Recombinant Carboxydothermus hydrogenoformans ATP synthase subunit c (atpE)

What is the fundamental structure and function of ATP synthase subunit c in Carboxydothermus hydrogenoformans?

ATP synthase subunit c (atpE) in C. hydrogenoformans is a critical component of the F1F0-ATP synthase complex. Similar to other bacterial systems, it likely forms a cylindrical oligomer within the membrane domain of ATP synthase. This oligomeric structure, often composed of 10-15 monomers (similar to the c10 oligomer observed in other species), creates a ring that rotates during ATP synthesis .

The function of subunit c involves direct participation in proton pumping across the membrane, working in conjunction with subunit a. This process couples the proton gradient generated by the respiratory chain to ATP synthesis. The protein contains a conserved carboxyl group that is essential for proton translocation during the rotation of the c-ring .

How does C. hydrogenoformans ATP synthase subunit c compare with homologs from other extremophilic bacteria?

While the search results don't provide direct comparative data, we can extrapolate based on general knowledge of extremophilic ATP synthases:

C. hydrogenoformans ATP synthase subunit c likely possesses adaptations that allow it to function optimally under the organism's growth conditions (temperatures up to 78°C, anaerobic environments rich in carbon monoxide). These adaptations may include:

• Enhanced thermostability through increased hydrophobic interactions, salt bridges, and compact packing

• Structural modifications that maintain proton translocation efficiency at high temperatures

• Potential adaptations to maintain functional coupling with other ATP synthase subunits under extreme conditions

Compared to mesophilic bacteria, the C. hydrogenoformans atpE may show higher intrinsic stability when expressed recombinantly, potentially making it valuable for structural studies and biotechnological applications requiring thermostable components.

What are the optimal expression systems for producing recombinant C. hydrogenoformans ATP synthase subunit c?

The optimal expression system for recombinant C. hydrogenoformans ATP synthase subunit c would need to address several challenges:

• Membrane protein expression: As a membrane protein, atpE requires specialized expression systems that can properly insert the protein into membranes.

• Thermophilic origin: The protein originates from a thermophile, which may affect folding when expressed in mesophilic hosts.

• Potential toxicity: Overexpression of membrane proteins can be toxic to host cells by disrupting membrane integrity.

Based on these considerations, several expression systems could be suitable:

E. coli-based systems:

• C41(DE3) or C43(DE3) strains specifically engineered for membrane protein expression

• Tunable expression systems with weak promoters to prevent toxicity

• Fusion with solubility-enhancing tags (MBP, SUMO) for initial purification

Alternative host systems:

• Bacillus subtilis for gram-positive expression

• Thermophilic expression hosts (e.g., Thermus thermophilus) for expression at elevated temperatures

For functional studies, co-expression with other ATP synthase subunits might be necessary to achieve proper assembly of the c-ring or larger ATP synthase subcomplexes.

The expression constructs should carefully consider inclusion or exclusion of any N-terminal signal sequences, as these could affect membrane insertion in heterologous hosts .

How can elementary flux mode analysis be applied to understand the role of ATP synthase in the carbon metabolism of C. hydrogenoformans?

Elementary flux mode analysis (EFMA) can be a powerful approach to understand the role of ATP synthase in C. hydrogenoformans metabolism:

• Model integration: ATP synthase reactions should be incorporated into the stoichiometric model of C. hydrogenoformans metabolism, particularly connecting it to the acetyl-CoA pathway and hydrogen production pathways .

• Software implementation: Tools like CellNetAnalyzer with MATLAB can be used to perform the analysis, as demonstrated for other C. hydrogenoformans pathways .

• Flux optimization: Linear programming (LP) can determine the optimal flux distributions that maximize ATP production under different substrate conditions.

The methodology would include:

Such analysis could reveal how ATP synthase activity influences the theoretical maximum hydrogen yield of 47.62 mmol/gCDW/h reported for C. hydrogenoformans under optimal conditions .

What are the potential effects of genetic modifications to atpE on hydrogen production in C. hydrogenoformans?

Genetic modifications of the ATP synthase subunit c could significantly impact hydrogen production in C. hydrogenoformans through several mechanisms:

• Efficiency of energy coupling: Mutations affecting the proton-binding site of subunit c could alter the stoichiometry of protons required for ATP synthesis, potentially redirecting more protons toward hydrogen production .

• Thermostability modifications: Enhancing the thermostability of ATP synthase through atpE modifications could improve hydrogen production at elevated temperatures by maintaining efficient energy conversion.

• Regulatory effects: Modifications to atpE expression levels could alter the balance between energy conservation (ATP synthesis) and hydrogen production.

