Recombinant Marinomonas sp. ATP synthase subunit a (atpB)
Description
Marinomonas as a Genus
Marinomonas is a genus of Gram-negative bacteria primarily found in marine environments. The species Marinomonas sp. strain MWYL1, from which the ATP synthase subunit a (atpB) in this study is derived, has been extensively characterized in genomic studies. Marinomonas sp. MWYL1 shares significant genomic similarity with other members of its genus, including Marinomonas sp. NFXS50, with an Average Nucleotide Identity (ANI) value of 84.57% . Other species within this genus include Marinomonas primoryensis, isolated from a brackish, ice-covered lake in Antarctica , and Marinomonas ostreistagni, a mesophilic bacterium isolated from pearl-oyster culture . These bacteria have evolved specialized adaptations to thrive in their respective marine environments, including efficient energy production systems like the ATP synthase complex.
ATP Synthase Complex Structure and Function
ATP synthase (F-type ATPase) is a remarkable molecular motor that functions as the primary enzyme responsible for ATP production in living organisms. This enzyme complex is ubiquitous across bacteria, chloroplasts, and mitochondria, with conserved structural and functional features despite minor variations . The complete ATP synthase complex consists of two main domains: a hydrophilic F₁ domain containing the catalytic sites for ATP synthesis, and a hydrophobic membrane-embedded F₀ domain that forms the proton translocation channel .
The F₁ domain comprises a hexameric structure of alternating α and β subunits (α₃β₃) arranged in a ring, along with central stalk subunits γ, δ, and ε . The F₀ domain typically includes the c-ring (composed of multiple c subunits), and several additional subunits including the critical subunit a (atpB), which is the focus of this report . In bacterial F-type ATP synthases, the complete structure can be represented as α₃:β₃:γ:δ:ε:a:b:b':c₉ , indicating the stoichiometry of each component.
Proton Translocation Mechanism
The ATP synthase subunit a (atpB) plays a critical role in proton translocation, which is essential for the generation of ATP through oxidative phosphorylation . The mechanism involves the movement of protons across the membrane through the F₀ domain, specifically through the interaction between subunit a and the c-ring .
According to the chemiosmotic theory proposed by Peter Mitchell, the difference in proton concentration and charge separation across the membrane generates the proton motive force (PMF) . This energy is harnessed when protons flow back through the ATP synthase complex. Specifically, subunit a provides the pathway for protons to access the c-ring, facilitating their movement across the membrane .
The two aqueous channels in subunit a are positioned strategically to allow protons to enter from one side of the membrane, bind to a critical site on the c-ring, and exit through the second channel after rotation . This proton flow drives the rotation of the c-ring, which in turn rotates the central stalk (γ subunit) within the F₁ domain, ultimately leading to ATP synthesis .
Rotational Catalysis and the Binding Change Mechanism
The proton translocation mediated by subunit a drives the rotation of the c-ring and the central γ shaft, which induces conformational changes in the catalytic sites located at the interface of α and β subunits in the F₁ domain . This process follows the "Binding Change Mechanism" postulated by Paul Boyer, which involves rotational motion and steady-state catalysis .
In this mechanism, each of the three catalytic sites of F₁ cycles through three conformational states: βE (empty or open), βDP (bound to ADP or loose), and βTP (bound to ATP or tight) . During ATP synthesis, a complete 360° rotation of the γ shaft results in the formation and subsequent release of three ATP molecules . The specific structure of subunit a ensures that proton flow occurs in the correct direction to drive this rotational mechanism.
Expression and Purification
Recombinant Marinomonas sp. ATP synthase subunit a (atpB) has been successfully expressed in Escherichia coli expression systems, allowing for the production of the protein for research and commercial purposes . The recombinant protein typically includes a His-tag fusion at the N-terminus to facilitate purification through affinity chromatography . The expression region covers the full length of the protein (residues 1-282) .
The purification process yields a high-purity product (greater than 90% as determined by SDS-PAGE) . The recombinant protein is usually supplied in a lyophilized powder form or in a storage buffer optimized for stability .
Antibody Production and Immunological Techniques
Recombinant atpB is utilized for the production of specific antibodies that can be employed in various immunological techniques, including:
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Western blotting to detect and quantify ATP synthase components in cell extracts.
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Immunoprecipitation to isolate intact ATP synthase complexes for functional studies.
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Immunohistochemistry to localize ATP synthase in cellular preparations.
The recombinant protein is particularly suitable for ELISA (Enzyme-Linked Immunosorbent Assay) applications, as indicated by several commercial suppliers .
Comparative Studies Across Species
The availability of recombinant atpB from Marinomonas sp. facilitates comparative studies of ATP synthase across different bacterial species and even across domains of life. Such comparative analyses provide insights into the evolution of this critical enzyme complex and how it has adapted to different environmental conditions, particularly in marine environments where Marinomonas species are found .
