Recombinant Caldicellulosiruptor saccharolyticus ATP synthase subunit c (atpE)

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Description

Introduction to Recombinant Caldicellulosiruptor saccharolyticus ATP Synthase Subunit c (atpE)

Recombinant Caldicellulosiruptor saccharolyticus ATP synthase subunit c (atpE) is a heterologously expressed, full-length protein derived from the thermophilic bacterium Caldicellulosiruptor saccharolyticus. This subunit is a core component of the ATP synthase complex, which catalyzes ATP synthesis by translocating protons across membranes. The recombinant protein is produced in E. coli, fused with an N-terminal histidine (His) tag for purification via affinity chromatography, and spans 70 amino acids (aa 1–70) .

FeatureDetail
Protein LengthFull-length (1–70 aa)
Expression HostE. coli
Purification TagHis-tag
FormLyophilized powder
Genome SourceCaldicellulosiruptor saccharolyticus (UniProt: A4XKX5)

Caldicellulosiruptor saccharolyticus is a Gram-positive, anaerobic bacterium renowned for its ability to degrade cellulose, hemicellulose, and pectin at extreme temperatures (~70–80°C) . Its ATP synthase system is integral to energy conservation during fermentation, particularly in biohydrogen production .

Protein Architecture

The recombinant subunit c is part of the F₀ subcomplex of ATP synthase, which facilitates proton translocation. In C. saccharolyticus, this subunit likely exhibits enhanced thermostability due to its thermophilic origin, making it valuable for studying extremophilic enzymes .

Functional Role

ATP synthase subunit c cooperates with subunit a to pump protons across the membrane, creating a proton gradient that drives ATP synthesis. In C. saccharolyticus, this process is linked to its high-yield biohydrogen production, where hydrogenases convert NADH and reduced ferredoxin into H₂ .

Biohydrogen Production

C. saccharolyticus produces hydrogen via fermentation of lignocellulosic biomass, with yields approaching the theoretical maximum of 4 mol H₂ per mol hexose . The ATP synthase subunit c plays a critical role in maintaining energy balance during this process.

Thermophilic Adaptation

The recombinant protein’s thermostability may enable its use in industrial bioprocesses requiring high-temperature conditions. For example, its stability could be leveraged in enzymatic studies or biofuel production .

Production and Purification

The recombinant subunit c is expressed in E. coli, which offers cost-effective scalability. The His-tag facilitates purification, yielding a high-purity protein suitable for structural studies or functional assays .

Production ParameterDetail
Host StrainE. coli BL21(DE3) or similar
Induction SystemIPTG (isopropyl β-D-1-thiogalactopyranoside)
Purification MethodNickel-affinity chromatography
YieldDependent on expression conditions (typical yields not explicitly reported)

Mitochondrial vs. Bacterial ATP Synthase

In mitochondria, ATP synthase subunit c isoforms (e.g., P1, P2, P3) differ in targeting peptides but share identical mature regions . By contrast, C. saccharolyticus subunit c lacks such isoforms, reflecting evolutionary divergence in thermophilic bacteria.

FeatureMitochondrial Subunit cC. saccharolyticus Subunit c
IsoformsP1, P2, P3 (variable targeting peptides)Single isoform (no targeting peptide)
ThermostabilityModerateHigh (thermophilic origin)
Proton TranslocationCooperates with subunit aSimilar mechanism

Key Challenges

  1. Structural Stability: Full characterization of the recombinant protein’s 3D structure is pending.

  2. Scalability: Optimizing E. coli expression yields for industrial use.

  3. Functional Integration: Investigating interactions with other ATP synthase subunits in C. saccharolyticus.

Potential Applications

  • Biofuel Production: Enhancing ATP synthase efficiency to improve H₂ yields.

  • Enzyme Engineering: Leveraging thermostability for biocatalytic processes.

