Recombinant Sulfurihydrogenibium sp. ATP synthase subunit c (atpE)

What is the basic structure and function of ATP synthase subunit c in Sulfurihydrogenibium sp.?

ATP synthase subunit c from Sulfurihydrogenibium sp. (strain YO3AOP1) is a 114-amino acid protein that forms part of the membrane-embedded F0 sector of ATP synthase. The protein has a highly hydrophobic character and contains transmembrane alpha-helical regions that allow it to be embedded in the membrane. Multiple c-subunits assemble to form a ring structure (cn) within the membrane, which functions as a rotor during ATP synthesis .

The amino acid sequence (MVKFSKVLMLMVLAGTVS AAFAAEGDPMARAVFYGALAIGAGVAIGAAAGGGA AGLGNAIRGVLEGMARNPNMGPKLLTTMFIGMALIETFV LYALLIAIIFIFTGIFDSKAGF) reveals the hydrophobic nature of this protein, which is essential for its membrane localization and function . The c-subunit ring plays a crucial role in the mechanical rotation coupled to proton translocation across the membrane, which ultimately drives ATP synthesis .

How does the c-subunit ring stoichiometry affect ATP synthase function?

The stoichiometry of c-subunits in the ring (referred to as cn) is a critical determinant of the ATP synthase coupling ratio—the number of protons required to synthesize one ATP molecule. Different organisms have varying numbers of c-subunits per ring, ranging from c10 to c15 .

This variation directly affects the bioenergetic efficiency of ATP synthesis. Since three ATP molecules are generated per complete rotation of the c-ring, and each c-subunit translocates one proton, the ratio of protons transported to ATP generated (coupling ratio) varies from 3.3 to 5.0 among different organisms, depending entirely on the number of c-subunits in the ring .

For Sulfurihydrogenibium sp., determining the exact c-ring stoichiometry would provide valuable insights into how this thermophilic bacterium has adapted its energy conversion machinery to its extreme environmental conditions.

What are the optimal expression systems for recombinant production of Sulfurihydrogenibium sp. ATP synthase subunit c?

Based on successful approaches with other ATP synthase c-subunits, Escherichia coli expression systems are recommended for recombinant production of Sulfurihydrogenibium sp. ATP synthase subunit c. For optimal expression:

• Use a synthetic gene with codons optimized for E. coli expression, similar to the approach used for spinach chloroplast ATP synthase .

• Consider expression vectors such as pMAL-c2x, pET-32a(+), or pFLAG-MAC, which have proven successful for similar membrane proteins .

• For this highly hydrophobic membrane protein, fusion tags can significantly improve expression and solubility. Maltose-binding protein (MBP) fusion has shown success with other ATP synthase c-subunits .

The expression protocol should include:

• Transformation into a suitable E. coli strain (e.g., BL21(DE3))

• Culture growth at 37°C to mid-log phase (OD600 of 0.6-0.8)

• Induction with IPTG (typically 0.5-1.0 mM)

• Continued culture at reduced temperature (16-30°C) for 3-4 hours or overnight

• Cell harvest by centrifugation and storage of pellets at -80°C until purification

What purification strategies are most effective for isolating recombinant ATP synthase subunit c?

Purification of recombinant Sulfurihydrogenibium sp. ATP synthase subunit c requires specialized approaches due to its hydrophobic nature. A recommended multi-step purification protocol includes:

• Cell Lysis: Resuspend cells in lysis buffer (e.g., 20 mM Tris-HCl pH 8.0) containing protease inhibitors, followed by lysozyme treatment (1 mg/mL) and sonication .

• Affinity Chromatography: If the recombinant protein includes a fusion tag, use the appropriate affinity resin (e.g., amylose resin for MBP-tagged proteins) .

• Tag Cleavage: If applicable, remove the fusion tag using a specific protease (e.g., Factor Xa or TEV protease).

• Secondary Purification: Further purify using size exclusion chromatography or ion exchange chromatography.

• Storage: Store the purified protein in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for extended storage .

It's crucial to maintain the protein in detergent-containing buffers throughout the purification process to prevent aggregation of this highly hydrophobic protein.

What are the most effective methods for analyzing the secondary structure of recombinant ATP synthase subunit c?

