Recombinant Heterosigma akashiwo ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in Heterosigma akashiwo?

ATP synthase subunit c in Heterosigma akashiwo is a hydrophobic membrane protein that forms part of the F0 sector of chloroplastic ATP synthase. It functions as a component of a multimeric ring (cn) embedded in the thylakoid membrane. The rotation of this c-subunit ring is mechanically coupled to ATP synthesis and is driven by proton translocation across the membrane along an electrochemical gradient. Each c-subunit contains two alpha-helical domains connected by a loop region, forming a hairpin-like structure in the membrane. The amino acid sequence (MDSIISAASVIAAGLSVGLAAIGPGIGQGNAAGQAVEGIARQPEAENKIRGTLLLSLAFMEALTIYGLVVALSLLFANPFTS) reveals its highly hydrophobic nature, consistent with its membrane-embedded location .

The c-subunit ring in chloroplast ATP synthase works in conjunction with the central stalk (γ-subunit) to convert the energy of proton flow into mechanical rotation. This rotation drives conformational changes in the catalytic F1 region where ATP synthesis occurs. For each complete rotation of the c-ring, three ATP molecules are synthesized at the α-β subunit interfaces in the F1 region .

How does the stoichiometry of the c-subunit ring in H. akashiwo compare to other organisms?

The stoichiometry of c-subunit rings varies across different organisms and has significant implications for bioenergetic efficiency. While the exact number of c-subunits in the H. akashiwo ATP synthase ring hasn't been definitively established, the variation in c-ring stoichiometry across species reflects evolutionary adaptations to different metabolic demands and environments. The ratio of protons translocated to ATP synthesized directly depends on the number of c-subunits (n) per oligomeric ring, with each complete rotation producing 3 ATP molecules for every n protons that pass from the lumen to the stroma .

Research using recombinant systems enables investigation into the factors that influence this stoichiometric variation. Comparative analysis between different photosynthetic organisms shows that this variation is inherently related to the metabolism of the organism, although the exact causes of this variability remain an active area of research .

What expression systems are most effective for producing recombinant H. akashiwo ATP synthase subunit c?

Based on successful approaches with similar proteins, the most effective expression system for recombinant H. akashiwo ATP synthase subunit c utilizes E. coli, particularly BL21 derivative strains. Due to the hydrophobic nature of this membrane protein, direct expression often results in inclusion bodies or toxicity to the host cells. Therefore, expression as a fusion protein with a solubility tag is highly recommended.

A proven approach involves cloning the codon-optimized atpH gene into expression vectors such as pMAL-c2x, which creates a fusion with maltose binding protein (MBP). This strategy has been demonstrated to produce significant quantities of soluble protein with spinach chloroplast ATP synthase subunit c, and can be adapted for H. akashiwo .

The expression protocol typically involves:

• Transformation of the recombinant plasmid into the E. coli expression strain

• Culture growth at optimal temperature (typically 30-37°C)

• Induction with IPTG when cultures reach appropriate density

• Post-induction growth at reduced temperature (16-25°C) to enhance proper folding

• Cell harvesting by centrifugation

Alternative vectors such as pET-32a(+) and pFLAG-MAC have also been tested for recombinant c-subunit expression and may be suitable alternatives depending on specific experimental requirements .

What are the critical factors to consider when designing a codon-optimized gene insert for H. akashiwo atpH expression?

When designing a codon-optimized gene insert for H. akashiwo atpH expression in E. coli, researchers should consider:

• Codon usage bias: Replace rare codons in the original sequence with synonymous codons preferred by E. coli to enhance translation efficiency. This is particularly important for membrane proteins like ATP synthase subunit c that may contain codons rarely used in E. coli.

• GC content adjustment: Optimize GC content to approximately 50-60% to ensure stable mRNA secondary structures and efficient translation.

• Removal of secondary structures: Eliminate potential RNA secondary structures, especially near the 5' end of the mRNA, which could impede translation initiation.

• Addition of appropriate restriction sites: Incorporate compatible restriction sites at both ends of the gene for directional cloning while ensuring these modifications don't interfere with the coding sequence.

• Fusion protein considerations: When designing for expression as a fusion protein, ensure proper reading frame maintenance and incorporate an appropriate protease cleavage site between the tag and the target protein.