Product Specs

Form
Lyophilized powder
Note: While we will prioritize shipping the format currently in stock, we are happy to accommodate specific format requests. Please indicate your preferred format when placing your order, and we will prepare accordingly.
Lead Time
Delivery time may vary depending on the purchase method and location. Please consult your local distributor for specific delivery time estimates.
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.
Notes
Repeated freezing and thawing is not recommended. Store working aliquots at 4°C for up to one week.
Reconstitution
We recommend briefly centrifuging the vial prior to opening to ensure the contents settle to the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, we suggest adding 5-50% glycerol (final concentration) and aliquoting the solution at -20°C/-80°C. Our standard glycerol concentration is 50%, which can serve as a reference for your own preparations.
Shelf Life
Shelf life is influenced by various factors, including storage conditions, buffer composition, temperature, and the inherent stability of the protein.
Generally, liquid form has a shelf life of 6 months at -20°C/-80°C. Lyophilized form has a shelf life of 12 months at -20°C/-80°C.
Storage Condition
Upon receipt, store at -20°C/-80°C. Aliquoting is necessary for multiple uses. Avoid repeated freeze-thaw cycles.
Tag Info
Tag type will be determined during the manufacturing process.
The tag type is determined during production. If you have a specific tag type requirement, please inform us, and we will prioritize developing the specified tag.
Synonyms
atpE; Csac_1975; ATP synthase subunit c; ATP synthase F(0 sector subunit c; F-type ATPase subunit c; F-ATPase subunit c; Lipid-binding protein
Buffer Before Lyophilization
Tris/PBS-based buffer, 6% Trehalose.
Datasheet
Please contact us to get it.
Expression Region
1-70
Protein Length
full length protein
Species
Caldicellulosiruptor saccharolyticus (strain ATCC 43494 / DSM 8903 / Tp8T 6331)
Target Names
atpE
Target Protein Sequence
MTALAAGIAMLAGLGVGIGIGIATAKAAESVGRQPEAYGRILPLFFIGAALAEAVAIYSF VIAILLVLKV
Uniprot No.

Target Background

Function
F(1)F(0) ATP synthase generates ATP from ADP in the presence of a proton or sodium gradient. This enzyme is composed of two structural domains: F(1), containing the extramembraneous catalytic core, and F(0), containing the membrane proton channel, connected by a central stalk and a peripheral stalk. ATP synthesis in the catalytic domain of F(1) is coupled to proton translocation through a rotary mechanism involving the central stalk subunits. This subunit plays a direct role in translocation across the membrane. A homomeric c-ring, composed of 10-14 subunits, forms the central stalk rotor element in conjunction with the F(1) delta and epsilon subunits.
Database Links
Protein Families
ATPase C chain family
Subcellular Location
Cell membrane; Multi-pass membrane protein.

Q&A

What is the basic structure of C. saccharolyticus ATP synthase subunit c (atpE)?

C. saccharolyticus ATP synthase subunit c (atpE) is a 70-amino acid protein that forms an α-helical hairpin structure. This highly hydrophobic peptide typically arranges into oligomeric complexes that span the lipid bilayer . The protein constitutes an integral part of the F0 complex of ATP synthase, where multiple copies form the c-ring structure essential for the rotary mechanism of ATP synthesis . The transmembrane nature of this protein allows it to participate in proton translocation across membranes, a critical function for energy conservation in this thermophilic bacterium.

What expression systems are most effective for producing recombinant C. saccharolyticus atpE?

Recombinant C. saccharolyticus atpE has been successfully expressed in E. coli expression systems with an N-terminal His-tag . For optimal expression of this membrane protein, researchers should consider:

  • Using specialized E. coli strains designed for membrane protein expression

  • Employing lower induction temperatures (18-25°C) to facilitate proper folding

  • Supplementing growth media with specific lipids or detergents to stabilize the protein during expression

  • Utilizing fusion tags (His-tag being common) to enhance solubility and facilitate purification

The hydrophobic nature of this protein presents challenges that require careful optimization of expression conditions to maximize yield while maintaining native structure.

What purification strategies yield the highest purity and activity of recombinant atpE?

