Recombinant Pelomedusa subrufa ATP synthase subunit a (MT-ATP6)
Description
Overview of Recombinant Pelomedusa subrufa ATP Synthase Subunit a (MT-ATP6)
Recombinant Pelomedusa subrufa ATP synthase subunit a (MT-ATP6) is a bioengineered protein derived from the African side-necked turtle (Pelomedusa subrufa). It corresponds to the mitochondrial ATP synthase subunit a (also known as F-ATPase protein 6), a critical component of the F₀ subcomplex in ATP synthase (Complex V). This protein facilitates proton translocation across the inner mitochondrial membrane, coupling this energy to ATP synthesis via rotary catalysis .
Role in ATP Synthesis
MT-ATP6 forms part of the F₀ subcomplex, interacting with subunit c to create a proton-conductive pore. During oxidative phosphorylation:
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Proton Translocation: Protons flow through the F₀ channel, driving rotation of the c-ring subunits.
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Energy Coupling: The central stalk (γδε subunits) transmits rotational energy to the F₁ catalytic domain, enabling ATP synthesis .
ELISA and Antibody Development
The recombinant MT-ATP6 protein is widely used as an antigen in enzyme-linked immunosorbent assays (ELISA) to detect antibodies against ATP synthase subunit a. This application is critical for studying autoimmune responses or mitochondrial disorders .
Disease Modeling and Pathogenicity Studies
While human MT-ATP6 variants (e.g., m.9016A>G, m.9025G>A) are linked to mitochondrial diseases like Leigh syndrome and retinitis pigmentosa, the Pelomedusa subrufa recombinant protein serves as a reference for comparative studies . For example:
Stability and Handling Guidelines
| Parameter | Recommendation |
|---|---|
| Storage | -20°C or -80°C; avoid repeated freeze-thaw cycles |
| Working Aliquots | Store at 4°C for ≤1 week |
| Buffer Compatibility | Tris-based buffer optimized for stability |
Comparative Analysis with Other MT-ATP6 Recombinant Proteins
| Species | Source | Tag | Key Application |
|---|---|---|---|
| Pelomedusa subrufa | E. coli | N/A | ELISA, structural studies |
| Petromyzon marinus | E. coli | His-tag | Biochemical assays |
| Bos taurus | E. coli | N/A | Disease modeling |
| Data compiled from . |
Research Challenges and Future Directions
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Heteroplasmy Studies: The recombinant protein could simulate heteroplasmic states in mitochondrial diseases, though current models (e.g., yeast) lack stable heteroplasmy .
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Evolutionary Insights: Comparing Pelomedusa subrufa MT-ATP6 with human homologs (e.g., OSCP subunit interactions) may reveal conserved mechanisms in ATP synthase evolution .
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Therapeutic Targeting: Mutations in MT-ATP6 highlight its potential as a target for drugs modulating proton translocation in mitochondrial disorders .
Product Specs
Note: While we prioritize shipping the format currently in stock, we are happy to accommodate any specific format requirements. Please indicate your preference in the order notes section, and we will prepare accordingly.
Note: All protein shipments are standardly packed with blue ice packs. If you require dry ice shipping, please inform us in advance, as additional fees will apply.
Generally, liquid forms have a shelf life of 6 months at -20°C/-80°C. Lyophilized forms typically have a shelf life of 12 months at -20°C/-80°C.
Target Background
Q&A
What is the functional significance of ATP synthase subunit a in mitochondrial energy production?
ATP synthase subunit a plays a crucial role in cellular bioenergetics as an essential component of the mitochondrial ATP synthase complex (Complex V). This subunit forms part of the membrane-embedded F0 domain that creates a proton channel through the inner mitochondrial membrane. The functional significance includes:
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Facilitates proton translocation down the electrochemical gradient established by the electron transport chain
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Couples proton movement to the rotational motion of the F1 domain
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Enables the conversion of ADP to ATP through conformational changes in the F1 catalytic sites
The importance of this subunit is underscored by the fact that mutations in the MT-ATP6 gene are associated with several severe neurological disorders, including neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome and maternally inherited Leigh syndrome .
What methodological approaches are recommended for reconstitution and storage of recombinant MT-ATP6 protein?
Successful work with recombinant MT-ATP6 requires careful attention to reconstitution and storage protocols. Based on established procedures for similar mitochondrial membrane proteins, researchers should follow these methodological guidelines:
| Parameter | Recommendation | Rationale |
|---|---|---|
| Initial Reconstitution | Briefly centrifuge vial before opening | Ensures protein collection at bottom of vial |
| Reconstitution Medium | Deionized sterile water, 0.1-1.0 mg/mL | Provides optimal protein concentration |
| Long-term Storage | -20°C/-80°C with 50% glycerol | Prevents protein degradation |
| Working Storage | 4°C for up to one week | Maintains short-term stability |
| Freeze-thaw | Avoid repeated cycles | Prevents protein denaturation |
| Aliquoting | Recommended for multiple use | Minimizes freeze-thaw exposure |
The commercially available protein is typically supplied in a storage buffer containing Tris-based buffer with 50% glycerol at pH 8.0, optimized for maintaining protein stability .