A well-designed codon-optimized gene has been shown to significantly increase expression levels of hydrophobic membrane proteins compared to native gene sequences .

What is the most effective purification strategy for recombinant H. akashiwo ATP synthase subunit c?

The most effective purification strategy for recombinant H. akashiwo ATP synthase subunit c involves a multi-step approach tailored to its hydrophobic nature:

• Initial affinity chromatography: For MBP-fusion proteins, amylose resin affinity chromatography provides a highly specific first purification step. The MBP-c-subunit fusion binds to the amylose resin while contaminants are washed away.

• Protease cleavage: The purified fusion protein is then subjected to site-specific protease treatment (commonly Factor Xa or TEV protease) to separate the c-subunit from its fusion partner.

• Reversed-phase HPLC: The cleaved protein mixture is further purified using reversed-phase chromatography, which is particularly effective for separating hydrophobic membrane proteins. A C4 or C18 column with an appropriate acetonitrile/water gradient containing trifluoroacetic acid (TFA) typically provides good separation of the c-subunit from the MBP and other contaminants.

• Final polishing: Size exclusion chromatography may be employed as a final polishing step to ensure homogeneity of the purified c-subunit.

This purification approach has proven effective for obtaining high-purity recombinant c-subunit from spinach chloroplast ATP synthase and can be adapted for H. akashiwo .

How can researchers verify the correct folding and secondary structure of purified recombinant H. akashiwo ATP synthase subunit c?

Verification of correct folding and secondary structure of the purified recombinant H. akashiwo ATP synthase subunit c is crucial before proceeding with functional studies. Several complementary techniques should be employed:

• Circular Dichroism (CD) Spectroscopy: CD spectroscopy is particularly valuable for confirming the alpha-helical secondary structure that is characteristic of ATP synthase c-subunits. The c-subunit should exhibit CD spectra typical of alpha-helical proteins with negative bands at 208 and 222 nm. This approach has been successfully used to confirm the correct secondary structure of recombinant spinach chloroplast c-subunit .

• FTIR Spectroscopy: Fourier-transform infrared spectroscopy provides complementary information about protein secondary structure, particularly useful for membrane proteins.

• NMR Spectroscopy: For more detailed structural analysis, NMR can be employed to examine the tertiary structure, especially when the protein is isotopically labeled.

• Mass Spectrometry: Electrospray ionization mass spectrometry (ESI-MS) can confirm the molecular weight of the purified protein and verify the absence of post-translational modifications or degradation.

• Functional Reconstitution Assays: Ultimate verification comes from functional reconstitution of the c-subunit into liposomes and demonstration of proton translocation activity or assembly into oligomeric rings.

These analytical techniques collectively provide confidence that the recombinant protein maintains its native structure and is suitable for downstream applications .

How can researchers reconstitute functional c-rings from recombinant H. akashiwo ATP synthase subunit c monomers?

Reconstitution of functional c-rings from recombinant H. akashiwo ATP synthase subunit c monomers involves carefully controlled in vitro assembly conditions:

• Detergent selection: Choose appropriate detergents that maintain the c-subunit in a soluble state while allowing for controlled oligomerization. Commonly used detergents include n-dodecyl-β-D-maltoside (DDM) or digitonin.

• Lipid incorporation: Add specific lipids (such as phosphatidylglycerol and phosphatidylcholine) that promote proper assembly and stability of the c-ring structure.

• Buffer optimization: Use buffers that mimic the native environment of the thylakoid membrane, typically containing:

  • 20-50 mM Tris or HEPES (pH 7.5-8.0)

  • 100-150 mM NaCl

  • 5-10% glycerol as a stabilizing agent

• Controlled oligomerization: Gradually induce oligomerization through carefully controlled changes in detergent concentration, often by dialysis against decreasing detergent concentrations.

• Verification of assembly: Confirm successful reconstitution through techniques such as:

  • Blue native PAGE to assess the formation of higher molecular weight complexes

  • Electron microscopy to visualize ring structures

  • Functional assays to test proton translocation capability

This approach enables investigation of the factors that influence c-ring stoichiometry and structure, which remain incompletely understood despite their importance in determining the bioenergetic efficiency of ATP synthesis .