Effective purification of recombinant C. saccharolyticus atpE requires a multi-step approach:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is the primary method for His-tagged protein

  • Detergent selection is critical - mild detergents like DDM or LMNG can maintain protein structure during extraction from membranes

  • Size exclusion chromatography as a polishing step to separate monomeric protein from aggregates

  • Buffer optimization to maintain stability, with recommended storage in Tris/PBS-based buffer containing 6% trehalose at pH 8.0

Purity greater than 90% can be achieved through these methods, as determined by SDS-PAGE analysis .

What are the optimal conditions for storing recombinant atpE?

Research demonstrates that recombinant C. saccharolyticus atpE requires specific storage conditions to maintain stability:

  • Store lyophilized powder at -20°C/-80°C upon receipt

  • Working aliquots should be maintained at 4°C for no more than one week

  • Reconstitution should be in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

  • Avoid repeated freeze-thaw cycles as they significantly reduce protein stability

The inclusion of trehalose (6%) in the storage buffer serves as a cryoprotectant that helps maintain protein structure during freeze-thaw processes .

How does calcium influence the conformational changes in atpE?

Experimental evidence demonstrates that calcium plays a critical role in the structural dynamics of ATP synthase subunit c:

  • Calcium induces conformational changes that promote transition from native α-helical structure to β-sheet formation

  • This structural transition is concentration-dependent and directly correlates with the protein's ability to self-assemble into different oligomeric states

  • In the presence of calcium, c subunit oligomers exhibit ion channel activity in lipid membranes with slight cation selectivity (PK/PCl = 6 ± 2)

  • Channel conductance ranges from 300-400 pS, with multiple conductance states that are voltage-dependent

These calcium-dependent properties suggest a mechanism similar to amyloidogenic proteins implicated in neurodegenerative disorders, where structural rearrangements lead to membrane permeabilization .

What methodologies can detect calcium-induced conformational changes in atpE?

Several complementary techniques can be employed to study calcium-induced structural changes:

TechniqueApplicationMeasurable Parameters
Fluorescence spectroscopyDetect conformational changesSecondary structure transitions, protein folding kinetics
Atomic force microscopy (AFM)Visualize oligomeric structuresFibril morphology, oligomer dimensions
Black lipid membrane methodsMeasure ion channel activityConductance, ion selectivity, voltage dependence
Circular dichroism (CD)Quantify secondary structureα-helix to β-sheet ratio
ThT fluorescence assaysMonitor amyloid formationKinetics of β-sheet-rich structure formation

These methods have confirmed that c subunit preparations with and without calcium exhibit different structural properties while maintaining ion channel activity .

How can researchers distinguish between physiological oligomeric states and pathological aggregation of atpE?

Distinguishing between functional oligomers and pathological aggregates involves several analytical approaches:

  • Structural analysis: Native oligomers typically maintain α-helical structure, while pathological aggregates show increased β-sheet content measurable by CD spectroscopy

  • Morphological examination: AFM and electron microscopy can differentiate between:

    • Ring-shaped native c-ring complexes

    • Amyloid-like fibrils formed under certain conditions

    • Unstructured aggregates resulting from denaturation

  • Functional assays: Electrophysiology measurements reveal differences in:

    • Ion channel properties

    • Voltage dependence

    • Selectivity profiles

    • Conductance states

  • Calcium dependence: Sensitivity to calcium concentration provides a key indicator, as pathological aggregation shows stronger calcium dependence than physiological assembly .

What techniques are most effective for studying atpE ion channel activity?

Research has established several methodologies for characterizing the ion channel properties of atpE:

  • Planar lipid bilayer reconstitution: Incorporating purified atpE into artificial membranes allows direct measurement of channel activity

  • Voltage clamp electrophysiology: This technique has revealed that atpE channels exhibit:

    • Multiple conductance states

    • Voltage-dependent behavior with tendency to switch to low conductance states at higher voltages

    • Average conductance ranging from 300-400 pS

  • Ion selectivity measurements: By manipulating ion compositions on either side of the membrane, researchers have determined that atpE channels exhibit slight cation selectivity (PK/PCl = 6 ± 2)

  • Single-channel analysis: Statistical analysis of channel conductances under different conditions provides insights into channel gating mechanisms and substate behavior

How does atpE contribute to the thermophilic nature of C. saccharolyticus?

