Recombinant Bacillus halodurans L-arabinose transport system permease protein AraQ (araQ)
Product Specs
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Generally, the shelf life of liquid form is 6 months at -20°C/-80°C. For lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: bha:BH0903
STRING: 272558.BH0903
Q&A
What is the biological role of AraQ in Bacillus halodurans L-arabinose transport?
AraQ is a transmembrane permease component of the ABC-type L-arabinose transporter in B. halodurans. It partners with AraP (substrate-binding lipoprotein) and AraEGH (ATPase components) to facilitate the energy-dependent uptake of arabinose and arabino-oligosaccharides . Methodologically, its function is validated through growth assays under arabinose-limiting conditions. For example, deletion of araQ reduces growth rates by 5-fold in media containing arabinotriose as the sole carbon source . Structural studies reveal that AraQ contains a conserved C-terminal tail (e.g., -GVKG motif) critical for interaction with the MsmX ATPase, which energizes the transport system .
What protocols are used to express and purify recombinant AraQ?
Recombinant AraQ is typically expressed in E. coli or yeast systems with affinity tags (e.g., His₆) for purification. Key steps include:
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Cloning: The araQ gene (BH0903, UniProt Q9KEE9) is amplified from B. halodurans genomic DNA and ligated into vectors like pET or pYES under inducible promoters .
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Expression: Induction with IPTG (for E. coli) or galactose (for yeast) at 20–25°C to minimize inclusion body formation .
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Purification: Immobilized metal affinity chromatography (IMAC) followed by size-exclusion chromatography to isolate monomeric or oligomeric forms .
Critical parameters include maintaining a Tris-based buffer (pH 7.5–8.0) with 50% glycerol for storage stability .
How is AraQ’s transport activity experimentally validated?
Functional validation involves uptake assays and growth phenotyping:
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Radiolabeled substrate uptake: Cells expressing AraQ are incubated with ³H-labeled L-arabinose or arabinotriose. Transport rates are quantified via scintillation counting .
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Growth curves: Strains lacking araQ (ΔaraQ) show impaired growth in minimal media with arabinose derivatives (Table 1) .
Table 1. Impact of araQ mutations on arabinotriose uptake in B. subtilis
| Strain | Doubling Time (min) | Arabinotriose Uptake Efficiency |
|---|---|---|
| Wild-type | 98.2 ± 10.0 | 100% (baseline) |
| ΔaraQ | 472.8 ± 22.6 | 17% |
| araQ<sup>D180A</sup> | 149.3 ± 12.8 | 32% |
How do specific mutations in AraQ alter substrate transport kinetics?
Site-directed mutagenesis of conserved residues (e.g., D180A in the C-terminal tail) disrupts MsmX ATPase binding, reducing transport efficiency . Methodology:
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Mutagenesis: Overlap PCR introduces point mutations (e.g., D180A) into araQ .
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Functional assays: Compare growth rates (Table 1) and substrate affinity (K<sub>m</sub>) via Michaelis-Menten kinetics.
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Structural analysis: Molecular dynamics simulations predict destabilization of helix-helix interactions in the transmembrane domain (TMD) .
What structural features enable AraQ-MsmX ATPase interaction?
AraQ’s C-terminal tail (-GVKG motif) binds the Q-loop of MsmX, as shown by:
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Truncation studies: Deleting the last four residues (ΔGVKG) reduces arabinotriose uptake by 83% .
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Cross-linking assays: Chemical cross-linkers (e.g., DSS) confirm physical interaction between AraQ and MsmX in membrane fractions .
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Homology modeling: AraQ’s cytoplasmic domain shares 64% similarity with E. coli MalG, which forms analogous ATPase interactions .
How does AraQ integrate into the broader arabinose regulon?
AraQ is transcriptionally regulated by AraR, which represses araQ expression in the absence of L-arabinose . Key findings:
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DNA footprinting: AraR binds cooperatively to two operator sites (OR<sub>A1</sub>, OR<sub>A2</sub>) upstream of araQ, inducing DNA looping .
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Induction mechanism: L-arabinose binding to AraR reduces its DNA affinity by 10<sup>3</sup>-fold, derepressing araQ transcription .
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Cross-regulation: AraQ also transports D-xylose and D-galactose, linking it to catabolic pathways beyond arabinose .
What discrepancies exist between in vitro and in vivo AraQ analyses?
Contradiction: In vitro binding assays show AraQ-MsmX interaction requires 2 mM ATP, but in vivo activity occurs at physiological ATP concentrations (0.5–1 mM) .
Resolution:
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Accessory proteins: In vivo, chaperones like GroEL stabilize the AraQ-MsmX complex .
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Membrane potential: Proton motive force enhances substrate affinity in vivo, compensating for lower ATP levels .
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Data reconciliation: Use B. subtilis membrane vesicles to replicate native lipid composition and electrochemical gradients .
