM | Substrate |
| Inorganic phosphate | 5 mM | Substrate |
| Luciferin/luciferase reagent | As per manufacturer | ATP detection |
| Buffers with varying salt concentrations | 0.1-1M NaCl/KCl | Mimicking halotolerant conditions |
These assays should be calibrated with ATP standards and include appropriate controls such as inhibitors like oligomycin to verify specificity.
How can site-directed mutagenesis be used to study D. hansenii ATP6 function?
Site-directed mutagenesis provides a powerful approach to investigate structure-function relationships in D. hansenii ATP6. Based on the protein sequence , several key regions can be targeted:
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Conserved transmembrane domains that likely form the proton channel
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Putative ion-binding sites that may be involved in salt tolerance
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Interface regions that interact with other ATP synthase subunits
Recommended Mutagenesis Protocol:
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Design primers for specific mutations based on sequence alignment with ATP6 from other species
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Perform PCR-based mutagenesis on the ATP6 expression vector
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Transform into an appropriate E. coli strain for protein expression
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Express and purify mutant proteins using the same methods as for wild-type
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Compare functional properties of mutants to wild-type using activity assays
Potential Key Residues for Mutagenesis:
| Region | Target Residues | Potential Function | Expected Outcome of Mutation |
|---|---|---|---|
| Transmembrane helices | Conserved charged residues | Proton translocation | Altered ATP synthesis rate |
| Lipid-facing surface | Hydrophobic residues | Membrane anchoring | Changed stability in high salt |
| Subunit interfaces | Polar/charged residues | Complex assembly | Disrupted ATP synthase formation |
How does the mitochondrial genome organization affect ATP6 expression in D. hansenii?
The mitochondrial genome of D. hansenii contains the ATP6 gene, and understanding its organization provides insights into ATP6 expression regulation. The assembled genome of D. hansenii strain TMW 3.1188 shows that contigs 10, 12, 13, and 26 align to the mitochondrial genome . This genomic context is important for understanding transcriptional regulation of ATP6.
In diploid strains of D. hansenii, such as TMW 3.1188, researchers should consider the presence of two alleles when analyzing ATP6 expression and function . Sequence variations between alleles may lead to slightly different protein isoforms, potentially contributing to functional diversity and adaptive capacity under varying environmental conditions.
What approaches can be used to study ATP6 in the context of protein-protein interactions?
ATP6 functions as part of the larger ATP synthase complex, making protein-protein interaction studies crucial for understanding its role. While not directly about ATP6, the methodologies used in studying Dbp5-Gle1 interactions in D. hansenii provide valuable insights applicable to ATP6 research .
Size Exclusion Chromatography (SEC):
Similar to the approach used for studying Dbp5-Gle1 interactions , SEC can be employed to analyze ATP6 complex formation with other ATP synthase subunits. This technique allows for:
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Verification of complex formation through co-migration of proteins
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Assessment of complex stability under different salt conditions
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Evaluation of how mutations affect complex assembly
Crosslinking Mass Spectrometry:
This technique can identify specific interaction points between ATP6 and other subunits:
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Use chemical crosslinkers to stabilize protein-protein interactions
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Digest complexes with proteases
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Identify crosslinked peptides by mass spectrometry
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Map interaction interfaces at amino acid resolution
What role does ATP6 play in D. hansenii's adaptation to varying environmental conditions?
D. hansenii is known for its ability to adapt to various environmental stresses, particularly high salt concentrations . As part of the ATP synthase complex, ATP6 is likely involved in energy metabolism adaptations that enable survival under these conditions.
Integrated multi-omics studies have shown that D. hansenii responds differently to sodium and potassium at both expression and protein activity regulation levels . Although the specific role of ATP6 in these responses hasn't been fully characterized, its function in maintaining mitochondrial ATP production suggests it plays a critical role in energy homeostasis under stress conditions.
Researchers investigating this aspect should consider:
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Comparing ATP6 expression levels under different salt stresses (NaCl vs. KCl)
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Analyzing ATP6 post-translational modifications in response to environmental changes
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Measuring ATP synthase activity in mitochondria isolated from D. hansenii grown under varying conditions
What are the best practices for reconstituting lyophilized D. hansenii ATP6?
For optimal results when working with lyophilized D. hansenii ATP6:
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Briefly centrifuge the vial before opening to ensure the protein powder is at the bottom
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Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL
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Add glycerol to a final concentration of 5-50% (typically 50%) as a cryoprotectant
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Aliquot the reconstituted protein to minimize freeze-thaw cycles
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For long-term storage, keep at -20°C or -80°C
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For working solutions, store aliquots at 4°C for no more than one week
Improper reconstitution can lead to protein aggregation, loss of activity, or increased susceptibility to degradation, compromising experimental results.
How can researchers optimize expression of recombinant D. hansenii ATP6?
Based on established protocols for similar membrane proteins, researchers can optimize expression using the following strategies:
coli Expression Optimization:
| Parameter | Optimization Strategy | Rationale |
|---|---|---|
| Expression strain | BL21(DE3), C41(DE3), or C43(DE3) | Strains tolerant to membrane protein expression |
| Induction temperature | 16-20°C | Slower expression reduces inclusion body formation |
| IPTG concentration | 0.1-0.5 mM | Lower concentrations favor proper folding |
| Growth media | TB or 2×YT with appropriate antibiotics | Rich media support higher biomass |
| Additives | 1% glucose, 5-10% glycerol | Stabilize membrane proteins |
| Induction time | OD600 of 0.6-0.8 | Mid-log phase optimal for induction |
| Post-induction time | 16-20 hours | Extended time at lower temperature |
What analytical methods are most effective for characterizing purified D. hansenii ATP6?
To ensure protein quality and integrity, several analytical methods should be employed:
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SDS-PAGE: Confirms protein size (expected ~27 kDa including His-tag) and assesses purity (should be >90%)
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Western blotting: Verifies protein identity using anti-His antibodies or specific ATP6 antibodies
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Mass spectrometry:
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Intact mass analysis to confirm molecular weight and post-translational modifications
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Peptide mapping to verify sequence and identify any modifications
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Circular dichroism (CD) spectroscopy: Assesses secondary structure content, particularly important for confirming proper folding of membrane proteins
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Dynamic light scattering (DLS): Evaluates protein homogeneity and detects aggregation
How can researchers integrate ATP6 studies with broader omics approaches in D. hansenii research?
Following the example of integrated multi-omics studies of D. hansenii , researchers can adopt a comprehensive approach:
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Transcriptomics: Analyze ATP6 gene expression under different conditions using RNA-Seq
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Proteomics: Quantify ATP6 protein levels using mass spectrometry-based approaches
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Phosphoproteomics: Identify potential phosphorylation sites on ATP6 that may regulate its function
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Metabolomics: Measure ATP/ADP ratios and other energy-related metabolites to correlate with ATP6 function
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Genomics: Compare ATP6 sequences across different D. hansenii strains to identify variants that may correlate with phenotypic differences
This integrated approach provides a comprehensive understanding of ATP6's role in the broader context of D. hansenii physiology and stress responses.
