Recombinant Pelodictyon phaeoclathratiforme ATP synthase subunit delta (atpH)

What is the structural role of the ATP synthase delta subunit in Pelodictyon phaeoclathratiforme?

In green sulfur bacteria like P. phaeoclathratiforme, the ATP synthase architecture has evolved to function optimally under specific environmental conditions, particularly in anoxic zones where these organisms typically inhabit. Unlike the delta subunits in some other bacteria, the P. phaeoclathratiforme delta subunit must maintain structural integrity under alkaline conditions common in stratified water columns.

How does the delta subunit contribute to ATP synthase regulation in photosynthetic bacteria?

The delta subunit plays a regulatory role in ATP synthase activity through its effects on complex stability and conformational changes during catalysis. In photosynthetic bacteria, the delta subunit's interactions with other components of the ATP synthase affect electron transport rates and energy conversion efficiency .

Studies in other photosynthetic organisms have shown that altered abundance of the delta subunit correlates directly with changes in linear electron flow (LEF) and non-photochemical quenching (NPQ). For example, when the delta subunit (AtpD) abundance was increased in rice, it resulted in enhanced ATP synthase activity and stimulated photosynthesis . In P. phaeoclathratiforme, which contains bacteriochlorophyll e and lives in low-light environments, the delta subunit likely plays a pivotal role in optimizing ATP production under energy-limited conditions .

What is known about pH-dependency of ATP synthase function in bacteria similar to P. phaeoclathratiforme?

While specific data for P. phaeoclathratiforme is limited, studies on bacterial ATP synthases have revealed significant pH-dependent activity. For instance, in Caldalkalibacillus thermarum, the binding affinity of ATP to the ε subunit changes 5.9-fold between pH 7.0 (weakest binding) and pH 8.5 (strongest binding) .

For green sulfur bacteria that inhabit different layers of stratified water bodies with varying pH, this pH-sensitivity likely represents an important regulatory mechanism. P. phaeoclathratiforme typically thrives in anoxic, potentially alkaline environments, suggesting its ATP synthase may have evolved specific pH adaptations. The presence of unique histidine residues, which can change protonation states depending on pH, might influence nucleotide binding and enzyme activity, similar to what has been observed in other bacterial ATP synthases .

What are the optimal expression systems for recombinant production of P. phaeoclathratiforme ATP synthase delta subunit?

For recombinant expression of the P. phaeoclathratiforme ATP synthase delta subunit, E. coli-based expression systems have proven most effective, particularly BL21(DE3) or Rosetta strains that can accommodate potential codon bias. The optimal expression protocol typically includes:

The expression of membrane-associated proteins like ATP synthase subunits often benefits from specialized approaches that consider their native environment. For P. phaeoclathratiforme, an anaerobic phototroph, expression under microaerobic conditions may improve protein folding and solubility.

What purification challenges are specific to the recombinant delta subunit from P. phaeoclathratiforme?

Purification of the recombinant delta subunit presents several challenges:

• The protein may form aggregates due to exposed hydrophobic surfaces that normally interface with other ATP synthase subunits.

• The native conformation may depend on interactions with other subunits, making the isolated recombinant protein potentially unstable.

• Contamination with host cell proteins that interact with ATP or other nucleotides can occur.

A multi-step purification strategy is recommended:

• Initial capture using immobilized metal affinity chromatography (IMAC)

• Intermediate purification using ion exchange chromatography

• Polishing step using size exclusion chromatography

Buffer optimization is critical, with the addition of 5-10% glycerol and 1-5 mM β-mercaptoethanol typically improving stability. Since ATP binding to subunits of the ATP synthase complex has been shown to be pH-dependent , maintaining optimal pH during purification (generally pH 7.5-8.0 for green sulfur bacterial proteins) is essential for structural integrity.

How can researchers verify the correct folding and functionality of recombinant P. phaeoclathratiforme delta subunit?

Multiple complementary approaches should be employed to verify proper folding and functionality:

• Circular Dichroism (CD) Spectroscopy: Compare the secondary structure profile with predicted models or known structures of delta subunits from related organisms.

• Thermal Shift Assays: Assess protein stability and potential ligand binding (e.g., using SYPRO Orange and monitoring the melting curve in the presence/absence of ATP).

• Limited Proteolysis: Properly folded proteins typically show distinct, reproducible proteolytic patterns compared to misfolded variants.

• Functional Reconstitution: Assemble the recombinant delta subunit with other ATP synthase components (either native or recombinant) and measure ATP synthesis/hydrolysis activities.

