Recombinant Prosthecochloris vibrioformis ATP synthase subunit beta (atpD)

What is the basic structure and function of P. vibrioformis ATP synthase subunit beta?

P. vibrioformis ATP synthase subunit beta (atpD) is a crucial component of the F₁F₀-ATP synthase complex in this green sulfur bacterium. The protein consists of 462 amino acids with a sequence that includes key functional domains for nucleotide binding and catalysis. The recombinant form maintains the essential structural features with >85% purity when analyzed by SDS-PAGE . As part of the F₁ sector, this subunit contains the catalytic sites responsible for ATP synthesis and hydrolysis, participating in the conversion of electrochemical energy into chemical energy during photosynthetic processes characteristic of green sulfur bacteria like Prosthecochloris, which performs anoxygenic photosynthesis .

How does P. vibrioformis atpD differ from other bacterial ATP synthase beta subunits?

P. vibrioformis atpD shares conserved functional domains with other bacterial ATP synthase beta subunits but exhibits species-specific variations in certain amino acid residues. Phylogenetic analyses show that proteins from the ATP synthase complex, including the beta subunit, maintain sufficient conservation to be used as reliable phylogenetic markers for green sulfur bacteria (GSB) classification . The protein's sequence in Prosthecochloris reflects its adaptation to the unique environmental niche of this organism, which is capable of anoxygenic photosynthesis and nitrogen fixation . Detailed comparison with other ATP synthase beta subunits from photosynthetic bacteria reveals conservation patterns that correspond to the evolutionary divergence of the four GSB genera: Chlorobium, Chlorobaculum, Prosthecochloris, and the fourth genus .

What storage and handling conditions are recommended for the recombinant protein?

The recombinant Prosthecochloris vibrioformis ATP synthase subunit beta should be stored at -20°C, with extended storage recommended at -20°C or -80°C to maintain protein stability and activity . For protein reconstitution, it is advisable to briefly centrifuge the vial before opening to bring contents to the bottom. The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) before aliquoting for long-term storage . The shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can maintain stability for up to 12 months under the same conditions . Repeated freezing and thawing should be avoided, with working aliquots stored at 4°C for up to one week to preserve structural integrity and functional activity .

What purification strategies can resolve common challenges with recombinant atpD inclusion bodies?

When expressing ATP synthase subunits in heterologous systems, researchers frequently encounter inclusion body formation, as observed with chloroplast ATP synthase beta subunit expression in E. coli . A successful purification strategy involves solubilizing these inclusion bodies with 4 M urea followed by stepwise dialysis to slowly remove the denaturant, which has been shown to restore more than fifty percent of the protein to a functional form with native nucleotide binding properties . For P. vibrioformis atpD specifically, purification protocols should aim to achieve >85% purity as verified by SDS-PAGE . The purification workflow may include:

• Cell lysis under conditions that preserve protein structure

• Inclusion body isolation by centrifugation

• Solubilization using appropriate denaturants (e.g., urea)

• Gradual refolding through controlled dialysis

• Affinity chromatography utilizing the protein's nucleotide binding properties

• Quality assessment using SDS-PAGE and activity assays

This approach maximizes both yield and functional recovery of the recombinant protein.

How can researchers assess the catalytic activity of recombinant P. vibrioformis atpD?

The catalytic activity of recombinant P. vibrioformis ATP synthase subunit beta can be assessed through several complementary approaches. Based on established methodologies for ATP synthase components, researchers can evaluate nucleotide binding properties using fluorescence-based assays, similar to those employed for the E. coli βY331W mutant ATP synthase, which utilized a specific tryptophan residue as a fluorescent probe for catalytic site nucleotide binding . ATP hydrolysis activity can be measured through phosphate release assays or coupled enzyme assays that track ADP production.

For more comprehensive functional characterization, researchers should consider:

• Nucleotide binding affinity measurements using isothermal titration calorimetry

• ATPase activity assays under varying pH and temperature conditions

• Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

• Assessment of proton translocation efficiency when reconstituted in liposomes

• Site-directed mutagenesis of key catalytic residues followed by activity measurements

These approaches provide a multi-dimensional understanding of the protein's functional properties and how they compare to ATP synthase beta subunits from other organisms.

What experimental approaches can determine protein-protein interactions involving P. vibrioformis atpD?

Investigating protein-protein interactions involving P. vibrioformis ATP synthase subunit beta requires multiple complementary techniques to establish biologically relevant associations. Based on the structural organization of ATP synthases and approaches used with related proteins, researchers should consider:

• Co-immunoprecipitation assays using antibodies against atpD or potential interacting partners

• Yeast two-hybrid screening to identify novel interacting proteins

• Chemical cross-linking followed by mass spectrometry to map interaction interfaces

• Surface plasmon resonance to determine binding kinetics and affinities

• Bioluminescence resonance energy transfer (BRET) for monitoring interactions in living cells

• Native gel electrophoresis to visualize intact complexes

These methods can reveal interactions with other ATP synthase subunits and potentially unexpected binding partners within the unique photosynthetic machinery of P. vibrioformis, which performs anoxygenic photosynthesis as a green sulfur bacterium .