Several complementary techniques can be employed to analyze the secondary structure of recombinant Sulfurihydrogenibium sp. ATP synthase subunit c:

• Circular Dichroism (CD) Spectroscopy: This technique provides information about the alpha-helical content of the protein. For ATP synthase subunit c, which should have a predominantly alpha-helical structure, CD spectroscopy in the far-UV region (190-250 nm) can confirm the proper folding of the recombinant protein .

• Fourier Transform Infrared Spectroscopy (FTIR): FTIR can provide additional confirmation of the alpha-helical structure and is particularly useful for membrane proteins.

• Nuclear Magnetic Resonance (NMR) Spectroscopy: For detailed structural analysis, NMR can provide atomic-level information about the protein structure in detergent micelles or lipid bilayers.

• X-ray Crystallography: Although challenging for membrane proteins, crystallography could provide high-resolution structural data if crystals can be obtained.

Comparison of the experimental data with theoretical predictions based on the amino acid sequence (MVKFSKVLMLMVLAGTVS AAFAAEGDPMARAVFYGALAIGAGVAIGAAAGGGA AGLGNAIRGVLEGMARNPNMGPKLLTTMFIGMALIETFV LYALLIAIIFIFTGIFDSKAGF) can confirm the proper folding and secondary structure of the purified protein .

How can researchers determine the oligomeric state of ATP synthase subunit c in membrane environments?

Determining the oligomeric state of ATP synthase subunit c in membrane environments is crucial for understanding its functional assembly. Several techniques are recommended:

• Blue Native PAGE: This technique allows analysis of native protein complexes and can help determine the size of the assembled c-ring.

• Analytical Ultracentrifugation: When combined with appropriate detergents, this method can provide information about the oligomeric state of membrane proteins.

• Crosslinking Experiments: Chemical crosslinking followed by SDS-PAGE analysis can reveal the oligomeric arrangement of subunit c.

• Single-particle Cryo-electron Microscopy: This advanced technique can provide structural information about the assembled c-ring without requiring crystallization.

• Atomic Force Microscopy (AFM): AFM can be used to visualize the c-ring structure in reconstituted membrane environments.

The c-subunit ring stoichiometry in different organisms ranges from c10 to c15, affecting the coupling ratio of protons transported to ATP generated . Determining the specific stoichiometry of Sulfurihydrogenibium sp. ATP synthase c-ring would provide valuable insights into its bioenergetic efficiency.

What methods can be used to assess the proton translocation function of recombinant ATP synthase subunit c?

Assessing the proton translocation function of recombinant Sulfurihydrogenibium sp. ATP synthase subunit c requires reconstitution into lipid membranes and several functional assays:

• Liposome Reconstitution: Purified recombinant subunit c should be reconstituted into liposomes along with other necessary ATP synthase subunits to form a functional complex.

• pH Gradient Monitoring: Using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) to monitor proton gradient formation across liposome membranes.

• Patch-Clamp Electrophysiology: For direct measurement of proton currents through the reconstituted ATP synthase complex.

• ATP Synthesis Assays: Measuring ATP production in response to an artificially imposed proton gradient can indirectly assess the functionality of the c-ring.

• Rotation Assays: Advanced single-molecule techniques can be used to directly observe c-ring rotation coupled to proton translocation.

These functional assays can help determine whether the recombinant subunit c can properly assemble into a functional c-ring capable of facilitating proton translocation, which is mechanically coupled to ATP synthesis .

How does the thermostability of Sulfurihydrogenibium sp. ATP synthase subunit c compare to mesophilic homologs?

Sulfurihydrogenibium sp. is a thermophilic bacterium, and its ATP synthase subunit c likely possesses adaptations for thermostability. Comparative analysis with mesophilic homologs can reveal these adaptations:

• Thermal Denaturation Assays: Using techniques like differential scanning calorimetry (DSC) or CD spectroscopy with temperature ramping to determine melting temperatures.

• Sequence Analysis: Comparison of amino acid composition with mesophilic homologs, focusing on features known to enhance thermostability:

  • Increased number of ionic interactions

  • Enhanced hydrophobic core packing

  • Higher proportion of certain amino acids (Arg, Glu, Pro)

  • Decreased number of thermolabile residues

• Structural Analysis: Examining structural features that may contribute to thermostability, such as shorter loops, additional salt bridges, or disulfide bonds.

• Functional Assays at Various Temperatures: Testing proton translocation or ATP synthesis activity across a temperature range to determine the temperature optimum and stability.