What are the key differences in experimental handling between recombinant H. akashiwo ATP synthase subunit c and other membrane proteins?

Recombinant H. akashiwo ATP synthase subunit c presents unique experimental challenges compared to other membrane proteins:

• Extreme hydrophobicity: The c-subunit is exceptionally hydrophobic even among membrane proteins, necessitating special handling procedures:

  • Use of high concentrations of detergents or chaotropic agents during purification

  • Avoidance of conditions that might cause precipitation

  • Storage in solutions containing sufficient detergent or solubilizing agents

• Small size: At approximately 8 kDa, the c-subunit is smaller than many membrane proteins, requiring adapted electrophoresis conditions:

  • Tricine-SDS-PAGE rather than standard Laemmli gels

  • Higher percentage (15-20%) acrylamide gels

  • Special staining procedures for visualization

• Oligomerization tendency: The c-subunit has a natural propensity to form oligomers, which can complicate experimental analysis. Researchers must carefully control:

  • Buffer conditions to either promote or prevent oligomerization

  • Sample preparation for analytical techniques

  • Interpretation of results from size-based analyses

• Limited immunogenicity: Due to high sequence conservation and small size, generating specific antibodies against the c-subunit can be challenging, often requiring:

  • Conjugation to carrier proteins

  • Careful epitope selection

  • Validation against recombinant protein standards

These special considerations must be accounted for when designing experiments involving the recombinant H. akashiwo ATP synthase subunit c .

How can recombinant H. akashiwo ATP synthase subunit c be used to investigate the molecular basis of c-ring stoichiometry variation?

Recombinant H. akashiwo ATP synthase subunit c provides a powerful tool for investigating the molecular determinants of c-ring stoichiometry variation through several advanced approaches:

• Site-directed mutagenesis: By introducing specific mutations into the recombinant atpH gene, researchers can:

  • Modify key residues at the interface between adjacent c-subunits

  • Alter the curvature-determining regions of the protein

  • Test hypotheses about which amino acids influence ring size determination

• Chimeric protein construction: Creating chimeric proteins that combine sequences from species with different known c-ring stoichiometries can pinpoint regions responsible for determining ring size.

• In vitro reconstitution with varying conditions: Systematic variation of reconstitution conditions allows investigation of environmental factors affecting ring assembly:

  • Lipid composition

  • Ionic strength

  • pH and proton concentration

  • Presence of other ATP synthase subunits

• Cross-linking studies: Chemical cross-linking of assembled c-rings followed by mass spectrometry can directly determine the number of subunits per ring under different conditions.

This research direction is particularly valuable since the ratio of protons translocated to ATP synthesized varies according to the number of c-subunits (n) per oligomeric ring, which is organism-dependent and has significant implications for bioenergetic efficiency .

What techniques are available for studying the interaction between recombinant H. akashiwo ATP synthase subunit c and other components of the ATP synthase complex?

Several sophisticated techniques are available to study interactions between recombinant H. akashiwo ATP synthase subunit c and other components of the ATP synthase complex:

• Surface Plasmon Resonance (SPR): Allows real-time, label-free monitoring of binding interactions between the c-subunit and other ATP synthase components, providing both kinetic and affinity data. The recombinant c-subunit can be immobilized on a sensor chip in a detergent environment compatible with membrane proteins.

• Isothermal Titration Calorimetry (ITC): Provides direct measurement of the thermodynamic parameters of binding interactions, including binding stoichiometry, which is particularly relevant for understanding assembly mechanisms.

• Microscale Thermophoresis (MST): A relatively new technique that can measure interactions in complex solutions and requires minimal sample amounts, making it suitable for difficult-to-express membrane proteins.

• Cryo-Electron Microscopy (Cryo-EM): Enables visualization of the assembled complex at near-atomic resolution, providing insights into structural arrangements and conformational changes.

• Native Mass Spectrometry: Advanced mass spectrometry approaches that maintain non-covalent interactions can determine subunit stoichiometry and identify specific interaction interfaces.

• FRET-Based Approaches: Fluorescence resonance energy transfer using labeled subunits can detect proximity and conformational changes in reconstituted complexes.

• Cross-linking Mass Spectrometry (XL-MS): Chemical cross-linking followed by mass spectrometry analysis identifies specific residues involved in subunit interactions.

