Recombinant Dehalococcoides sp. ATP synthase subunit c (atpE)

What is Dehalococcoides sp. ATP synthase subunit c (atpE) and what is its function?

ATP synthase subunit c (atpE) in Dehalococcoides sp. is a critical component of the F0 portion of the F1F0-ATP synthase complex. This protein forms the c-ring in the membrane-embedded sector of ATP synthase, which functions as a proton channel. The rotation of this c-ring, driven by proton translocation across the membrane, is coupled to ATP synthesis in the F1 portion of the complex.

Dehalococcoides ethenogenes 195, one of the few isolated strains that can completely dechlorinate tetrachloroethene (PCE) to ethene, possesses a specialized metabolism that relies on hydrogen as an electron donor and chlorinated compounds as electron acceptors . The ATP synthase in this organism is therefore essential for energy conservation in this unique metabolic pathway.

Methodological approach: To study the function of atpE in Dehalococcoides, researchers should consider:

• Sequence alignment with well-characterized ATP synthase subunit c proteins from model organisms

• Gene expression analysis under various dechlorinating conditions

• Membrane isolation and ATP synthase activity assays

Why is Dehalococcoides sp. ATP synthase of particular interest to researchers?

Dehalococcoides sp. ATP synthase is of significant interest because of the organism's specialized energy metabolism. Unlike most bacteria, Dehalococcoides has a remarkably narrow metabolic repertoire, exclusively using hydrogen as an electron donor and chlorinated compounds as electron acceptors . This metabolic specialization makes its energy conservation systems, including ATP synthase, potentially unique.

The relatively small genome of Dehalococcoides ethenogenes 195 compared to other dehalorespiring bacteria such as Desulfitobacterium hafniense Y51 suggests evolutionary streamlining focused on its specialized niche . Understanding how ATP synthase functions within this constrained metabolic framework provides insights into:

• Adaptation of essential cellular machinery to specialized metabolic pathways

• Minimal requirements for energy conservation in obligate dehalorespirers

• Potential unique structural or functional adaptations of ATP synthase components

What expression systems are most effective for producing recombinant Dehalococcoides sp. ATP synthase subunit c?

Several expression systems can be employed for recombinant production of Dehalococcoides sp. ATP synthase subunit c, each with distinct advantages:

Methodological approach: When selecting an expression system, researchers should:

• Analyze the codon usage in the atpE gene and optimize for the expression host

• Include affinity tags (His6 or Strep-tag) for purification, preferably with a cleavable linker

• Test expression using a reporter fusion (such as GFP) to monitor proper folding

• Evaluate expression in membrane fractions using Western blotting

Recombinant protein production facilitates a scalable and reliable source of proteins that would otherwise be scarce when extracted from natural sources . This is particularly important for Dehalococcoides sp. proteins due to the challenging cultivation requirements of this organism.

How can experimental design be optimized for functional studies of recombinant Dehalococcoides sp. ATP synthase subunit c?

Designing robust experiments for functional studies of recombinant Dehalococcoides sp. ATP synthase subunit c requires careful consideration of multiple variables:

Independent Variables to Consider:

• Expression conditions (temperature, induction time, inducer concentration)

• Detergent types for membrane protein extraction

• Reconstitution lipid composition

• Buffer pH and ionic strength

• Presence of specific ions (Mg²⁺, Ca²⁺)

Dependent Variables to Measure:

• Protein yield and purity

• Proper folding (circular dichroism analysis)

• Oligomerization state (native PAGE, size exclusion chromatography)

• Proton translocation activity

• ATP synthesis/hydrolysis rates

A true experimental design should incorporate randomization and proper controls . For example, when testing different detergents for optimal extraction of functional ATP synthase subunit c:

• Randomly assign cultures to different detergent treatment groups

• Include positive controls (detergents known to work for similar membrane proteins)

• Implement negative controls (no detergent or detergents known to denature membrane proteins)

• Perform replicate experiments to ensure statistical validity

This approach helps isolate the effect of detergent choice from other variables that might influence protein functionality .

What structural analysis techniques provide the most valuable insights into Dehalococcoides sp. ATP synthase subunit c?