What structural determination techniques are most appropriate for P. vibrioformis atpD?

For comprehensive structural characterization of P. vibrioformis ATP synthase subunit beta, researchers should employ a multi-technique approach:

• X-ray crystallography remains the gold standard for high-resolution structural determination, though crystallization of membrane-associated proteins presents challenges

• Cryo-electron microscopy (cryo-EM) has revolutionized structural biology of large complexes and could reveal atpD's position within the intact ATP synthase

• Nuclear magnetic resonance (NMR) spectroscopy for analyzing specific domains or ligand interactions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe conformational dynamics

• Small-angle X-ray scattering (SAXS) for solution-state structural information

Researchers should note that structural analysis might require removal of the tag used during recombinant expression, as the tag type may vary during the manufacturing process . Comparative modeling based on homologous proteins can provide initial structural insights while experimental structures are being determined.

How can advanced spectroscopic methods enhance understanding of P. vibrioformis atpD structure-function relationships?

Advanced spectroscopic methods offer valuable insights into the structure-function relationships of P. vibrioformis ATP synthase subunit beta beyond static structural models. Fluorescence spectroscopy, particularly when combining site-directed mutagenesis to introduce tryptophan residues at strategic positions, can serve as specific probes for nucleotide binding events, similar to the approach used with E. coli βY331W mutant ATP synthase . Circular dichroism spectroscopy provides information about secondary structure content and stability under varying conditions.

Additional advanced spectroscopic approaches include:

• Förster resonance energy transfer (FRET) to measure distances between specific labeled residues

• Electron paramagnetic resonance (EPR) spectroscopy with site-directed spin labeling to track conformational changes

• Infrared spectroscopy to monitor structural changes during catalytic cycles

• Raman spectroscopy for probing local environments around specific amino acids

• Single-molecule FRET to capture conformational dynamics during ATP synthesis/hydrolysis

These methodologies can reveal the molecular mechanisms of catalysis, conformational changes during the rotary motion of ATP synthase, and the specific adaptations in P. vibrioformis that support its unique ecological niche as an anoxygenic photosynthetic bacterium .

How can P. vibrioformis atpD be used in phylogenetic and evolutionary studies of green sulfur bacteria?

P. vibrioformis ATP synthase subunit beta serves as an excellent phylogenetic marker for evolutionary studies of green sulfur bacteria. Research has demonstrated that house-keeping proteins, including ATP synthase components, produce phylogenetic trees congruent with those based on SSU rRNA genes, validating their use in evolutionary analyses . For researchers interested in leveraging atpD for phylogenetic studies, the following methodological approach is recommended:

• Sequence alignment of atpD from multiple green sulfur bacteria species using MUSCLE or CLUSTALW

• Identification of conserved and variable regions to design PCR primers for environmental sampling

• Construction of phylogenetic trees using maximum likelihood, neighbor-joining, or Bayesian methods

• Comparison with trees generated from other markers (e.g., rRNA genes, cytochrome b) to confirm evolutionary patterns

• Analysis of selection pressures on different domains using dN/dS ratios

This approach has successfully supported the division of green sulfur bacteria into four distinct genera: Chlorobium, Chlorobaculum, Prosthecochloris, and a fourth genus, with phylogenies based on ATP synthase components showing congruence with other markers at the genus level .

What insights can comparative analysis of P. vibrioformis atpD with other bacterial ATP synthases provide?

Comparative analysis of P. vibrioformis ATP synthase subunit beta with homologs from diverse bacteria offers insights into both conserved catalytic mechanisms and adaptive variations. The ATP synthase beta subunit contains highly conserved regions essential for nucleotide binding and catalysis, alongside variable regions that reflect adaptation to specific ecological niches and energetic requirements. For green sulfur bacteria like Prosthecochloris, which perform anoxygenic photosynthesis and nitrogen fixation , these adaptations may relate to functioning under low-oxygen conditions.

A systematic comparative approach should include:

• Multiple sequence alignment of atpD sequences from diverse bacterial phyla

• Identification of residues under positive or negative selection pressure

• Mapping of conserved and variable regions onto known structural domains

• Correlation of sequence variations with ecological niches and metabolic strategies

• Functional analysis of key residues through site-directed mutagenesis and activity assays

This comparative approach has revealed evolutionary relationships among photosynthetic bacteria, with ATP synthase components providing consistent phylogenetic signals comparable to those from ribosomal RNA and other house-keeping genes .