Functional Studies in Native Environments
Understanding how the ATP synthase functions in the native marine environment of Marinomonas sp. represents another important research direction. Studies examining how the enzyme adapts to changing environmental conditions, including variations in salinity, temperature, and pH, will contribute to our understanding of bacterial bioenergetics in marine ecosystems.
Biotechnological Applications
The unique properties of Marinomonas sp. atpB may have potential applications in biotechnology, including the development of bioenergy systems inspired by natural ATP synthases. The structural and functional insights gained from studying this protein could inform the design of artificial energy conversion systems with improved efficiency.
Product Specs
Note: We prioritize shipping the format we currently have in stock. However, if you have a specific format requirement, please indicate it during order placement, and we will fulfill your request.
Note: All our proteins are shipped with standard blue ice packs by default. If you require dry ice shipping, please inform us in advance. Additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. The shelf life of the lyophilized form is 12 months at -20°C/-80°C.
Target Background
KEGG: mmw:Mmwyl1_4468
STRING: 400668.Mmwyl1_4468
Q&A
What is the basic structure of Marinomonas sp. ATP synthase subunit a (atpB)?
Marinomonas sp. ATP synthase subunit a (atpB) is a 282-amino acid transmembrane protein (UniProt accession: A6W3T4) that forms part of the F0 domain of the ATP synthase complex. The available recombinant form contains an N-terminal His-tag to facilitate purification . The a-subunit contains transmembrane helices that form critical portions of the proton translocation pathway. Based on homology with other bacterial ATP synthases, the a-subunit interacts with the c-ring rotor and provides the proton path from outside the membrane surface to the carboxylates of interacting c-subunits .
What role does the a-subunit play in ATP synthase function?
The a-subunit serves as a critical stator component that facilitates proton movement across the membrane. It provides the proton pathway from the periplasmic (or exterior) side of the membrane to the c-subunits of the rotor complex . This subunit contains essential residues, including a highly conserved arginine (equivalent to Arg-210 in E. coli) in transmembrane helix 4 (TMH4), which prevents proton short-circuiting and helps modulate the pKa of essential carboxylates in the c-subunits . The a-subunit is therefore integral to the coupling mechanism that converts proton movement to rotational motion and ultimately ATP synthesis.
What expression systems are effective for recombinant Marinomonas sp. ATP synthase subunit a (atpB)?
E. coli is the demonstrated expression system for recombinant Marinomonas sp. ATP synthase subunit a . When expressing membrane proteins like atpB, specialized E. coli strains such as C41(DE3) or C43(DE3) may provide better yields compared to standard BL21(DE3) strains. These strains are designed to handle the potential toxicity associated with membrane protein overexpression. Expression should be optimized by testing various induction conditions (IPTG concentration, temperature, and duration) to maximize protein yield while maintaining proper folding.
What purification strategy is recommended for His-tagged atpB protein?
For His-tagged Marinomonas sp. atpB purification, a multi-step approach is recommended:
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Cell lysis using either sonication or high-pressure homogenization in a buffer containing detergent (commonly DDM or LDAO) to solubilize the membrane protein
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Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin
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Size exclusion chromatography (SEC) for further purification and buffer exchange
Table 1: Recommended Buffer Compositions for atpB Purification
| Purification Step | Buffer Composition | Purpose |
|---|---|---|
| Lysis | 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1% DDM, protease inhibitors | Membrane solubilization |
| IMAC Binding | 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% DDM, 20 mM imidazole | Initial binding |
| IMAC Wash | 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% DDM, 50 mM imidazole | Remove non-specific binding |
| IMAC Elution | 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 0.05% DDM, 300 mM imidazole | Elute target protein |
| SEC | 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 0.03% DDM | Final purification |
What approaches can be used to study the structure-function relationship of atpB?
Several complementary techniques can be employed to investigate structure-function relationships of Marinomonas sp. atpB:
How can researchers assess the membrane topology of Marinomonas sp. atpB?
Membrane topology can be investigated using:
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Cysteine scanning mutagenesis: Introduce single cysteines throughout the protein sequence and assess their accessibility to membrane-impermeable sulfhydryl reagents
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PhoA/LacZ fusion approach: Generate fusions with reporter enzymes to determine which segments are cytoplasmic or periplasmic
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Protease protection assays: In reconstituted proteoliposomes to identify protected fragments
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Computational prediction: Using algorithms like TMHMM, Phobius, or TOPCONS, then validating experimentally
What methods can be used to assess proton translocation activity of recombinant atpB?
To measure the proton translocation function of atpB when incorporated into the ATP synthase complex:
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Reconstitution into liposomes: Purified atpB can be co-reconstituted with other ATP synthase subunits into liposomes
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pH-sensitive fluorescent dyes: Measure proton translocation using dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) or pyranine
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Membrane potential measurements: Using voltage-sensitive dyes like oxonol VI or DiSC3(5)
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Patch-clamp electrophysiology: For direct measurement of proton currents through the reconstituted complex
How can researchers investigate the interaction between atpB and c-subunits in the ATP synthase complex?