Multiple complementary structural analysis techniques should be employed to comprehensively characterize Dehalococcoides sp. ATP synthase subunit c:

Methodological approach:

• Begin with homology modeling based on closely related bacterial ATP synthase subunit c structures

• Perform circular dichroism spectroscopy to confirm secondary structure content

• Use cross-linking mass spectrometry to validate predicted subunit interactions

• For high-resolution structures, pursue either X-ray crystallography or cryo-EM depending on protein stability and homogeneity

• Validate structural findings with functional assays

How can researchers address data inconsistencies in ATP synthase activity measurements from Dehalococcoides sp.?

Inconsistencies in ATP synthase activity measurements from Dehalococcoides sp. often stem from multiple experimental variables. A systematic approach to resolving these contradictions includes:

Sources of Data Inconsistency:

• Protein denaturation during purification

• Incomplete reconstitution of multisubunit complexes

• Variation in lipid composition affecting activity

• Experimental conditions not mimicking the anaerobic environment of Dehalococcoides

• Contaminating ATPase activities

Resolution Strategy:

• Standardize purification protocols: Document detergent types, concentrations, and exposure times. Compare activity measurements across different purification methods to identify optimal conditions.

• Implement multiple activity assays:

• Control for contaminating activities: Include specific inhibitors:

  • N,N'-dicyclohexylcarbodiimide (DCCD) for F-type ATP synthases

  • Oligomycin as a secondary control

  • Compare activity with and without inhibitors to quantify specific contribution

• Ensure complete reconstitution: Use analytical techniques (BN-PAGE, electron microscopy) to verify intact complex assembly before activity measurements.

• Reproduce native conditions: Maintain strict anaerobic conditions during purification and activity measurements, as exposure to oxygen may alter protein conformation and activity in this strictly anaerobic organism.

What are the most effective approaches for studying the interaction between recombinant Dehalococcoides sp. ATP synthase subunit c and electron transport chain components?

Studying interactions between recombinant Dehalococcoides sp. ATP synthase and electron transport chain (ETC) components requires specialized techniques due to the membrane-embedded nature of these complexes and the strict anaerobic requirements of Dehalococcoides.

Recommended Approaches:

• In vitro reconstitution systems:

  • Co-reconstitute purified ATP synthase and ETC components into liposomes

  • Measure coupled electron transport and ATP synthesis

  • Vary component ratios to determine optimal stoichiometry

• Protein-protein interaction studies:

  • Chemical cross-linking followed by mass spectrometry

  • Surface plasmon resonance with immobilized components

  • Förster resonance energy transfer (FRET) with fluorescently labeled subunits

• Respiratory chain super-complex analysis:

  • Blue native PAGE to isolate intact super-complexes

  • Proteomic analysis of isolated complexes

  • Activity measurements of isolated super-complexes

Experimental Design Considerations:

• Maintain strict anaerobic conditions throughout all procedures

• Use native lipids extracted from Dehalococcoides when possible

• Include appropriate electron donors (H₂) and acceptors (chlorinated compounds)

• Control for non-specific interactions with appropriate negative controls

This integrated approach allows researchers to determine whether Dehalococcoides sp. ATP synthase forms super-complexes with ETC components, which could explain the efficient energy conservation in this metabolically specialized organism.

What purification strategies yield the highest quality recombinant Dehalococcoides sp. ATP synthase subunit c?

Purification of recombinant membrane proteins like ATP synthase subunit c presents significant challenges related to maintaining native structure and function. For Dehalococcoides sp. ATP synthase subunit c, the following purification workflow has proven effective:

• Membrane isolation and solubilization:

  • Lyse cells using French press or sonication

  • Isolate membranes by ultracentrifugation

  • Solubilize using mild detergents (n-dodecyl-β-D-maltoside or digitonin)

• Affinity chromatography:

  • Utilize His-tag affinity in immobilized metal affinity chromatography (IMAC)

  • Include low concentrations of detergent in all buffers

  • Elute with imidazole gradient to minimize protein denaturation

• Size exclusion chromatography:

  • Separate oligomeric states

  • Remove aggregates

  • Buffer exchange to remove imidazole

Quality assessment should include:

• SDS-PAGE for purity

• Western blotting for identity confirmation

• Circular dichroism for secondary structure integrity

• Mass spectrometry for accurate mass determination

These steps address many of the challenges in producing high-quality recombinant proteins, including proper folding, stability, and avoiding contamination .

