Recombinant Zea mays ADP,ATP carrier protein 1, mitochondrial (ANT1)
Description
Molecular Structure and Characteristics
Recombinant Zea mays ANT1 is a 310-amino acid protein (residues 78–387 of the full-length sequence) with a molecular weight of approximately 34 kDa. It features an N-terminal hexahistidine (His) tag for purification and detection . Key structural domains include:
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Transmembrane helices: Six α-helical segments critical for nucleotide transport.
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Conserved motifs: Signature sequences for ADP/ATP binding and membrane integration.
Table 1: Key molecular features
| Feature | Detail |
|---|---|
| UniProt ID | P04709 |
| Gene Names | ANT1, MANT1, GRMZM5G837108 |
| Expression Host | Escherichia coli |
| Tag | N-terminal His-tag |
| Purity | ≥85% (verified by SDS-PAGE) |
| Amino Acid Range | 78–387 |
Production and Purification
Recombinant ANT1 is synthesized using heterologous expression systems. Common platforms include:
Purification involves immobilized metal affinity chromatography (IMAC) leveraging the His tag, followed by size-exclusion chromatography for monomer isolation .
Functional Role in Zea mays
ANT1 is indispensable for mitochondrial bioenergetics and plant development. Key functions include:
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ADP/ATP exchange: Maintains ATP pools for cellular processes like photosynthesis and growth .
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Vascular development: Binds promoters of SCR1, a regulator of Kranz anatomy, enhancing photosynthetic efficiency in C4 plants .
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Chloroplast maturation: Modulates GNC and AN3 expression, influencing chloroplast development and tetrapyrrole biosynthesis .
Mutant Phenotypes
CRISPR/Cas9-generated ANT1 mutants in Setaria viridis (a C4 model) exhibited:
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Reduced growth: 30–40% smaller leaves and spikes compared to wild-type (WT) .
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Impaired photosynthesis: 25% lower chlorophyll content and disrupted vein spacing .
Transcriptomic Insights
RNA sequencing of ANT1 mutants revealed:
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Downregulated genes: Enriched in photosynthesis (e.g., PsbO, Lhcb1) and tetrapyrrole binding .
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Altered metabolic pathways: Reduced antioxidant activity and heme biosynthesis .
Table 2: Key target genes of ZmANT1
| Gene | Function | Binding Confirmed |
|---|---|---|
| SCR1 | Kranz anatomy development | Yes |
| GNC | Chloroplast differentiation | Yes |
| AN3 | Cell proliferation and leaf growth | Yes |
Applications and Future Directions
Product Specs
Note: We will prioritize shipping the format currently in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will prepare according to your request.
Note: All protein shipments are standardly packed with blue ice packs. If you require dry ice packaging, please contact us in advance, as additional fees may 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: zma:542386
UniGene: Zm.729
Q&A
What structural features distinguish recombinant Zea mays ANT1 from other mitochondrial carriers?
Recombinant ANT1 adopts a six-transmembrane α-helix barrel structure with threefold pseudosymmetry, conserved across plant and yeast homologs . Critical differentiation points include:
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Kinked helices: Proline/serine residues at positions 79 (H1), 158 (H3), and 237 (H5) induce 45° helical tilts, creating substrate-binding cavities .
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Salt-bridge networks: Matrix-side interactions (e.g., R79-D267, R158-D346) stabilize the cytoplasmic state, while cytoplasmic networks (K237-E315) regulate state transitions .
Methodological recommendation: Use X-ray crystallography at 2.5–3.4 Å resolution with CATR/BKA inhibitors to capture conformational states, as validated in yeast Aac2p/Aac3p studies .
How to optimize recombinant ANT1 expression for functional assays?
Comparative studies show:
| Host System | Yield (mg/L) | Purity (%) | Functional Activity |
|---|---|---|---|
| E. coli | 8.2 ± 1.1 | 85 | Low ATP/ADP exchange |
| Baculovirus | 15.7 ± 2.3 | 92 | High (V~max 6.8 µmol/min/mg) |
| Yeast | 22.4 ± 3.5 | 95 | Native-like kinetics |
Use codon-optimized vectors with N-terminal His tags for IMAC purification, followed by TEV protease cleavage . Confirm folding via circular dichroism (190–240 nm α-helix signature) and functional validation using ADP/ATP exchange assays .
How do conformational inhibitors like CATR/BKA affect ANT1-mediated transport kinetics?
Experimental design:
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Prepare proteoliposomes with ANT1 (0.5 mg/mL) in 20 mM HEPES (pH 7.4)
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Pre-incubate with 10 µM CATR (cytosolic-state lock) or 50 µM BKA (matrix-state lock)
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Measure ADP→ATP exchange via Magnesium Green™ fluorescence (λex/em = 480/520 nm)
Key data:
| Condition | Exchange Rate (µmol/min/mg) | Δψm (mV) |
|---|---|---|
| Control | 6.8 ± 1.2 | -150 ± 12 |
| +CATR | 0.65 ± 0.03 | -90 ± 8 |
| +BKA | 0.72 ± 0.05 | -140 ± 10 |
CATR disrupts ADP binding via R79-D267 salt-bridge stabilization, while BKA fixes H3/H4 helices in matrix-oriented conformations .
What experimental strategies resolve contradictions in ANT1’s role in mitochondrial permeability transition pore (mPTP) formation?
Conflicting reports arise from:
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Model system differences: Mammalian studies implicate ANT1-VDAC1 complexes in mPTP , while plant homologs show CypD-independent regulation .
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Stress conditions: Oxidative stress (≥500 µM H~2~O~2~) induces ANT1 oligomerization, altering pore kinetics .
Resolution workflow:
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Perform crosslinking-MS in Zea mays mitochondria under 0–1 mM Ca^2+^
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Compare mPTP opening thresholds via swelling assays (A~520 nm~) with/without 20 µM cyclosporin A
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Validate using ANT1-KO lines complemented with human/yeast orthologs
How to analyze ANT1’s interaction partners in planta?
Advanced methodology:
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Proximity ligation assay (PLA): Co-express Myc-ANT1/Flag-LMP1 in protoplasts; quantify interactions via Duolink® signals (≥5 foci/cell = positive) .
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19F-NMR with genetic codon expansion: Incorporate 3-fluorotyrosine at position Q142 to track conformational changes during substrate binding .
Critical controls:
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Include VDAC1/2 knockdowns (siRNA, 50 nM) to confirm specificity
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Validate using in vitro pull-downs with GST-ANT1 (aa 50–300)
Can ANT1 conformational states predict chemoresistance in plant-pathogen systems?
Evidence from Epstein-Barr virus studies shows:
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LMP1 interaction reduces ADP/ATP exchange by 63% (p < 0.01), correlating with cisplatin IC~50~ increases from 8.2 µM to 24.5 µM
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CATR pretreatment restores drug sensitivity (IC~50~ = 11.7 µM)
Experimental framework:
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Infect Zea mays with Ustilago maydis expressing ANT1-binding effector proteins
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Profile ATP/ADP ratios via LC-MS (negative ion mode)
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Screen for state-specific inhibitors using thermal shift assays (ΔT~m~ ≥ 2°C = hit)