Potential genetic modification approaches include:

• Site-directed mutagenesis of conserved residues involved in proton translocation

• Modifications to optimize c-ring assembly and interaction with other subunits

• Promoter engineering to regulate expression levels of atpE relative to other metabolic enzymes

These modifications should be evaluated within the context of the entire metabolic network. Elementary flux mode analysis could predict the effects of such modifications before experimental implementation . The gene knockout studies performed for other metabolic genes in C. hydrogenoformans provide a methodological framework for similar studies with atpE .

What are the recommended protocols for purification and functional characterization of recombinant C. hydrogenoformans ATP synthase subunit c?

Purification protocol:

• Extraction from membranes:

  • Solubilize membranes using mild detergents (DDM, LDAO, or C12E8)

  • Alternatively, extract using organic solvents (chloroform/methanol mixture) for c-subunit specifically

• Chromatographic purification:

  • Ni-NTA affinity chromatography if His-tagged

  • Size exclusion chromatography to separate monomeric from oligomeric forms

  • Ion exchange chromatography for final polishing

• Oligomer preparation:

  • Reconstitution of purified monomers into liposomes using defined lipid compositions

  • Dialysis-mediated detergent removal to promote native oligomer formation

Functional characterization methods:

• Proton translocation assays:

  • Reconstitution into liposomes with pH-sensitive fluorescent dyes

  • Measurement of proton transport upon establishment of membrane potential

• ATP synthesis activity:

  • Co-reconstitution with essential ATP synthase subunits

  • Measurement of ATP synthesis coupled to artificial proton gradient

  • Adaptation of established cytochrome c oxidase-dependent ATP synthesis assays

• Structural characterization:

  • Native gel electrophoresis to assess oligomeric state

  • Mass spectrometry to confirm molecular weight and post-translational modifications

  • Circular dichroism to evaluate secondary structure and thermal stability

For thermostability assessment, all functional assays should be performed across a temperature range (25-80°C) to determine temperature optima and stability profiles relevant to the thermophilic nature of C. hydrogenoformans.

How can researchers effectively measure ATP synthase activity in the context of hydrogen production by C. hydrogenoformans?

Measuring ATP synthase activity in relation to hydrogen production requires specialized methodologies that link these two processes:

• In vivo measurements:

  • Simultaneous monitoring of intracellular ATP levels (using luciferase-based assays) and hydrogen production rates (using gas chromatography)

  • Correlation analysis between ATP synthesis rates and hydrogen evolution under varying conditions

  • Inhibitor studies using specific ATP synthase inhibitors (oligomycin, DCCD) to determine the relationship between ATP synthesis inhibition and hydrogen production

• In vitro coupled assays:

  • Development of reconstituted systems containing both hydrogenase and ATP synthase components

  • Measurement of ATP synthesis driven by hydrogen oxidation or hydrogen production coupled to ATP hydrolysis

  • Use of Clark-type electrodes to measure oxygen consumption as an indirect measure of electron transfer activities

• Metabolic flux analysis:

  • Application of isotope labeling (13C-labeled CO or pyruvate) to trace carbon flow

  • Integration of ATP synthesis rates into elementary flux mode analysis models

  • Quantification of flux distributions under different growth conditions

The optimal experimental design would include controls to distinguish ATP produced via substrate-level phosphorylation from that produced by ATP synthase, possibly by using specific inhibitors or genetic variants lacking functional ATP synthase.

What structural analysis techniques are most informative for studying C. hydrogenoformans ATP synthase subunit c oligomers?

Several complementary structural techniques can provide insights into the structure and dynamics of C. hydrogenoformans ATP synthase subunit c oligomers:

• Cryo-electron microscopy (cryo-EM):

  • Particularly suitable for membrane protein complexes

  • Can resolve c-ring structure and interactions with other ATP synthase subunits

  • Sample preparation in nanodiscs or amphipols can maintain native-like environment

• X-ray crystallography:

  • If diffraction-quality crystals can be obtained, provides high-resolution structural details

  • Particularly informative for the c-ring in detergent or lipidic cubic phase

  • Has been successfully applied to c-rings from other species

• Solid-state nuclear magnetic resonance (ssNMR):

  • Provides atomic-level insights into structure and dynamics

  • Can be performed in lipid bilayers, maintaining a native-like environment

  • Particularly informative for proton binding sites and conformational changes

• Mass spectrometry approaches:

  • Native mass spectrometry to determine oligomeric state

  • Hydrogen-deuterium exchange to probe dynamics and accessibility

  • Cross-linking mass spectrometry to map interactions with other subunits

• Molecular dynamics simulations:

  • Complement experimental data to understand dynamics at atomic resolution

  • Particularly valuable for modeling proton translocation mechanisms

  • Can predict effects of temperature on structure and function

For thermophilic proteins like C. hydrogenoformans atpE, structural studies should ideally be performed at physiologically relevant temperatures (70-78°C) when the techniques allow, or complemented with studies of temperature effects on structure.

