Recombinant Desulfobacterium autotrophicum ATP synthase subunit delta (atpH)

What is the role of ATP synthase subunit delta (atpH) in Desulfobacterium autotrophicum?

ATP synthase subunit delta (atpH) is a key component of the F1F0 ATP synthase complex in Desulfobacterium autotrophicum. This protein forms part of the F1 region of the ATP synthase and plays a crucial role in energy conservation during sulfidogenesis. The F1F0 ATP synthase produces ATP from ADP in the presence of a proton or sodium gradient across the membrane. In Db. autotrophicum, the ATP synthase is critical for energy generation in sulfate-reducing conditions . The delta subunit specifically helps connect the membrane-embedded F0 domain to the catalytic F1 domain, contributing to the structural stability of the complex while enabling the rotary mechanism necessary for ATP synthesis.

How does the structure of atpH compare between Desulfobacterium autotrophicum and other bacteria?

The ATP synthase subunit delta in Db. autotrophicum consists of 178 amino acids with a sequence that exhibits specific adaptations to the organism's environmental niche. When comparing the sequence of Db. autotrophicum atpH (P41011) with that of other bacteria like E. coli (P0ABA5), there are notable differences in amino acid composition that may reflect adaptations to different energy metabolism pathways. For example, the Db. autotrophicum sequence begins with "MNQEVIAKRYASALFQIALEQGQLDRIEED..." while the E. coli sequence starts with "MSEFITVARPYAKAAFDFAVEHQSVERWQD..." . These differences likely contribute to the specific structural and functional properties of the protein in sulfate-reducing bacteria versus facultative anaerobes.

What are the expression regions and properties of recombinant atpH?

The recombinant Desulfobacterium autotrophicum atpH protein has the following properties:

• Full-length protein spanning amino acids 1-178

• Molecular weight of approximately 20-22 kDa

• Purity >85% as determined by SDS-PAGE

• Typically produced in yeast expression systems

• Stable in Tris-based buffer with 50% glycerol

• Optimal storage at -20°C to -80°C with shelf life of 6-12 months depending on form (liquid vs. lyophilized)

The protein may be tagged for purification purposes, though the tag type varies depending on the manufacturing process. The recombinant protein maintains the structural features necessary for functional studies related to ATP synthase activity.

What are the optimal conditions for expression and purification of recombinant atpH?

For optimal expression and purification of recombinant Desulfobacterium autotrophicum atpH, researchers should consider the following protocol:

• Expression System Selection:

  • Yeast systems (particularly Pichia pastoris) are preferred for expression of bacterial ATP synthase components due to their ability to handle complex proteins with proper folding

  • E. coli BL21(DE3) can also be used with optimized codon usage

• Expression Conditions:

  • For yeast expression: Cultivation at 28-30°C in buffered minimal medium with methanol induction

  • For E. coli expression: Induction with 0.1-0.5 mM IPTG at OD600 of 0.6-0.8, followed by cultivation at 25-30°C for 4-6 hours

• Purification Strategy:

  • Single-step Ni-affinity chromatography for His-tagged constructs

  • Recommended buffer: 50 mM Tris-HCl pH 7.5-8.0, 150-300 mM NaCl, 5-10% glycerol

  • Gradient elution with imidazole (50-250 mM)

• Storage:

  • Final protein should be stored in Tris-based buffer with 50% glycerol

  • Aliquot and store at -20°C for short-term or -80°C for long-term storage

  • Avoid repeated freeze-thaw cycles

How can I design experiments to study the function of atpH in Desulfobacterium autotrophicum?

To study the function of atpH in Desulfobacterium autotrophicum, you might employ the following experimental approaches:

• In vitro ATP synthesis/hydrolysis assays:

  • Reconstruct the ATP synthase complex with purified subunits

  • Measure ATP synthesis rates in proteoliposomes with established pH/ion gradients

  • Compare wild-type and mutant atpH proteins to determine functional contributions

• Structural studies:

  • Use cryo-EM techniques similar to those applied for other bacterial ATP synthases

  • Employ cross-linking studies to map interaction partners of atpH within the complex

  • Investigate the role of atpH in maintaining structural integrity during rotational catalysis

• Mutagenesis approaches:

  • Create point mutations in key residues predicted to be involved in subunit interactions

  • Design chimeric proteins by swapping domains between atpH from different bacterial species

  • Perform deletion analysis to determine essential regions for function

• Split-plot experimental design for comparative studies:

  • Use a design similar to that described in reference , with replicates organized in a 12 × 7 α-design

  • This allows for multiple experimental factors to be tested simultaneously while controlling for variation

• Energy coupling experiments:

  • Investigate the relationship between sulfate reduction and ATP synthesis

  • Study Na+-dependent processes in relation to ATP synthase function

What analytical methods are most effective for characterizing recombinant atpH?