Understanding the thermostable properties of Sulfurihydrogenibium sp. ATP synthase subunit c could provide insights for engineering thermostable variants of ATP synthase for biotechnological applications.

How can site-directed mutagenesis of Sulfurihydrogenibium sp. ATP synthase subunit c inform structure-function relationships?

Site-directed mutagenesis of the recombinant Sulfurihydrogenibium sp. ATP synthase subunit c provides a powerful approach for investigating structure-function relationships:

• Key Residues for Mutagenesis:

  • Proton-binding residue (typically a conserved acidic residue)

  • Residues involved in c-c subunit interactions

  • Residues at the interface with other ATP synthase subunits

  • Residues potentially involved in thermostability

• Functional Analysis of Mutants:

  • Assessing assembly into the c-ring structure

  • Measuring proton translocation efficiency

  • Evaluating effects on ATP synthesis rates

  • Testing thermostability changes

• Structural Analysis of Mutants:

  • Determining whether mutations affect the alpha-helical structure

  • Analyzing changes in oligomeric assembly

  • Examining alterations in protein stability

The results from such mutagenesis studies would provide insights into the molecular mechanisms of proton translocation, c-ring assembly, and the determinants of thermostability in this thermophilic ATP synthase.

What factors influence the stoichiometry of the c-ring in ATP synthase, and how can this be investigated using the recombinant system?

The factors influencing c-ring stoichiometry in ATP synthase remain poorly understood, despite its significance for bioenergetic efficiency. Using recombinant Sulfurihydrogenibium sp. ATP synthase subunit c, researchers can investigate these factors:

• Environmental Conditions:

  • Test whether temperature, pH, or ionic conditions affect c-ring assembly

  • Examine if lipid composition influences stoichiometry

• Protein Sequence Determinants:

  • Create chimeric proteins with c-subunits from organisms with different known stoichiometries

  • Use site-directed mutagenesis to alter residues at subunit interfaces

• Assembly Factors:

  • Identify and characterize proteins that may assist in c-ring assembly

  • Test whether other ATP synthase subunits influence c-ring stoichiometry

• Methodological Approaches:

  • In vitro reconstitution of c-rings from purified recombinant subunits

  • Analysis of c-ring size and stability using the techniques described in section 3.2

  • Comparative analysis with c-subunits from organisms with known stoichiometries

The development of recombinant expression systems enables the application of molecular biology techniques that cannot be applied to native c-rings, facilitating investigations into the factors influencing stoichiometric variation .

What strategies can address poor expression or insolubility of recombinant ATP synthase subunit c?

The hydrophobic nature of ATP synthase subunit c often leads to expression and solubility challenges. Here are strategies to address these issues:

• Fusion Tags:

  • MBP tag has shown success with other ATP synthase c-subunits

  • Other solubility-enhancing tags (SUMO, Trx) may be effective

  • Consider testing multiple fusion constructs in parallel

• Expression Conditions:

  • Lower induction temperature (16-20°C)

  • Reduced IPTG concentration (0.1-0.5 mM)

  • Extended expression time (overnight)

  • Alternative media formulations (e.g., terrific broth)

• Specialized Host Strains:

  • C41(DE3) or C43(DE3) strains designed for membrane protein expression

  • Strains with additional tRNAs for rare codons

  • Strains with altered membrane properties

• Solubilization Methods:

  • Screen multiple detergents (DDM, LDAO, Triton X-100)

  • Test different detergent concentrations

  • Consider lipid-detergent mixed micelles

• Codon Optimization:

  • Design synthetic gene with codons optimized for E. coli expression

  • Avoid rare codons, especially at the N-terminus

Implementing these strategies systematically can significantly improve the yield and solubility of recombinant Sulfurihydrogenibium sp. ATP synthase subunit c.

How can researchers verify the correct folding and functional integrity of purified recombinant ATP synthase subunit c?

Verifying the correct folding and functional integrity of purified recombinant Sulfurihydrogenibium sp. ATP synthase subunit c is essential before proceeding with advanced experiments:

• Structural Verification:

  • CD spectroscopy to confirm alpha-helical secondary structure

  • Size exclusion chromatography to assess homogeneity

  • Native PAGE to evaluate oligomeric state

• Functional Verification:

  • Reconstitution into liposomes

  • Proton translocation assays (as described in section 4.1)

  • Assembly with other ATP synthase subunits

• Thermal Stability Analysis:

  • CD spectroscopy with temperature ramping

  • Differential scanning calorimetry

  • Thermal shift assays

• Comparison with Native Protein:

  • Immunological cross-reactivity with antibodies against native protein

  • Comparative structural analysis

  • Functional complementation in appropriate model systems

• Mass Spectrometry Analysis:

  • Confirm protein identity and integrity

  • Detect any post-translational modifications

  • Analyze protein-lipid interactions

These complementary approaches provide a comprehensive assessment of whether the recombinant protein has maintained its native structure and functional capabilities.