How can site-directed mutagenesis be applied to study functional residues in Dehalococcoides sp. ATP synthase subunit c?

Site-directed mutagenesis provides powerful insights into structure-function relationships of ATP synthase subunit c. For Dehalococcoides sp. atpE, a systematic mutagenesis approach should focus on:

Priority Target Residues:

• The conserved carboxylic acid residue (typically Asp or Glu) in the ion-binding site

• Residues lining the proton translocation pathway

• Residues at subunit-subunit interfaces

• Residues at the interface with F₁ subunits

Mutagenesis Strategy:

• Generate a library of single amino acid substitutions:

  • Conservative substitutions (maintaining similar chemical properties)

  • Non-conservative substitutions (altering chemical properties)

  • Alanine scanning of targeted regions

• Functional analysis of mutants:

  • Expression and assembly assessment

  • ATP hydrolysis/synthesis activity

  • Proton translocation efficiency

  • Oligomeric state analysis

Data Analysis and Interpretation:

• Compare activity parameters (Vmax, Km) between wild-type and mutants

• Correlate activity changes with structural position

• Generate structure-function maps

Example Data Table - Effects of Key Mutations:

Note: This table presents hypothetical data based on similar studies in other ATP synthases

This systematic approach allows researchers to determine which residues are essential for function and which may be targets for engineering enhanced properties.

How can recombinant Dehalococcoides sp. ATP synthase subunit c contribute to bioremediation research?

Recombinant Dehalococcoides sp. ATP synthase subunit c has significant potential applications in bioremediation research, particularly for chlorinated solvent contamination:

• Energy metabolism engineering:

  • Understanding ATP synthase function could enable engineering of Dehalococcoides strains with enhanced energy efficiency

  • Improved growth rates would accelerate bioremediation processes

  • Engineered strains may function better in suboptimal field conditions

• Biomarker development:

  • ATP synthase subunit c expression levels could serve as a metabolic activity indicator

  • Monitoring atpE transcripts can help assess Dehalococcoides activity in contaminated sites

  • Antibodies against the recombinant protein could be used for in situ detection

• Synthetic biology approaches:

  • Recombinant expression of Dehalococcoides ATP synthase in more robust bacterial hosts

  • Creation of chimeric ATP synthases with enhanced stability

  • Development of cell-free systems incorporating recombinant ATP synthase and dehalogenase components

Dehalococcoides ethenogenes 195 is particularly valuable in bioremediation as it can completely dechlorinate tetrachloroethene (PCE) to non-toxic ethene, while having a highly specialized metabolism limited to using hydrogen as an electron donor and chlorinated compounds as electron acceptors . Engineering efforts focused on its energy conservation machinery could significantly enhance its bioremediation capabilities.

What future research directions might yield breakthrough insights in understanding Dehalococcoides sp. ATP synthase?

Several promising research directions could substantially advance our understanding of Dehalococcoides sp. ATP synthase:

• Single-molecule studies:

  • Real-time observation of c-ring rotation

  • Force measurements during ATP synthesis/hydrolysis

  • Conformational dynamics under varying conditions

• Cryo-electron tomography:

  • Visualization of ATP synthase in native membrane environment

  • Determination of supramolecular organization

  • Potential discovery of unique structural features

• Systems biology approaches:

  • Integration of ATP synthase function with whole-cell metabolic models

  • Multi-omics analysis correlating ATP synthase expression with dechlorination activity

  • Flux balance analysis to quantify energy conservation efficiency

• Comparative analysis across Dehalococcoides strains:

  • Correlation of ATP synthase sequence variations with dechlorination capabilities

  • Identification of strain-specific adaptations in energy conservation

  • Potential discovery of environmental adaptation mechanisms

• Synthetic biology applications:

  • Engineering minimal ATP synthase systems based on Dehalococcoides design principles

  • Creating hybrid systems with enhanced efficiency or stability

  • Developing biosensors based on ATP synthase activity

These research directions combine cutting-edge technological approaches with fundamental questions about energy conservation in specialized bacteria, potentially yielding insights that extend well beyond Dehalococcoides to our broader understanding of bioenergetics and membrane protein function.