How should researchers address contradictory results in ATP synthase activity measurements?

When facing contradictory results in ATP synthase activity measurements, researchers should implement a systematic approach to resolve inconsistencies:

• Methodological validation:

  • Verify assay conditions are appropriate for a thermophilic enzyme

  • Ensure all reagents are functional and not degraded

  • Test known ATP synthase samples as positive controls

• Context-dependent variation analysis:

  • As noted in result #4 regarding focus group data analysis, apparent contradictions may arise because "the context has changed"

  • Document all experimental conditions precisely

  • Determine if contradictions appear under specific conditions that might reveal regulatory mechanisms

• Multiple analytical approaches:

  • Apply different, complementary methods to measure the same parameter

  • For example, ATP synthesis can be measured by luciferase assay, HPLC, or 31P-NMR

  • Correlation between methods can reveal systematic errors

• Statistical robustness assessment:

  • Perform sufficient biological and technical replicates

  • Apply appropriate statistical tests to determine significance of differences

  • Consider using content analysis approaches as described in result #4 to identify patterns in complex datasets

• Integrated data analysis:

  • Combine direct activity measurements with other data types (structural, genetic, metabolic)

  • Use mathematical modeling to reconcile apparently contradictory results

  • Consider whether contradictions might reveal novel regulatory mechanisms

The key principle is to distinguish between true biological variation and technical artifacts, recognizing that ATP synthase activity may legitimately vary under different conditions due to regulatory processes or experimental variables.

What computational approaches can be used to predict the impact of atpE mutations on ATP synthase function?

Several computational approaches can predict the functional impact of mutations in C. hydrogenoformans atpE:

These computational predictions should guide experimental design by identifying high-priority mutations for laboratory testing, creating a feedback loop between computational prediction and experimental validation.

How can researchers integrate ATP synthase data with broader metabolic models of C. hydrogenoformans?

Integration of ATP synthase data into broader metabolic models requires several methodological steps:

• Stoichiometric model development:

  • Incorporate ATP synthase reactions with correct stoichiometry

  • Link ATP synthesis to proton motive force generation

  • Connect to acetyl-CoA pathway and hydrogen production pathways

• Constraint-based modeling approaches:

  • Apply flux balance analysis to predict optimal metabolic states

  • Use experimentally measured ATP synthesis rates as constraints

  • Perform elementary flux mode analysis as described in results #2 and #3

• Kinetic model integration:

  • Develop kinetic models of ATP synthase activity

  • Integrate with kinetic models of connected pathways

  • Simulate dynamic responses to changing conditions

• Multi-omics data integration:

  • Correlate ATP synthase activity with transcriptomic, proteomic, and metabolomic data

  • Identify regulatory relationships affecting ATP synthase expression and activity

  • Build gene regulatory networks including atpE and related genes

The integration methodology should follow similar approaches to those used for acetyl-CoA pathway analysis in C. hydrogenoformans :

• Collect reaction stoichiometry from databases and literature

• Fill gaps based on homology and context analysis

• Define compartments and boundary conditions

• Apply appropriate software tools (CellNetAnalyzer, MATLAB, etc.)

• Validate model predictions against experimental data

This integrated approach allows researchers to understand ATP synthase function not in isolation but as part of the complex metabolic network that enables C. hydrogenoformans to convert carbon monoxide to hydrogen with high efficiency.

What are the most promising research directions for C. hydrogenoformans ATP synthase subunit c studies?

The most promising research directions for C. hydrogenoformans ATP synthase subunit c include:

• Structure-function relationships under extreme conditions:

  • High-resolution structural studies of the c-ring at elevated temperatures

  • Identification of thermostability determinants that could be transferred to other systems

  • Characterization of proton binding and release mechanisms under thermophilic conditions

• Synthetic biology applications:

  • Engineering C. hydrogenoformans atpE for enhanced thermostability or altered ion specificity

  • Creation of chimeric ATP synthases combining features from different extremophiles

  • Development of minimal ATP synthase systems for bioenergetic applications

• Integration with hydrogen production optimization:

  • Engineering ATP synthase to alter the energy conservation/hydrogen production balance

  • Optimization of electron flux distribution between ATP synthesis and hydrogen production

  • Development of strains with modified ATP synthases for enhanced hydrogen yields beyond the currently predicted maximum

• Comparative studies across extremophiles:

  • Systematic comparison of ATP synthase c-subunits across thermophilic, acidophilic, and alkaliphilic organisms

  • Identification of convergent adaptations to extreme environments

  • Evolutionary analysis of ATP synthase adaptation mechanisms