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) .
Feature | Detail |
---|---|
Protein Length | Full-length (1–70 aa) |
Expression Host | E. coli |
Purification Tag | His-tag |
Form | Lyophilized powder |
Genome Source | Caldicellulosiruptor 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 .
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 .
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 .
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.
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 .
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 .
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.
Feature | Mitochondrial Subunit c | C. saccharolyticus Subunit c |
---|---|---|
Isoforms | P1, P2, P3 (variable targeting peptides) | Single isoform (no targeting peptide) |
Thermostability | Moderate | High (thermophilic origin) |
Proton Translocation | Cooperates with subunit a | Similar mechanism |
Structural Stability: Full characterization of the recombinant protein’s 3D structure is pending.
Scalability: Optimizing E. coli expression yields for industrial use.
Functional Integration: Investigating interactions with other ATP synthase subunits in C. saccharolyticus.
Biofuel Production: Enhancing ATP synthase efficiency to improve H₂ yields.
Enzyme Engineering: Leveraging thermostability for biocatalytic processes.
KEGG: csc:Csac_1975
STRING: 351627.Csac_1975
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.
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.
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 .
Research demonstrates that recombinant C. saccharolyticus atpE requires specific storage conditions to maintain stability:
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 .
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 .
Several complementary techniques can be employed to study calcium-induced structural changes:
Technique | Application | Measurable Parameters |
---|---|---|
Fluorescence spectroscopy | Detect conformational changes | Secondary structure transitions, protein folding kinetics |
Atomic force microscopy (AFM) | Visualize oligomeric structures | Fibril morphology, oligomer dimensions |
Black lipid membrane methods | Measure ion channel activity | Conductance, ion selectivity, voltage dependence |
Circular dichroism (CD) | Quantify secondary structure | α-helix to β-sheet ratio |
ThT fluorescence assays | Monitor amyloid formation | Kinetics 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 .
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:
Functional assays: Electrophysiology measurements reveal differences in:
Calcium dependence: Sensitivity to calcium concentration provides a key indicator, as pathological aggregation shows stronger calcium dependence than physiological assembly .
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:
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
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
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
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:
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.
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
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