For comprehensive characterization of recombinant atpH, researchers should employ multiple complementary techniques:

• Biochemical Characterization:

  • SDS-PAGE for purity assessment (>85% is standard)

  • Western blotting with anti-atpH antibodies (1:10,000 dilution recommended)

  • Size exclusion chromatography to confirm monomeric state

  • Circular dichroism (CD) spectroscopy to analyze secondary structure elements

• Functional Analysis:

  • ATPase activity assays (colorimetric phosphate release measurement)

  • Proton translocation assays in reconstituted proteoliposomes

  • Binding affinity measurements for interaction partners (KD determination)

• Structural Analysis:

  • X-ray crystallography or cryo-EM for high-resolution structural information

  • Hydrogen/deuterium exchange mass spectrometry (HDX-MS) for conformational dynamics

  • Limited proteolysis combined with mass spectrometry to identify flexible regions

• Interaction Studies:

  • Pull-down assays with other ATP synthase subunits

  • Surface plasmon resonance (SPR) to measure binding kinetics

  • Native mass spectrometry for intact complex analysis

• Quality Control Metrics:

  • Endotoxin testing for research applications

  • Thermostability assays using differential scanning fluorimetry

  • Long-term stability assessment under various storage conditions

How does ATP synthase from Desulfobacterium autotrophicum compare with other sulfate-reducing bacteria?

The ATP synthase from Desulfobacterium autotrophicum shows distinct features when compared to other sulfate-reducing bacteria (SRB):

The ATP synthase in Db. autotrophicum must specifically be adapted to function in conjunction with the organism's dissimilatory sulfate reduction pathway. While the basic structure of F-type ATP synthases is conserved, the specific sequences and certain functional properties reflect adaptations to the metabolic and environmental niche of this organism .

What evolutionary insights can be gained from studying atpH in Desulfobacterium autotrophicum?

Studying atpH in Desulfobacterium autotrophicum offers several evolutionary insights:

• Adaptation to sulfate-reducing lifestyle:

  • The sequence and structure of atpH reflect adaptations to the energy conservation requirements of dissimilatory sulfate reduction

  • These adaptations may include modifications to optimize ATP synthesis under the specific redox conditions present during sulfate respiration

• Genome evolution and gene organization:

  • The key enzymes of dissimilatory sulfate reduction, including ATP synthase components, are scattered around the chromosome in Db. autotrophicum

  • This scattered arrangement contrasts with the clustered gene organization observed in some uncultured prokaryotes, suggesting evolutionary divergence in gene organization patterns

• Thermoadaptation mechanisms:

  • While Db. autotrophicum is not a thermophile, comparative analysis with thermophilic ATP synthases reveals that increased ionic interactions rather than tighter packing or shorter loops contribute to thermal stability

  • This information provides insights into the evolutionary mechanisms of protein thermostability

• Functional convergence and divergence:

  • The ATP synthase in Db. autotrophicum must interface with a different electron transport chain compared to aerobic organisms

  • Studying these differences can illuminate how similar protein complexes have been adapted to diverse bioenergetic strategies through evolution

• Horizontal gene transfer:

  • The high number of genome plasticity elements (>100 transposon-related genes) in Db. autotrophicum suggests potential horizontal gene transfer events that may have influenced ATP synthase evolution

  • This provides perspective on the mobility of bioenergetic genes across bacterial lineages

How do the structural adaptations of atpH relate to the ecological niche of Desulfobacterium autotrophicum?

The structural adaptations of atpH in Desulfobacterium autotrophicum are closely tied to the organism's ecological niche:

• Anoxic marine sediment adaptations:

  • The ATP synthase must function effectively in the low-energy environment of anoxic marine sediments

  • Sequence features likely optimize proton/ion translocation under the specific pH and salt conditions of these environments

• Integration with sulfate respiration:

  • The structure of atpH facilitates efficient energy conservation during sulfate reduction

  • Specific residues may be adapted to optimize the coupling between proton translocation and ATP synthesis under the redox conditions present during sulfidogenesis

• Na⁺ dependence in marine strains:

  • Marine sulfate-reducing bacteria like Db. autotrophicum show Na⁺-dependent accumulation of sulfate and thiosulfate

  • This Na⁺ dependency may extend to the ATP synthase, with structural adaptations in atpH that facilitate interaction with Na⁺-coupled processes

• Metabolic versatility support:

  • Db. autotrophicum can grow chemolithoautotrophically

  • The ATP synthase must be structurally adapted to function effectively under variable metabolic modes, including both heterotrophic and autotrophic growth conditions

• Tolerance to sulfide:

  • Given that the organism produces sulfide as an end product of metabolism, the ATP synthase components must be structurally resilient to the potential inhibitory effects of this compound

  • Specific amino acid compositions may contribute to sulfide resistance

How can recombinant atpH be used to study bioenergetics in sulfate-reducing bacteria?

