Recombinant Galdieria sulphuraria ATP synthase subunit c, chloroplastic (atpH)

Basic Research Questions

• What is the structure and function of ATP synthase subunit c in extremophilic algae like Galdieria sulphuraria?

  ATP synthase subunit c (atpH) forms a ring structure embedded in the thylakoid membrane of chloroplasts. In algae like Galdieria sulphuraria, this multimeric protein complex is responsible for generating adenosine triphosphate (ATP) required for photosynthetic metabolism. The synthesis of ATP is mechanically coupled to the rotation of this c-subunit ring, which is driven by proton translocation across the membrane along an electrochemical gradient . The c-subunit ring's structure consists of alpha-helical secondary structures that span the membrane, and this structural conformation is critical for its functional role in ATP synthesis .

  The extremophilic nature of G. sulphuraria likely influences its ATP synthase properties, allowing the organism to maintain energy production under harsh conditions such as high temperatures and extremely acidic environments (pH 2.0-3.0) .

• How can researchers optimize recombinant expression of G. sulphuraria ATP synthase subunit c?

  Based on successful recombinant expression of similar proteins, researchers should consider the following methodological approach:

  • Use a codon-optimized gene insert for the G. sulphuraria c-subunit to improve expression in bacterial systems

  • Express the hydrophobic c-subunit as a fusion protein (e.g., with maltose binding protein) to improve solubility

  • Use BL21 derivative Escherichia coli cells as the expression system

  • Employ a plasmid vector with appropriate promoters for regulated expression

  • Include detergents during protein isolation to maintain stability of this membrane protein

  This approach has been successful for other chloroplastic ATP synthase c-subunits, yielding milligram quantities of purified protein with proper folding .

• What purification strategies are recommended for obtaining high-purity recombinant G. sulphuraria ATP synthase subunit c?

  A multi-step purification protocol is recommended:

  Purification Step	Methodology	Purpose
  Initial Capture	Affinity chromatography (if expressed as fusion protein)	Isolate fusion protein from bacterial lysate
  Cleavage	Protease treatment	Separate c-subunit from fusion partner
  Final Purification	Reversed phase column chromatography with ethanol as eluent	Obtain highly purified c-subunit

  This protocol has been effective for similar chloroplastic ATP synthase c-subunits, resulting in protein preparations that maintain their native alpha-helical secondary structure as confirmed by circular dichroism spectroscopy .

• How do culture conditions affect G. sulphuraria growth and protein expression?

  Galdieria sulphuraria can be cultured under various conditions that might affect protein expression:

  • Temperature: Optimal growth at 37°C

  • pH: Naturally grows at pH 2.0, but can adapt to different pH environments

  • Growth mode: Can be grown heterotrophically in the dark (2-L culture volumes are suitable for laboratory research)

  • Culture age: Cellular metabolism changes significantly between logarithmic and stationary phases, which may affect protein expression

  When designing expression systems for G. sulphuraria proteins, researchers should consider that different strains may respond differently to culture conditions. For instance, G. maxima ACUF551 (a related species) shows good growth performance under nitrate at pH 5, which could inform approaches for G. sulphuraria cultivation .

• How can researchers verify the structural integrity of recombinant G. sulphuraria ATP synthase subunit c?

  Multiple complementary techniques should be employed:

  • Circular dichroism (CD) spectroscopy to confirm alpha-helical secondary structure

  • SDS-PAGE to verify protein purity and molecular weight

  • Western blotting with anti-c-subunit antibodies for identity confirmation

  • Mass spectrometry for precise molecular mass determination

  • Functional reconstitution assays to verify ability to form oligomeric rings

  These analytical methods collectively provide strong evidence that the recombinant protein maintains its native structure and can potentially assemble into functional complexes.

Advanced Research Questions

• What factors affect the stoichiometry of the c-subunit ring in ATP synthase, and how might this be investigated in G. sulphuraria?

  The number of c-subunits per oligomeric ring (cn) varies among organisms (known to range from 8 to 15) and directly influences the H+/ATP ratio, thereby affecting the organism's bioenergetic efficiency . This variation is particularly interesting in extremophiles like G. sulphuraria.

