Recombinant Cellvibrio japonicus ATP synthase subunit c (atpE)

What is the basic structure of Cellvibrio japonicus ATP synthase subunit c (atpE)?

Cellvibrio japonicus ATP synthase subunit c (atpE) is a small membrane protein consisting of 79 amino acids (1-79aa). The full amino acid sequence is MTEAAALIILAGALVIGLGAIGAAIGVALLGGKFLEGAARQPELLPMLRTQFFVVMGLVDAIPMIGVGIGMYILFALGA. It is identified in UniProt database as B3PIT2 and has several synonyms including CJA_3814, ATP synthase F(0) sector subunit c, F-type ATPase subunit c, and Lipid-binding protein . As typical for c-subunits, it likely forms part of an oligomeric ring structure within the F₀ sector of the ATP synthase complex.

How does the c-subunit contribute to ATP synthase function?

The c-subunit forms an oligomeric ring in the F₀ domain of ATP synthase that is essential for proton translocation and energy conversion. Recent studies with similar ATP synthases have demonstrated that proton translocation through F₀ drives rotation of the c-subunit oligomeric ring relative to the a-subunit . This rotational motion is mechanically coupled to conformational changes in the F₁ sector, ultimately driving ATP synthesis. Key acidic residues in c-subunits (typically glutamic acid) participate in proton release to and uptake from the a-subunit during the rotational catalysis mechanism .

What expression systems are recommended for producing recombinant Cellvibrio japonicus atpE?

Based on research protocols, E. coli expression systems have been successfully employed for the recombinant production of Cellvibrio japonicus atpE . When expressed with an N-terminal His-tag, the protein can be effectively purified to greater than 90% purity using standard immobilized metal affinity chromatography (IMAC) techniques. For optimal expression, codon optimization for E. coli may be beneficial, especially considering that Cellvibrio japonicus has a different codon usage pattern than E. coli.

What are the optimal conditions for reconstituting lyophilized atpE protein?

For optimal reconstitution of lyophilized Cellvibrio japonicus atpE protein, it is recommended to:

• Briefly centrifuge the vial before opening to ensure the protein powder is at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard) for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store aliquots at -20°C or -80°C for long-term preservation

The reconstituted protein should be handled carefully as repeated freeze-thaw cycles can lead to denaturation and loss of activity.

What methods can be used to assess the functional integrity of purified atpE protein?

To evaluate the functional integrity of purified atpE protein, researchers can employ several complementary approaches:

• Circular Dichroism (CD) Spectroscopy: To confirm proper secondary structure formation

• Size Exclusion Chromatography: To assess oligomeric state and aggregation

• Liposome Reconstitution Assays: To evaluate membrane integration capability

• Proton Translocation Assays: Using pH-sensitive fluorescent dyes to measure proton transport activity when reconstituted in proteoliposomes

• ATP Synthesis Coupling Assays: When co-reconstituted with other ATP synthase components

These assays should be performed under conditions that mimic the native membrane environment, often requiring the use of appropriate detergents or lipid compositions.

How can site-directed mutagenesis of atpE be used to study proton translocation mechanisms?

Site-directed mutagenesis of atpE provides valuable insights into the proton translocation mechanism. Based on studies with similar ATP synthases, researchers should consider:

• Targeting conserved acidic residues (particularly glutamic acid residues) that likely participate in the proton channel

• Creating mutations analogous to the E56D mutation studied in Bacillus PS3 ATP synthase, which demonstrated decreased ATP synthesis and proton pump activities

• Designing experiments to test single versus multiple mutations across different c-subunits to investigate cooperativity

• Using molecular dynamics simulations in parallel with biochemical assays to interpret experimental results

Research has shown that ATP synthesis and proton pump activities decrease significantly with single mutations in key residues and decrease further when multiple c-subunits are mutated, suggesting functional cooperation among c-subunits .

What techniques can be used to study the oligomeric ring formation of c-subunits?

To investigate c-subunit oligomeric ring formation, researchers can employ:

• Blue Native PAGE: To preserve native protein complexes during electrophoresis

• Crosslinking Studies: Using bifunctional reagents to stabilize protein-protein interactions

• Cryo-Electron Microscopy: For high-resolution structural analysis of the intact c-ring

• Genetic Fusion Approaches: Similar to the single-chain c-ring strategy used in Bacillus PS3 studies, where multiple c-subunits are expressed as a single polypeptide to control stoichiometry

• AFM (Atomic Force Microscopy): For topographical analysis of membrane-embedded c-rings

These approaches can reveal critical information about the stoichiometry, stability, and structural dynamics of the c-ring during ATP synthase operation.

What strategies can address poor solubility when working with recombinant atpE?

The hydrophobic nature of atpE can lead to solubility challenges. Researchers can implement these strategies:

• Optimized Detergent Selection: Test various detergents (DDM, LDAO, C12E8) at different concentrations to find optimal solubilization conditions

• Fusion Tags: Beyond His-tags, consider solubility-enhancing tags like MBP or SUMO

• Expression Temperature Modulation: Lower temperatures (16-20°C) often improve proper folding of membrane proteins

• Co-expression with Chaperones: GroEL/GroES or other chaperone systems can improve folding

• Cell-Free Expression Systems: Consider lipid-supplemented cell-free systems that can directly incorporate membrane proteins into liposomes

A systematic approach testing multiple conditions is recommended, with protein quality assessed at each step using techniques like SEC-MALS (Size Exclusion Chromatography with Multi-Angle Light Scattering).