Recombinant atpH from Desulfobacterium autotrophicum can serve as a valuable tool for investigating bioenergetic mechanisms in sulfate-reducing bacteria through several advanced applications:

What are the challenges in studying the function of atpH in the context of the complete ATP synthase complex?

Studying atpH function within the complete ATP synthase complex presents several significant challenges:

• Membrane protein complex reconstitution:

  • The ATP synthase is a large, multi-subunit membrane complex

  • Reconstituting the functional complex requires all subunits in the correct stoichiometry

  • The hydrophobic nature of the F₀ domain poses technical difficulties in maintaining structural integrity during purification

• Maintaining native lipid environment:

  • The lipid composition of Db. autotrophicum membranes differs from model organisms

  • ATP synthase function is sensitive to the surrounding lipid environment

  • Reconstructing the appropriate lipid context for functional studies is technically challenging

• Coupling with electron transport components:

  • In vivo, the ATP synthase functions in concert with respiratory complexes

  • Recreating this integrated electron/proton transfer network in vitro is difficult

  • Understanding how components like the Rnf complex and hydrogenases interface with ATP synthase requires specialized approaches

• Measuring rotational dynamics:

  • ATP synthesis involves rotational catalysis

  • Measuring rotational dynamics in real-time requires sophisticated biophysical techniques

  • The specific contribution of atpH to these dynamics is difficult to isolate experimentally

• Anaerobic experimental conditions:

  • As a strict anaerobe, Db. autotrophicum proteins may be oxygen-sensitive

  • Maintaining anaerobic conditions throughout purification and experimentation adds technical complexity

  • Special equipment and protocols are needed to preserve protein function

How might atpH be engineered for biotechnological applications?

Engineering atpH from Desulfobacterium autotrophicum for biotechnological applications presents several intriguing possibilities:

• Biosensor development:

  • Engineered atpH variants could be developed as biosensors for:

    • Monitoring sulfide levels in environmental samples

    • Detecting proton/sodium gradients in cellular systems

    • Assessing ATP synthesis capacity in microbial communities

• Enhanced ATP production systems:

  • Engineering atpH to optimize ATP synthesis under specific conditions could enhance:

    • Biofuel production processes that require ATP

    • Bioremediation applications involving sulfate-reducing bacteria

    • Microbial electrosynthesis systems where energy conversion efficiency is critical

• Protein fusion strategies:

  • Creating fusion proteins with atpH could facilitate:

    • Targeting of enzymes to membrane interfaces

    • Creation of artificial electron transport chains

    • Development of self-assembling protein networks for nanobiotechnology

• Extremophile adaptations:

  • Incorporating structural features from Db. autotrophicum atpH into ATP synthases from other organisms could enhance:

    • Stability under high sulfide conditions

    • Function in low-energy environments

    • Adaptability to varying ionic conditions

• Design of experiments approach:

  • Applying the central composite circumscribed (CCC) design methodology used in recombinant protein production optimization:

    • Identifying optimal expression and purification conditions

    • Systematically exploring protein engineering variables

    • Developing predictive models for protein function

What are common problems encountered when working with recombinant atpH and how can they be addressed?

Researchers working with recombinant Desulfobacterium autotrophicum atpH commonly encounter several challenges:

• Protein solubility issues:

  • Problem: Recombinant atpH may form inclusion bodies during expression

  • Solution: Optimize expression conditions by lowering temperature to 18-25°C, using lower inducer concentrations, or adding solubility-enhancing tags like SUMO or MBP

  • Alternative approach: If inclusion bodies persist, develop refolding protocols using gradual dialysis from denaturing to native conditions

• Protein stability concerns:

  • Problem: atpH may show degradation during purification or storage

  • Solution: Add protease inhibitors throughout purification, optimize buffer conditions (try including 5-10% glycerol), and store at -80°C in single-use aliquots

  • Monitoring approach: Check protein stability using analytical size exclusion chromatography at different time points

• Functional activity loss:

  • Problem: Purified atpH loses ability to interact with other ATP synthase subunits

  • Solution: Maintain native-like conditions during purification, avoid harsh elution conditions, and validate functionality using binding assays

  • Preservation strategy: Consider co-purification with interaction partners to maintain structural integrity

• Protein yield limitations:

  • Problem: Low expression yields of functional protein

  • Solution: Optimize codon usage for expression host, test different promoter systems, and screen various cell lines

  • Scale-up consideration: Implement fed-batch fermentation with optimized media formulations

• Verification challenges:

  • Problem: Difficulty confirming proper folding and functionality

  • Solution: Employ multiple complementary techniques including circular dichroism, limited proteolysis, and interaction studies

  • Activity assessment: Develop in vitro binding assays with other ATP synthase components

How do experimental conditions affect the structure and function of atpH during in vitro studies?