These methods collectively provide a comprehensive toolkit for investigating how the c-subunit interacts with other components of the ATP synthase complex, contributing to our understanding of the molecular mechanisms underlying ATP synthesis .

What are common challenges in recombinant expression of H. akashiwo ATP synthase subunit c and how can they be addressed?

Researchers frequently encounter several challenges when attempting recombinant expression of H. akashiwo ATP synthase subunit c. Here are the common issues and recommended solutions:

When expressing the c-subunit, a strategy that has proven successful is to express it as a fusion with MBP, which significantly enhances solubility. After initial purification of the fusion protein, the target can be cleaved and further purified using reversed phase chromatography. This approach has been demonstrated to yield significant quantities of highly purified c-subunit with correct alpha-helical secondary structure .

How can researchers distinguish between monomeric and oligomeric forms of recombinant H. akashiwo ATP synthase subunit c during purification and analysis?

Distinguishing between monomeric and oligomeric forms of recombinant H. akashiwo ATP synthase subunit c requires specialized analytical techniques:

• Size Exclusion Chromatography (SEC):

  • Use detergent-compatible SEC columns (Superdex or Superose)

  • Calibrate with membrane protein standards in the same detergent

  • Monitor elution profiles to identify different oligomeric species

  • Consider using SEC coupled with multi-angle light scattering (SEC-MALS) for more accurate molecular weight determination

• Blue Native PAGE (BN-PAGE):

  • Particularly useful for resolving different oligomeric states while maintaining native protein interactions

  • Use gradient gels (4-16% or 3-12%) for optimal resolution

  • Compare migration with known standards

  • Follow with second-dimension SDS-PAGE to confirm subunit composition

• Analytical Ultracentrifugation (AUC):

  • Provides detailed information about the size and shape of protein-detergent complexes

  • Sedimentation velocity experiments can resolve different oligomeric species

  • Sedimentation equilibrium provides absolute molecular weight information

• Cross-linking Studies:

  • Use bifunctional cross-linkers with different spacer lengths

  • Analyze cross-linked products by SDS-PAGE to identify oligomeric states

  • Follow with mass spectrometry for detailed interaction mapping

• Electron Microscopy:

  • Negative staining can visualize ring structures vs. monomers

  • Single-particle analysis provides structural information about oligomeric assemblies

Researchers should note that detergent choice significantly influences oligomerization state, and systematic screening of different detergents may be necessary to stabilize specific forms. Additionally, careful control of protein concentration, pH, and ionic strength can help manipulate the equilibrium between monomeric and oligomeric states .

How does the expression and purification of H. akashiwo ATP synthase subunit c compare with that from other photosynthetic organisms?

The expression and purification of H. akashiwo ATP synthase subunit c presents both similarities and notable differences compared to those from other photosynthetic organisms:

The amino acid sequence of H. akashiwo ATP synthase subunit c (MDSIISAASVIAAGLSVGLAAIGPGIGQGNAAGQAVEGIARQPEAENKIRGTLLLSLAFMEALTIYGLVVALSLLFANPFTS) reveals organism-specific features that may influence expression and purification strategies .

What structural and functional differences exist between H. akashiwo ATP synthase subunit c and its counterparts in other organisms?

Heterosigma akashiwo ATP synthase subunit c exhibits several structural and functional differences compared to its counterparts in other organisms, reflecting evolutionary adaptations to its unique ecological niche:

• Sequence variations: The H. akashiwo c-subunit (82 amino acids) contains specific residues that differ from those in other photosynthetic organisms. Key differences include:

  • Variations in the proton-binding glutamate residue region crucial for proton translocation

  • Organism-specific residues at the interface between adjacent subunits that may influence c-ring size

  • Unique patterns of hydrophobic residues that determine membrane integration

• c-ring stoichiometry: While the exact number of c-subunits in the H. akashiwo ATP synthase ring isn't definitively established, variations in c-ring stoichiometry across organisms reflect adaptations to different bioenergetic demands:

  • Bacterial ATP synthases: 10-15 c-subunits per ring

  • Chloroplast ATP synthases: typically 14 c-subunits per ring

  • Mitochondrial ATP synthases: 8-10 c-subunits per ring

  These differences directly affect the H+/ATP ratio and thus the bioenergetic efficiency of ATP synthesis .