How can researchers distinguish between functional and non-functional forms of reconstituted atpE?

Distinguishing functional from non-functional forms requires multi-parameter analysis:

• Proton Gradient Formation: Monitor the ability to establish a proton gradient using pH-sensitive fluorescent dyes

• ATP Hydrolysis-Driven Proton Pumping: Measure ATP-dependent proton translocation when co-reconstituted with F₁ components

• Proteoliposome Membrane Potential Measurements: Using voltage-sensitive dyes

• Electron Microscopy: To visualize proper incorporation into membranes and ring formation

• Thermal Stability Assays: Functional protein typically exhibits cooperative unfolding transitions

Correlation between multiple functional parameters provides stronger evidence of proper reconstitution than any single measurement.

What methods are effective for studying the interaction between atpE and other ATP synthase components?

To study interactions between atpE and other ATP synthase components, researchers can apply:

• Co-immunoprecipitation: Using antibodies against tagged versions of different subunits

• Surface Plasmon Resonance (SPR): To measure binding kinetics between purified components

• FRET (Förster Resonance Energy Transfer): For analyzing proximity relationships in reconstituted systems

• Hydrogen-Deuterium Exchange Mass Spectrometry: To identify interaction interfaces

• Crosslinking Mass Spectrometry: To map spatial relationships between components

Studies with similar ATP synthases have demonstrated that proper interactions between c-subunits and the a-subunit are critical for proton translocation and rotational catalysis .

How can researchers investigate the cooperativity among c-subunits within the oligomeric ring?

Investigating cooperativity among c-subunits requires specialized approaches:

• Genetically Fused Single-Chain c-Rings: Creating constructs where multiple c-subunits are expressed as a single polypeptide, enabling precise control of mutation positions

• Functional Assays with Variable Mutation Patterns: Systematic introduction of mutations at different positions within the c-ring to analyze positional effects

• Molecular Dynamics Simulations: To model proton transfer and energetics across multiple c-subunits

• Single-Molecule FRET: To detect conformational changes during rotation

• Site-Specific Labeling: For tracking movement of specific residues during function

Research with similar ATP synthases has shown that activity decreases further as mutations are positioned at increasing distances within the c-ring, suggesting long-range cooperativity effects .

What are the recommended approaches for determining the high-resolution structure of Cellvibrio japonicus atpE?

For high-resolution structural analysis of Cellvibrio japonicus atpE, researchers should consider:

• X-ray Crystallography: Requiring detergent screening and crystallization optimization

• Cryo-Electron Microscopy: Particularly effective for membrane protein complexes

• Solid-State NMR: Provides atomic-resolution data in a lipid environment

• Molecular Dynamics Simulations: To complement experimental structures with dynamic information

• Integrative Structural Biology: Combining multiple techniques (SAXS, crosslinking MS, etc.)

The choice of approach should consider the oligomeric state of the protein and whether the goal is to study the isolated c-subunit or the assembled c-ring.

How can computational approaches enhance our understanding of atpE function?

Computational approaches provide valuable insights into atpE function:

• Molecular Dynamics Simulations: To model proton transfer mechanisms, especially prolonged proton uptake events observed in mutated c-subunits

• Quantum Mechanics/Molecular Mechanics (QM/MM): For precise modeling of proton transfer energetics

• Coarse-Grained Simulations: To study longer timescale rotational movements of the c-ring

• Homology Modeling: To predict structural features based on better-characterized ATP synthase c-subunits

• Network Analysis: To identify allosteric communication pathways between distant c-subunits

Research has shown that computational approaches can reveal cooperative mechanisms in proton transfer that explain experimental observations of decreased activity when multiple c-subunits are mutated .

How does Cellvibrio japonicus atpE compare structurally and functionally to ATP synthase c-subunits from other species?

Comparative analysis reveals important evolutionary and functional insights:

The functional equivalence of key acidic residues across species suggests a conserved proton translocation mechanism, though ring stoichiometry varies significantly and likely affects the bioenergetic efficiency of ATP production .

What can researchers learn from studying the effects of mutations in Cellvibrio japonicus atpE compared to similar mutations in other species?

Comparative mutation studies provide insights into evolutionary conservation and adaptation:

• Mutations of conserved acidic residues (like those equivalent to E56D in Bacillus PS3) are expected to show similar decreases in ATP synthesis and proton pumping activities

• The degree of functional impairment may correlate with c-ring stoichiometry differences between species

• Species-specific compensatory mutations may exist that maintain function despite variations in primary sequence

• Environmental adaptations (temperature, pH, salt) may be reflected in differential sensitivity to equivalent mutations

• Cooperative effects between mutated c-subunits observed in Bacillus PS3 are likely conserved in Cellvibrio japonicus, with potential variations in the distance-dependence of this cooperation

Cross-species comparison of mutation effects serves as a powerful tool for identifying conserved functional principles versus species-specific adaptations in ATP synthase operation.