How does Sulfurihydrogenibium sp. ATP synthase subunit c compare to homologs from other extremophiles?

Comparative analysis of Sulfurihydrogenibium sp. ATP synthase subunit c with homologs from other extremophiles can provide insights into adaptive mechanisms:

Specific comparisons should include:

• Sequence alignment and phylogenetic analysis

• Structural modeling and comparison

• Analysis of conserved functional residues

• Examination of species-specific adaptations

This comparative approach can reveal how ATP synthase has evolved to function under various extreme conditions, with potential applications in protein engineering and biotechnology.

What insights can be gained from studying the c-ring stoichiometry across diverse species?

The study of c-ring stoichiometry across diverse species, including Sulfurihydrogenibium sp., provides valuable insights into bioenergetic adaptation:

Research questions that can be addressed include:

• Is c-ring stoichiometry correlated with environmental conditions?

• How does stoichiometry affect the energy conversion efficiency?

• What molecular mechanisms determine the number of c-subunits per ring?

• Has stoichiometry evolved as an adaptation to specific energy constraints?

Determining the c-ring stoichiometry of Sulfurihydrogenibium sp. ATP synthase would add valuable data to this comparative framework, potentially revealing adaptations specific to thermophilic environments .

What emerging technologies could advance our understanding of ATP synthase subunit c structure and function?

Several emerging technologies hold promise for advancing our understanding of Sulfurihydrogenibium sp. ATP synthase subunit c:

• Cryo-Electron Microscopy (Cryo-EM):

  • High-resolution structural determination of the entire ATP synthase complex

  • Visualization of conformational changes during catalysis

  • Analysis of the c-ring in its native lipid environment

• Single-Molecule Techniques:

  • Direct observation of c-ring rotation

  • Measurement of proton translocation at the single-molecule level

  • Real-time analysis of conformational dynamics

• Advanced Computational Methods:

  • Molecular dynamics simulations of the c-ring in membrane environments

  • Quantum mechanical calculations of proton transfer

  • Machine learning approaches for predicting structure-function relationships

• Synthetic Biology Approaches:

  • Creation of minimal ATP synthase systems

  • Engineering of c-rings with altered stoichiometry

  • Development of biohybrid energy conversion devices

• Native Mass Spectrometry:

  • Direct measurement of intact c-ring mass to determine stoichiometry

  • Analysis of lipid-protein interactions

  • Characterization of subunit interfaces

These technologies, applied to recombinant Sulfurihydrogenibium sp. ATP synthase subunit c, could provide unprecedented insights into the molecular mechanisms of biological energy conversion.

How might understanding the structure and function of thermophilic ATP synthase subunit c contribute to biotechnological applications?

Understanding the structure and function of Sulfurihydrogenibium sp. ATP synthase subunit c has several potential biotechnological applications:

• Bioenergy Applications:

  • Development of thermostable ATP synthases for biofuel production

  • Creation of artificial photosynthetic systems for solar energy conversion

  • Design of bio-inspired molecular motors

• Nanobiotechnology:

  • Engineering of c-rings as nanoscale rotary motors

  • Development of molecular pumps for targeted drug delivery

  • Creation of biosensors based on conformational changes

• Protein Engineering:

  • Transfer of thermostability features to mesophilic proteins

  • Design of pH-resistant membrane proteins

  • Engineering of proteins with altered ion selectivity

• Therapeutic Applications:

  • ATP synthase is a potential drug target for antibiotics and antiparasitics

  • Understanding c-subunit structure could facilitate drug design

  • Development of inhibitors specific to pathogen ATP synthases

• Synthetic Cell Development:

  • Incorporation of ATP synthase into artificial cell systems

  • Minimal energy conversion modules for synthetic biology

  • Bottom-up assembly of artificial organelles

The thermostable properties of Sulfurihydrogenibium sp. ATP synthase subunit c make it particularly valuable for applications requiring stability under harsh conditions or elevated temperatures.