How can site-directed mutagenesis of P. vibrioformis atpD advance understanding of ATP synthase catalytic mechanisms?

Site-directed mutagenesis of P. vibrioformis ATP synthase subunit beta provides a powerful approach for dissecting the catalytic mechanism of ATP synthesis and hydrolysis. By strategically modifying specific amino acid residues and assessing the functional consequences, researchers can map the roles of individual residues in substrate binding, catalysis, and conformational changes. A comprehensive mutagenesis strategy should target:

• Residues in the Walker A and B motifs essential for nucleotide binding

• Catalytic arginine finger residues involved in transition state stabilization

• Residues at subunit interfaces that facilitate conformational changes during catalysis

• Residues unique to green sulfur bacteria that may reflect adaptation to anoxygenic photosynthesis

• Introduction of reporter residues (e.g., tryptophan) for spectroscopic studies, similar to the approach used with E. coli βY331W mutant

Each mutant should undergo rigorous functional characterization using nucleotide binding assays, ATPase activity measurements, and structural analyses to determine how specific amino acid substitutions affect catalytic efficiency and mechanism.

What role might P. vibrioformis atpD play in understanding bioenergetic adaptations to extreme environments?

P. vibrioformis ATP synthase subunit beta represents an excellent model for investigating bioenergetic adaptations to specialized ecological niches. As a component of a photosynthetic bacterium capable of anoxygenic photosynthesis and nitrogen fixation , this protein likely possesses structural and functional adaptations that optimize ATP production under the specific energy constraints of its environment. Research in this area should focus on:

• Comparative analysis of ATP synthesis efficiency under varying light conditions, redox states, and pH environments

• Investigation of structural features that may confer stability under the conditions where green sulfur bacteria thrive

• Analysis of regulatory mechanisms that coordinate ATP synthesis with photosynthetic electron transport

• Examination of potential interactions with unique metabolic pathways, such as those involving the novel N-methyl-bacillithiol thiol found in Chlorobiaceae

• Assessment of temperature-dependent activity profiles to understand thermal adaptation

Such research contributes to our broader understanding of how core bioenergetic machinery evolves in response to specific environmental pressures, with potential applications in synthetic biology and bioenergy production systems.

How can researchers overcome protein solubility and stability challenges with recombinant P. vibrioformis atpD?

Recombinant production of ATP synthase components frequently encounters solubility and stability challenges. Based on established approaches with similar proteins, researchers working with P. vibrioformis atpD should consider the following strategies:

• Expression optimization: Modifying induction conditions (temperature, inducer concentration, duration) can significantly impact inclusion body formation. Lower temperatures (15-25°C) and reduced inducer concentrations often promote soluble expression.

• Solubilization and refolding: For inclusion bodies, a controlled solubilization with 4 M urea followed by stepwise dialysis has proven effective for ATP synthase beta subunits, restoring more than 50% of the protein to a functional form .

• Fusion partners: Addition of solubility-enhancing tags such as SUMO, MBP, or TrxA can improve folding and solubility.

• Buffer optimization: Systematic screening of pH, ionic strength, and additives (glycerol, arginine, detergents) can identify conditions that maximize stability.

• Storage considerations: Addition of 5-50% glycerol and storage at -20°C or -80°C extends shelf life, with lyophilized preparations maintaining stability for up to 12 months .

Successful implementation of these approaches should yield protein with >85% purity as assessed by SDS-PAGE and preserved functional properties.

What strategies can address data inconsistencies in functional assays of P. vibrioformis atpD?

When conducting functional assays with recombinant P. vibrioformis ATP synthase subunit beta, researchers may encounter inconsistent results due to various factors. A systematic troubleshooting approach should include:

• Protein quality assessment: Verify protein integrity through SDS-PAGE, native PAGE, and circular dichroism before functional studies. Degradation or improper folding can significantly impact assay reproducibility.

• Assay standardization: Establish rigorous controls including:

  • Positive controls using commercially available ATP synthase

  • Negative controls with heat-denatured protein

  • Internal standards to normalize between experimental batches

• Environmental variables: Systematically evaluate the impact of:

  • pH (test range 6.0-9.0)

  • Temperature (test range 25-50°C)

  • Ionic strength (100-300 mM)

  • Divalent cation concentration (Mg²⁺, Ca²⁺)

• Data analysis approaches: Apply statistical methods to:

  • Identify and exclude outliers

  • Implement curve fitting with appropriate models

  • Conduct replicate experiments to establish variability metrics

• Assay methodology validation: When inconsistencies persist, validate the assay using multiple methodologies (e.g., complement ATPase activity measurements with binding affinity determinations) to triangulate accurate functional parameters.

This comprehensive approach addresses the common sources of variability in functional characterization of recombinant proteins, particularly those from specialized organisms like the green sulfur bacterium Prosthecochloris vibrioformis .