  To investigate c-ring stoichiometry in G. sulphuraria:

  • Express and purify recombinant c-subunits as described previously

  • Reconstitute the c-subunits in liposomes to promote ring formation

  • Analyze the resulting oligomeric structures using:

    • Atomic force microscopy

    • Electron microscopy

    • Cross-linking followed by mass spectrometry

    • Native gel electrophoresis

  Researchers should examine whether G. sulphuraria's adaptation to extreme conditions correlates with a unique c-ring stoichiometry that might optimize energy production under these conditions .

• How does pH affect the function and reconstitution of G. sulphuraria ATP synthase subunit c?

  Given G. sulphuraria's natural habitat in acidic environments (pH 2.0), the relationship between pH and ATP synthase function is particularly relevant. Research indicates:

  • The organism has evolved to function optimally in acidic conditions

  • Different G. sulphuraria strains may have varying abilities to acidify or alkalize their growth medium depending on nitrogen source and initial pH

  • The proton gradient that drives ATP synthase is influenced by environmental pH and the cell's ability to maintain appropriate internal pH

  For reconstitution experiments, researchers should test multiple pH conditions to determine:

  1. Optimal pH for c-subunit ring assembly

  2. pH dependency of proton translocation

  3. Structural stability of the c-ring at different pH values

  This information would provide insights into how this extremophile has adapted its ATP synthase to function in acidic environments.

• What methodological approaches are most effective for studying proton translocation through the G. sulphuraria ATP synthase c-ring?

  To investigate the unique proton translocation properties of G. sulphuraria ATP synthase c-ring, researchers should consider:

  • Liposome reconstitution with purified c-subunits to create proteoliposomes

  • Fluorescent pH indicators to monitor proton movement across membranes

  • Patch-clamp electrophysiology to measure ion conductance

  • Site-directed mutagenesis of key residues involved in proton binding and release

  • Structural studies (X-ray crystallography or cryo-EM) to identify unique features of the proton channel

  These techniques would help elucidate how G. sulphuraria ATP synthase has adapted to function efficiently in extreme pH environments and potentially identify novel mechanisms of proton translocation.

• How does oxidative stress affect ATP synthase function in G. sulphuraria, and what experimental approaches can address this question?

  G. sulphuraria contains antioxidant compounds that may protect cellular components, including ATP synthase, from oxidative damage. Studies with G. sulphuraria indicate that it can reduce oxidative damage and mitochondrial dysfunction .

  To investigate the relationship between oxidative stress and ATP synthase function:

  • Expose G. sulphuraria cultures to controlled oxidative stress conditions

  • Isolate thylakoid membranes and measure ATP synthase activity

  • Compare ATP synthesis rates between stressed and unstressed samples

  • Analyze oxidative modifications to the c-subunit using mass spectrometry

  • Measure membrane integrity and proton gradient formation under oxidative stress

  This research could reveal unique adaptations that allow G. sulphuraria ATP synthase to function under conditions that would damage similar proteins in non-extremophilic organisms.

• What are the most effective approaches for reconstituting functional G. sulphuraria ATP synthase complexes for biophysical studies?

  For successful reconstitution of functional ATP synthase complexes:

  Step	Methodology	Considerations for G. sulphuraria
  Expression	Recombinant expression of individual subunits	May require codon optimization and fusion partners
  Purification	Multi-step chromatography	Must maintain native protein structure
  Assembly	In vitro reconstitution of subunits	May require specific lipids and pH conditions
  Validation	ATP synthesis assays	Should test function across pH and temperature ranges
  Biophysical Characterization	Single-molecule techniques, EM, AFM	May reveal unique adaptations to extreme conditions

  Researchers have successfully reconstituted recombinant c-subunits from spinach chloroplast ATP synthase into liposomes, forming structures similar to native oligomeric rings . Similar approaches could be applied to G. sulphuraria, with specific adaptations to account for the extremophilic nature of this organism. The reconstituted complexes would provide valuable systems for studying the unique bioenergetic properties of this extremophile.