• Binding Studies: Use isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to assess interaction with other ATP synthase subunits and nucleotides across different pH values (pH 7.0-8.5) to determine if the recombinant protein exhibits the expected pH-dependent behavior observed in other bacterial ATP synthases .

How does pH affect the activity and stability of recombinant P. phaeoclathratiforme delta subunit?

Based on studies of other bacterial ATP synthases, the P. phaeoclathratiforme delta subunit likely exhibits pH-dependent activity profiles. The optimal protocol for investigating this includes:

• Prepare the purified recombinant protein in a series of buffers spanning pH 6.5-9.0.

• Assess protein stability at each pH using thermal shift assays or intrinsic fluorescence.

• Measure nucleotide binding affinity using microscale thermophoresis or ITC across the pH range.

Critical findings from similar studies on other bacteria reveal that ATP binding affinity can change up to 5.9-fold between pH 7.0 and 8.5 . For P. phaeoclathratiforme, which inhabits environments where pH gradients exist, the delta subunit may have evolved specific histidine residues that sense pH changes and modulate protein function accordingly.

Researchers should pay particular attention to histidine residues in the protein sequence, as these have been implicated in pH-sensing mechanisms in ATP synthase subunits from other species . Molecular dynamics simulations suggested that protonation states of histidine residues may influence ATP binding site stability, even when these residues lie outside the direct binding pocket.

What methodologies are most effective for studying interactions between the delta subunit and other components of the ATP synthase complex?

Several complementary approaches are recommended:

• Crosslinking Mass Spectrometry (XL-MS): This technique can identify interaction interfaces between the delta subunit and other components by creating covalent bonds between closely positioned residues followed by MS analysis.

• Surface Plasmon Resonance (SPR): Immobilize the delta subunit on a sensor chip and measure real-time binding kinetics with other purified subunits under varying conditions (pH, ion concentration).

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This method reveals regions of the protein that become protected upon complex formation, providing detailed maps of interaction surfaces.

• Cryo-EM Analysis: For structural characterization of reconstituted sub-complexes containing the delta subunit.

• Förster Resonance Energy Transfer (FRET): Fluorescently labeled delta subunit and partner proteins can reveal conformational changes and binding dynamics in real-time.

When applying these methods to P. phaeoclathratiforme delta subunit research, special consideration should be given to the native low-light, anoxic environment of this organism. Interaction studies should ideally include conditions that mimic these natural environments, including appropriate pH, redox potential, and light conditions.

How can researchers assess the impact of mutations in the delta subunit on ATP synthase assembly and function?

A comprehensive mutational analysis should include:

• Site-Directed Mutagenesis: Target conserved residues, potential pH-sensing residues (particularly histidines), and residues at predicted interaction interfaces.

• In vitro Reconstitution Assays: Assess the ability of mutant delta subunits to associate with other ATP synthase components compared to wild-type protein.

• ATP Synthesis/Hydrolysis Measurements: Quantify the functional impact of mutations using reconstituted proteoliposomes.

• Thermal Stability Analysis: Compare melting temperatures of wild-type and mutant proteins to assess structural impacts.

As demonstrated in other bacterial ATP synthases, mutations in "second shell" residues outside the direct binding or interaction sites can have profound effects on function through allosteric mechanisms . For example, in Bacillus subtilis, changing a glutamate to arginine (E102R) resulted in a ~54-fold increase in ATP binding affinity to the ε subunit .

How does the P. phaeoclathratiforme delta subunit differ from those of other photosynthetic bacteria?

The P. phaeoclathratiforme delta subunit likely contains adaptations specific to its ecological niche. Comparative analysis should examine:

• Sequence Conservation: Align delta subunit sequences from diverse photosynthetic bacteria, focusing on green sulfur bacteria, purple bacteria, and cyanobacteria.

• pH Adaptation Signatures: Identify unique residues, particularly histidines, that may facilitate function in P. phaeoclathratiforme's natural alkaline environment.

• Structural Predictions: Compare predicted secondary and tertiary structures to identify conserved structural elements versus species-specific adaptations.

Green sulfur bacteria like P. phaeoclathratiforme contain bacteriochlorophyll e and thrive in deep anoxic layers of stratified water bodies . Their ATP synthase components, including the delta subunit, have likely evolved distinct features to optimize energy conversion under extreme low-light conditions. For instance, their ATP synthase may have adapted to maintain stability and function efficiently at the alkaline pH values often found in their natural habitats.

What insights can be gained by comparing ATP synthase regulation between P. phaeoclathratiforme and plant chloroplast ATP synthases?

This comparative analysis reveals fundamental differences and similarities in ATP synthase regulation across evolutionary diverse photosynthetic systems:

• In chloroplast ATP synthase, the delta subunit (AtpD) forms part of the peripheral stalk alongside subunits b and b', creating a stator that prevents unproductive rotation .