Interactions between atpB and c-subunits can be studied using:
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Co-immunoprecipitation: Using antibodies against atpB or the c-subunit
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Chemical cross-linking: Followed by mass spectrometry to identify interaction interfaces
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FRET (Förster Resonance Energy Transfer): By labeling the subunits with appropriate fluorophores
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Surface plasmon resonance (SPR): To measure binding kinetics between purified components
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Isothermal titration calorimetry (ITC): To determine thermodynamic parameters of the interaction
How does the Marinomonas sp. atpB compare to those from extremophiles in terms of proton pathway adaptation?
Unlike the ATP synthase a-subunits from alkaliphiles, which contain a distinctive lysine residue in their proton uptake pathway , Marinomonas sp. atpB likely possesses adaptations specific to its marine environment. Comparative analysis with extremophilic ATP synthases can reveal how this protein has evolved for function in its native conditions. Researchers should conduct multiple sequence alignments focusing on the transmembrane helices involved in proton translocation, particularly examining conserved charged residues. Site-directed mutagenesis can then be used to test the functional significance of identified unique residues.
What approaches can be used to study the role of atpB in ATP synthase latent ATPase activity?
Methods to investigate atpB's role in regulating ATPase activity include:
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Enzymatic assays: Measure ATP hydrolysis rates using colorimetric phosphate release assays
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Genetic complementation: Express Marinomonas sp. atpB in ATP synthase-deficient bacterial strains
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Chimeric proteins: Create fusion proteins with regions from other bacterial a-subunits to identify functional domains
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Inhibitor studies: Test the effects of known ATP synthase inhibitors on complexes containing wild-type or mutant atpB
How can researchers investigate the energy coupling mechanism involving atpB?
Energy coupling in ATP synthase involving atpB can be investigated through:
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Mutation of conserved residues: Similar to the approach used with mycobacterial F1-ATPase
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Proton pump assays: Using inside-out membrane vesicles or reconstituted proteoliposomes
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Single-molecule techniques: Such as optical tweezers to measure rotational torque
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Molecular dynamics simulations: To model proton movement through the a-subunit channel
Table 2: Key Residues for Mutagenesis Studies in atpB Based on Homology
| Position in E. coli | Proposed Function | Equivalent Position in Marinomonas sp.* | Mutation Strategy |
|---|---|---|---|
| Arg-210 | Prevents proton short-circuit | To be determined by alignment | R→K, R→Q, R→A |
| Glu-219 | Proton exit pathway | To be determined by alignment | E→D, E→Q, E→A |
| His-245 | Proton transfer | To be determined by alignment | H→A, H→R, H→F |
| Asn-214 | Stabilizes arginine | To be determined by alignment | N→D, N→A, N→Q |
*Exact positions should be determined through sequence alignment
How can researchers address protein stability issues with recombinant atpB?
Membrane proteins like atpB are often challenging to work with due to stability issues. Strategies to enhance stability include:
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Detergent screening: Test multiple detergents (DDM, LMNG, LDAO, etc.) to identify optimal solubilization conditions
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Lipid supplementation: Add specific lipids like cardiolipin or phosphatidylethanolamine during purification
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Thermostability assays: Use differential scanning fluorimetry to identify stabilizing buffer conditions
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Protein engineering: Introduce disulfide bonds or remove flexible regions to enhance stability
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Nanodiscs or amphipols: Reconstitute into more native-like membrane environments
What are potential solutions for low expression yields of recombinant atpB?
To address low expression yields:
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Codon optimization: Adapt the gene sequence for expression in E. coli
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Fusion partners: Use fusion tags like MBP or SUMO to enhance solubility
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Reduced induction temperature: Express at 16-20°C to slow down production and allow proper folding
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Cell-free expression systems: Consider membrane protein-compatible cell-free systems
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Alternative host organisms: Test expression in Bacillus subtilis or Pseudomonas species
How should researchers interpret discrepancies in experimental results when studying atpB function?
Discrepancies may arise from:
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Detergent effects: Different detergents can significantly alter protein behavior
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Incomplete complexes: atpB may function differently in isolation versus within the complete ATP synthase complex
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Post-translational modifications: Check for possible modifications affecting function
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Experimental conditions: pH, ionic strength, and temperature can all affect results
Researchers should systematically test these variables and report all conditions in detail when publishing results.
What statistical approaches are appropriate for analyzing data from ATP synthase functional assays?
For robust data analysis:
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Replicate experiments: Minimum of three biological replicates and three technical replicates
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Appropriate controls: Include positive controls (known functional ATP synthase) and negative controls (known inactive mutants)
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Multiple analytical techniques: Confirm findings using complementary methods
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Statistical tests: Use ANOVA for comparing multiple conditions or t-tests for pairwise comparisons
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Curve fitting: For kinetic data, use appropriate enzyme kinetics models and software like GraphPad Prism