• Membrane environment adaptation: As a marine alga, H. akashiwo has evolved to function in saline environments, potentially requiring:

  • Adaptations to maintain stability in higher ionic strength conditions

  • Modified lipid interactions compared to freshwater or terrestrial organisms

  • Structural features that enable function under the specific light conditions experienced by marine algae

• Associated bioactive properties: Studies of H. akashiwo have identified production of bioactive compounds, suggesting potential unique functional aspects of its cellular components, including the ATP synthase complex .

Understanding these differences is crucial for research applications and may provide insights into the evolutionary adaptations of ATP synthase to different ecological niches.

What are promising research applications for recombinant H. akashiwo ATP synthase subunit c beyond basic structural studies?

Recombinant H. akashiwo ATP synthase subunit c offers several promising research applications that extend beyond basic structural studies:

• Bioenergetic efficiency studies: As the c-ring stoichiometry determines the H+/ATP ratio, recombinant H. akashiwo c-subunit can be used to investigate how marine algae have optimized their energy conversion processes compared to terrestrial plants. This has implications for understanding adaptation to different light environments and carbon fixation efficiency.

• Bionanotechnology applications: The self-assembling properties of c-subunits into precisely structured rings make them attractive building blocks for:

  • Nanoscale rotary motors

  • Artificial proton channels

  • Template structures for nanomaterials

  • Components in synthetic minimal cells

• Red tide toxicity research: As H. akashiwo is associated with fish kills during red tide events, investigating whether components of its energy production machinery (including ATP synthase) contribute to its ecological impact could provide insights into harmful algal bloom mechanisms .

• Bioproduction of valuable compounds: H. akashiwo produces various bioactive compounds, and understanding its energy metabolism through ATP synthase studies could inform strategies for optimizing production of:

  • Polyunsaturated fatty acids (PUFAs)

  • Carotenoids (particularly fucoxanthin)

  • Exopolysaccharides with potential bioactivity

• Evolutionary biology: Comparative studies between H. akashiwo and other species can reveal evolutionary adaptations in the highly conserved ATP synthase complex, providing insights into the diversification of energy metabolism across the tree of life.

These applications represent valuable directions for future research utilizing recombinant H. akashiwo ATP synthase subunit c, extending its utility well beyond basic structural characterization .

What emerging technologies might enhance our ability to study the structure and function of H. akashiwo ATP synthase subunit c?

Several cutting-edge technologies show promise for advancing our understanding of H. akashiwo ATP synthase subunit c:

• Cryo-electron microscopy (Cryo-EM) advances:

  • Recent breakthroughs in single-particle cryo-EM now enable near-atomic resolution of membrane protein complexes

  • Time-resolved cryo-EM could potentially capture different conformational states during c-ring rotation

  • Application to recombinant H. akashiwo c-rings could reveal precise structural details influencing stoichiometry

• Integrative structural biology approaches:

  • Combining multiple techniques (X-ray crystallography, NMR, cryo-EM, mass spectrometry)

  • Computational modeling and molecular dynamics simulations

  • These integrated approaches provide comprehensive structural insights impossible with any single method

• Advanced biophysical techniques:

  • High-speed atomic force microscopy (HS-AFM) for observing c-ring dynamics in near-native conditions

  • Single-molecule FRET to track conformational changes during rotation

  • Nanodiscs and other membrane mimetics for maintaining native-like environments during analysis

• Genome editing in algal systems:

  • CRISPR-Cas9 adaptation for H. akashiwo could enable direct modification of the native atpH gene

  • Site-specific incorporation of unnatural amino acids for specialized biophysical studies

  • Development of inducible expression systems in the native organism

• Artificial intelligence and machine learning:

  • Prediction of protein-protein interactions within the ATP synthase complex

  • Automated analysis of cryo-EM data for more efficient structure determination

  • Identification of subtle patterns in sequence-structure-function relationships across species

These emerging technologies will likely transform our ability to study the structure and function of H. akashiwo ATP synthase subunit c, potentially revealing new insights into the molecular mechanisms of ATP synthesis and the evolutionary adaptations of this essential enzymatic complex in different organisms .