• Studies in plants have shown that altered AtpD abundance directly affects photosynthetic electron transport rates and non-photochemical quenching .

• P. phaeoclathratiforme, as an anoxygenic phototroph, lacks the oxygen-evolving photosystem II but must still coordinate ATP synthesis with electron transport.

Key research questions to explore:

• Does the P. phaeoclathratiforme delta subunit respond to similar regulatory inputs as plant AtpD?

• How do differences in photosynthetic machinery influence ATP synthase regulation?

• What can the comparison reveal about convergent evolution of energy regulation mechanisms?

The unique ecological niche of P. phaeoclathratiforme likely drives specific adaptations in its ATP synthase regulatory mechanisms that differ from those of plants, particularly in response to light intensity, oxygen levels, and pH.

What are the common pitfalls in functional studies of recombinant ATP synthase subunits, and how can they be addressed?

Several challenges frequently arise in ATP synthase subunit research:

• Protein Aggregation: The delta subunit may aggregate when expressed without its normal binding partners.

  • Solution: Co-express with interacting subunits or use solubility-enhancing tags; optimize buffer conditions with detergents or stabilizing agents.

• Loss of Native Conformation: Isolated subunits may not maintain their physiologically relevant conformation.

  • Solution: Validate structural integrity using CD spectroscopy; compare activity with the same subunit in the context of larger complexes.

• Background ATP Hydrolysis/Synthesis Activity: Contaminating ATPases can confound activity measurements.

  • Solution: Include appropriate controls; use specific inhibitors to distinguish ATP synthase activity from contaminants.

• pH-Dependent Effects: Assays performed at non-physiological pH may not reflect native activity.

  • Solution: Characterize activity across a pH range (6.5-9.0) relevant to P. phaeoclathratiforme's natural environment .

• Oxidation Sensitivity: Cysteine residues in the protein may be prone to oxidation during purification.

  • Solution: Maintain reducing conditions throughout purification and storage; consider site-directed mutagenesis of non-essential cysteines.

How can researchers accurately measure the ATP binding affinity of the recombinant delta subunit under various conditions?

For precise measurement of ATP binding to the P. phaeoclathratiforme delta subunit:

• Isothermal Titration Calorimetry (ITC):

  • Directly measures thermodynamic parameters of binding

  • Requires significant amounts of purified protein (typically 50-100 μM)

  • Can distinguish between enthalpy- and entropy-driven interactions

• Microscale Thermophoresis (MST):

  • Requires smaller protein quantities

  • Uses fluorescently labeled protein and measures changes in thermophoretic mobility upon ligand binding

  • Suitable for determining Kd values across different pH conditions

• Fluorescence-Based Methods:

  • Utilize intrinsic tryptophan fluorescence or extrinsic fluorescent ATP analogs

  • Enable real-time monitoring of binding events

  • Can detect conformational changes associated with nucleotide binding

Based on studies with other bacterial ATP synthase subunits, researchers should expect pH-dependent changes in binding affinity, with potential 5-10 fold differences across physiologically relevant pH ranges .

What advanced imaging techniques can reveal the subcellular localization and dynamics of ATP synthase in P. phaeoclathratiforme?

Several cutting-edge imaging approaches can provide insights into ATP synthase localization and dynamics:

• Super-Resolution Microscopy (STORM/PALM):

  • Achieves resolution below the diffraction limit (~20-30 nm)

  • Requires fluorophore-conjugated antibodies against the delta subunit or expression of fluorescent protein fusions

  • Can resolve individual ATP synthase complexes within bacterial membranes

• Cryo-Electron Tomography:

  • Provides 3D structural information in the native cellular context

  • Can visualize ATP synthase arrangement in the bacterial membrane

  • Reveals spatial relationships with other photosynthetic complexes

• Live-Cell Single-Particle Tracking:

  • Monitors movement and dynamics of individual ATP synthase complexes

  • Requires minimally invasive labeling strategies compatible with living cells

  • Can reveal potential spatial and temporal dynamics of ATP synthase

• Spatial Proteomics Combined with Mass Spectrometry:

  • Fractionates cellular compartments followed by MS analysis

  • Provides landscape of protein localization in cells

  • Can identify potential novel localizations of ATP synthase components

For P. phaeoclathratiforme specifically, these techniques may reveal how ATP synthase complexes are arranged in relation to chlorosomes (the light-harvesting structures containing bacteriochlorophyll e) and other components of the photosynthetic apparatus, providing insights into energy transfer and utilization in this specialized phototroph .