While specific data on atpE's contribution to thermophilia in C. saccharolyticus is limited in the search results, structural analysis suggests:

  • The amino acid composition likely contains features that enhance thermostability, such as increased hydrophobicity and specific salt bridge formations

  • The compact nature of the protein (only 70 amino acids) may contribute to stability at high temperatures

  • As part of the ATP synthase complex, atpE would need specific adaptations to maintain functionality at the high temperatures (up to 70°C) at which C. saccharolyticus grows optimally

  • The ability of C. saccharolyticus to produce biohydrogen at thermophilic temperatures suggests that all components of its energy metabolism, including ATP synthase, are thermally adapted

What is the relationship between atpE function and biohydrogen production in C. saccharolyticus?

C. saccharolyticus has been recognized as an excellent candidate for biological hydrogen production . The relationship between atpE function and this capability involves:

  • ATP synthase (containing atpE) plays a crucial role in maintaining cellular energy balance during fermentative growth conditions that lead to hydrogen production

  • The ATP/ADP ratio regulated by ATP synthase activity influences metabolic flux through hydrogen-producing pathways

  • Electron flow systems in C. saccharolyticus require coordinated activity of membrane-bound complexes, including those involved in establishing proton gradients that drive ATP synthase

  • Transcriptomic studies of C. saccharolyticus grown on different substrates provide insights into how energy conservation mechanisms (including ATP synthase components) are regulated during conditions conducive to hydrogen production

How can site-directed mutagenesis of atpE advance understanding of ATP synthesis in thermophiles?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in atpE:

  • Target residues for mutation:

    • Conserved residues involved in proton binding/translocation

    • Interface residues that mediate c-ring assembly

    • Residues potentially involved in thermostability

  • Experimental design:

    • Generate mutant constructs using standard molecular biology techniques

    • Express and purify mutant proteins following established protocols

    • Reconstitute into liposomes or nanodiscs for functional studies

  • Functional analysis:

    • Measure ATP synthesis rates at different temperatures

    • Assess proton translocation efficiency

    • Determine thermal stability profiles of wild-type versus mutant proteins

  • Structural consequences:

    • Examine effects on oligomerization using analytical ultracentrifugation

    • Assess structural changes using CD spectroscopy and thermal denaturation studies

This approach could reveal specific adaptations that allow C. saccharolyticus ATP synthase to function optimally under thermophilic conditions.

What genomic context approaches can be used to better understand atpE function in C. saccharolyticus?

Genomic context analysis provides valuable insights beyond traditional homology-based methods:

  • Whole genome re-annotation, as performed for C. saccharolyticus, can identify previously unrecognized functional relationships involving atpE

  • Comparative genomics across Caldicellulosiruptor species reveals conservation patterns and potential co-evolution of atpE with other energy metabolism components

  • Transcriptomic analysis during growth on different substrates identifies co-regulated genes and metabolic networks involving ATP synthase components

  • Non-homology based functional prediction methods can assign cellular processes or physical complexes for hypothetical proteins that may interact with atpE

  • Gene neighborhood analysis can identify functionally related genes that are co-located on the genome, providing insights into metabolic modules involving ATP synthase components

What are the major unresolved questions regarding C. saccharolyticus atpE structure and function?

Despite advances in understanding atpE, several knowledge gaps remain:

  • The exact stoichiometry of the c-ring in C. saccharolyticus ATP synthase has not been determined

  • The specific adaptations that allow atpE to function at thermophilic temperatures are not fully characterized

  • The potential dual role of atpE in both ATP synthesis and membrane permeabilization (as suggested by studies on other c subunits) requires further investigation in C. saccharolyticus

  • The regulatory mechanisms controlling atpE expression and function under different growth conditions remain to be elucidated

  • The potential for biotechnological applications of this thermostable protein has not been fully explored

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