How do mutations in the MT-ATP6 gene affect ATP synthesis efficiency and contribute to disease pathogenesis?
Mutations in the MT-ATP6 gene can significantly impact ATP synthesis through various molecular mechanisms. A detailed study of the novel m.8839G>C mutation associated with NARP syndrome revealed multiple cellular consequences:
Interestingly, despite showing several markers of mitochondrial dysfunction, direct measurement of ATP synthesis showed no differences between wild-type and mutated cybrids. Researchers proposed that the m.8839G>C mutation may "lower the efficiency between proton translocation within F0 and F1 rotation, required for ATP synthesis" .
This complex relationship between genotype and phenotype highlights the need for comprehensive functional characterization of MT-ATP6 mutations beyond simple ATP synthesis measurements.
What experimental approaches are most effective for characterizing novel mutations in the MT-ATP6 gene?
Characterization of novel MT-ATP6 mutations requires a multi-faceted experimental approach to establish pathogenicity. Based on published research, the following methodological workflow is recommended:
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Genetic Analysis
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Sequencing to identify the mutation
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Quantification of heteroplasmy in different tissues
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Comparison of mutation load between symptomatic and asymptomatic individuals
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Evolutionary Conservation Analysis
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Multiple sequence alignment across species
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Identification of conserved functional domains
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In silico prediction tools to classify mutation impact
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Functional Studies in Cybrid Models
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Generation of transmitochondrial cybrids (cells with patient mtDNA but control nuclear DNA)
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Assessment of:
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Cell proliferation rates
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mtDNA copy number (compensatory response)
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Steady-state levels of oxidative phosphorylation proteins
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Mitochondrial membrane potential
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ATP synthesis capacity
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Population Genetics
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Screening for mutation presence in control populations
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Haplogroup analysis to rule out polymorphic variants
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This systematic approach was successfully employed to characterize the m.8839G>C mutation, providing strong evidence for its pathogenicity despite the absence of direct effects on ATP synthesis rates .
How does the amino acid sequence of Pelomedusa subrufa ATP synthase subunit a compare with homologs from other species?
Comparative analysis of ATP synthase subunit a across evolutionarily distant species reveals important insights into functional conservation. Below is a comparison between Pelomedusa subrufa (turtle) and Petromyzon marinus (sea lamprey):
This high degree of sequence conservation, particularly in the central functional domain responsible for proton translocation, underscores the critical evolutionary constraints on this protein. The conserved regions likely represent essential structural elements for proper ATP synthase function, while variable regions may reflect species-specific adaptations .
What biochemical techniques can effectively differentiate between normal and pathogenic variants of ATP synthase subunit a?
Distinguishing normal from pathogenic variants of ATP synthase subunit a requires sophisticated biochemical approaches that assess various aspects of protein function:
Researchers investigating the m.8839G>C mutation employed several of these techniques and found that mutant cybrids exhibited decreased mitochondrial membrane potential despite normal ATP synthesis rates. This suggests that biochemical phenotypes of pathogenic variants can be subtle and may require multiple complementary approaches for detection .
What considerations are critical when designing experimental controls for studies involving recombinant ATP synthase subunit a?
Rigorous experimental design for studies involving recombinant ATP synthase subunit a requires careful consideration of controls to ensure valid and reproducible results:
| Control Type | Purpose | Implementation |
|---|---|---|
| Tag Controls | Assess tag interference with function | Compare tagged vs. untagged versions or alternative tag positions |
| Species Controls | Address evolutionary differences | Include human MT-ATP6 when studying disease-relevant mutations |
| Heteroplasmy Controls | Mimic in vivo conditions | Generate cybrid lines with varying mutation loads (0-100%) |
| Isogenic Controls | Eliminate nuclear genetic variation | Use transmitochondrial cybrids with identical nuclear background |
| Complementation Controls | Verify causality | Express wild-type protein in mutant cells to rescue phenotype |
| Environmental Controls | Account for metabolic adaptation | Standardize culture conditions (glucose vs. galactose media) |
| Technical Controls | Ensure assay reliability | Include positive and negative controls for each biochemical assay |
The importance of proper controls is exemplified in the study of the m.8839G>C mutation, where researchers compared isogenic wild-type and mutant cybrid lines to isolate the effects of the mutation from other genetic variables. Additionally, heteroplasmy analysis comparing symptomatic patients with asymptomatic carriers provided crucial evidence for pathogenicity .
