Recombinant Brachypodium distachyon ATP synthase subunit b, chloroplastic (atpF)
Product Specs
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Target Background
F1F0 ATP synthase synthesizes ATP from ADP using a proton or sodium gradient. This enzyme comprises two domains: the F1 domain, containing the extramembrane catalytic core, and the F0 domain, containing the membrane proton channel. These domains are linked by a central and peripheral stalk. ATP synthesis in the F1 catalytic domain is coupled, via a rotary mechanism of the central stalk subunits, to proton translocation. This protein is a component of the F0 channel, forming part of the peripheral stalk and linking F1 to F0.
KEGG: bdi:6439854
STRING: 15368.BRADI5G08883.1
Q&A
Basic Research Questions
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What is Brachypodium distachyon ATP synthase subunit b, chloroplastic (atpF) and what is its significance in research?
Brachypodium distachyon ATP synthase subunit b, chloroplastic (atpF) is a 183-amino acid protein component of the ATP synthase F0 sector located in chloroplasts. This protein (UniProt ID: B3TN47) plays a crucial role in energy transduction during photosynthesis by contributing to the formation of the peripheral stalk of the ATP synthase complex. The full amino acid sequence is: MKNVTHSFVFLAHWPSAGSFGLNTDILATNLINLTVVVGVLIFFGKGVLKDLLDNRKQRILSTIRNSEELRRGTFEQLEKARIRLQKVELEADEYRMNGYSEIEREKENLINATSISLEQLEKSKNETLYFEKQRAMNQVRQRVFQQAVQGALGTLNSCLNTELHFRTIRANIGILGSMEWKR . Its significance in research stems from its central role in photosynthetic energy production, making it relevant for studies on plant bioenergetics, chloroplast function, and potential biotechnological applications in crop improvement.
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How is recombinant Brachypodium distachyon atpF protein produced and purified for research applications?
The production of recombinant atpF protein follows a systematic methodology that includes:
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Gene cloning: The atpF coding sequence (1-183 amino acids) is amplified or synthesized and inserted into an expression vector with an N-terminal His-tag .
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Host selection: E. coli is typically used as the expression host due to its efficiency and scalability .
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Protein expression: Transformed bacteria are cultured and protein expression is induced under optimized conditions.
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Cell lysis and extraction: Bacterial cells are harvested and lysed to release the recombinant protein.
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Affinity purification: His-tagged atpF protein is purified using nickel affinity chromatography.
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Quality assessment: SDS-PAGE analysis confirms protein purity (typically >90%) .
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Formulation: The purified protein is formulated in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 .
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Final preparation: The protein is typically provided as a lyophilized powder for improved stability .
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What are the optimal storage and handling conditions for recombinant atpF protein?
Preserving the structural integrity and biological activity of recombinant atpF requires specific storage and handling protocols:
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Long-term storage: Store lyophilized protein at -20°C to -80°C for extended stability .
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Reconstitution protocol: Before opening, briefly centrifuge the vial to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .
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Aliquoting strategy: Following reconstitution, add glycerol to a final concentration of 5-50% (optimal: 50%) and create single-use aliquots to avoid repeated freeze-thaw cycles .
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Working storage: Working aliquots can be stored at 4°C for up to one week .
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Stability considerations: Repeated freeze-thaw cycles significantly reduce protein activity and should be strictly avoided .
These protocols ensure maximum retention of structural and functional integrity throughout experimental workflows.
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How does atpF protein structure relate to its function in the ATP synthase complex?
The atpF protein structure exhibits specialized domains that directly correlate with its function:
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N-terminal domain: Contains a chloroplast transit peptide that directs the protein to its subcellular location.
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Transmembrane domain (approximately residues 26-48): Forms an alpha-helical structure that anchors the protein in the thylakoid membrane. The sequence VFLAHWPSAGSFGLNTDILATNLINLTVVVGVLIFFGK contains predominantly hydrophobic residues optimized for membrane integration .
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Central stalk region: Contains the highly conserved LSTIRN motif essential for interactions with other ATP synthase subunits and the formation of the peripheral stalk.
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C-terminal domain: Rich in charged and polar amino acids that facilitate interactions with the F1 sector of ATP synthase.
This structure-function relationship enables atpF to serve as both a structural component that stabilizes the ATP synthase complex and a functional element that contributes to the rotational mechanism driving ATP synthesis during photosynthesis.
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