Experimental conditions significantly impact the structure and function of atpH during in vitro studies:

• pH effects:

  • Structural impact: pH extremes can disrupt the native conformation of atpH

  • Functional relevance: ATP synthase normally operates across a pH gradient; maintaining appropriate pH is critical for functional studies

  • Recommendation: Buffer systems should maintain pH 6.5-8.0 for most applications, with careful consideration of the specific question being addressed

• Ionic strength considerations:

  • Observation: As a protein from a marine bacterium, atpH function is influenced by salt concentration

  • Effect on interactions: High or low ionic strength can disrupt interactions with other ATP synthase subunits

  • Optimal conditions: 150-300 mM NaCl is typically suitable, but specific applications may require optimization

• Temperature sensitivity:

  • Stability window: While not from a thermophile, recombinant atpH shows reasonable stability at room temperature for short periods

  • Activity correlation: Temperature affects both binding affinity and catalytic rates in reconstituted systems

  • Experimental design: Include temperature controls and conduct assays at physiologically relevant temperatures (25-30°C)

• Redox environment:

  • Cysteine considerations: Oxidation of cysteine residues can affect protein folding and function

  • Preventive measures: Include reducing agents (0.5-1 mM DTT or 2-5 mM β-mercaptoethanol) in buffers

  • Alternative approach: Consider using degassed buffers for oxygen-sensitive applications

• Detergent effects:

  • Membrane association: When studying atpH in the context of the complete ATP synthase, detergent choice is critical

  • Structural preservation: Mild detergents (DDM, LMNG) better preserve native interactions

  • Concentration importance: Detergent concentrations should be maintained above CMC but minimized to prevent destabilization

• Buffer composition impact:

  • Component considerations: Phosphate buffers should be avoided for ATP hydrolysis studies

  • Stabilizing additives: Glycerol (5-10%) generally improves stability

  • Metal ions: Divalent cations (especially Mg²⁺) are essential for ATP synthase function

What are the technical considerations for designing structure-function studies of atpH?

When designing structure-function studies of Desulfobacterium autotrophicum atpH, researchers should consider these technical aspects:

• Mutagenesis strategy design:

  • Targeted approach: Focus on residues predicted to be at interfaces with other subunits

  • Conservation analysis: Compare sequences across diverse species to identify functionally critical residues

  • Systematic approach: Consider creating an alanine-scanning library covering key regions

  • Controls: Include both positive (known functional) and negative (known non-functional) controls

• Structural analysis techniques:

  • Resolution considerations: Cryo-EM has proven effective for bacterial ATP synthases at resolutions of 3.0-3.2 Å

  • Sample requirements: Protein purity >90% is typically needed for structural studies

  • Complementary approaches: Combine high-resolution techniques with lower-resolution methods like SAXS for comprehensive analysis

  • Dynamic information: Consider HDX-MS to capture conformational dynamics

• Functional assay selection:

  • Direct vs. indirect measurements: Choose between direct (ATP synthesis/hydrolysis) and indirect (binding/conformational) assays

  • Scale considerations: Develop high-throughput screening assays for mutant libraries

  • Sensitivity requirements: Ensure assays can detect subtle functional differences

  • Environmental variables: Control temperature, pH, and ionic conditions precisely

• Interaction mapping approach:

  • Binary interactions: Test direct interactions with neighboring subunits (alpha, gamma)

  • Complex assembly: Assess the role of atpH in complete complex formation

  • Quantitative measurements: Determine binding affinities and kinetic parameters

  • In vitro vs. in vivo: Complement in vitro studies with in vivo approaches when possible

• Experimental design considerations:

  • Statistical power: Design experiments with sufficient replicates for statistical significance

  • Controls: Include appropriate controls for each experimental variable

  • Randomization: Use randomized block designs to minimize systematic errors

  • Factorial approach: Consider design of experiments (DoE) approaches for multi-parameter optimization

• Data integration strategy:

  • Multi-technique correlation: Integrate structural, functional, and computational data

  • Visualization methods: Develop effective ways to visualize structure-function relationships

  • Model building: Use experimental data to build and refine mechanistic models

  • Validation approaches: Implement independent validation experiments for key findings