Recombinant Pelobacter carbinolicus ATP synthase subunit c 1 (atpE1)

What is the structural composition of Pelobacter carbinolicus ATP synthase subunit c 1?

The ATP synthase subunit c 1 (atpE1) from Pelobacter carbinolicus is a full-length protein of 88 amino acids. Its amino acid sequence is: MDFFTWVIITAGFGMAFGSLGTAIGQGLAVKSALEGVARNPGASGKILTTMMIGLAMVESLAIYVFVVSMIILFANPFKDVVLELVAG . The protein is encoded by the atpE1 gene (locus Pcar_0016) and is part of the F-type ATP synthase complex. As a lipid-binding protein, it plays a crucial role in the membrane-spanning component (F0) of the ATP synthase, which forms the proton channel .

Recombinant forms of this protein are typically produced with various tags (determined during the production process) and are available for research purposes . The protein must be properly stored in Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage .

How does ATP synthase subunit c 1 differ from other ATP synthase components in P. carbinolicus?

P. carbinolicus contains multiple isozymes of ATP synthase with different structural and functional properties. The genome analysis of P. carbinolicus has revealed that it contains at least two distinct ATP synthase subunit c proteins: subunit c 1 (atpE1) and subunit c 2 (atpE2) .

Key differences between these subunits include:

These multiple isozymes of ATP synthase are differentiated and assigned roles according to their structural properties and genomic contexts . The subtle differences in amino acid sequences may reflect adaptations to different metabolic conditions or energy requirements within the bacterium.

What are the optimal conditions for expressing recombinant P. carbinolicus atpE1 in heterologous systems?

When designing experiments to express recombinant P. carbinolicus atpE1, researchers should consider several methodological approaches:

• Expression System Selection: Recombinant atpE1 can be produced in various expression systems including:

  • E. coli (most common and cost-effective)

  • Yeast

  • Baculovirus

  • Mammalian cells

  The choice depends on research goals - E. coli is suitable for basic structural studies, while mammalian systems may provide more relevant post-translational modifications for functional studies.

• Tag Selection: The tag type should be determined based on downstream applications:

  • His-tag for simple purification

  • Avi-tag for biotinylation in vivo (using BirA technology)

  • GST or MBP tags for improved solubility

• Buffer Optimization: For optimal stability, use Tris-based buffer with 50% glycerol .

• Storage Conditions: Store at -20°C for short-term use or -80°C for extended storage. Working aliquots can be stored at 4°C for up to one week .

The experimental design should include appropriate controls and validation steps to confirm protein identity and functionality. For instance, SDS-PAGE should confirm >85% purity , and functional assays may be designed to assess ATP synthase activity in reconstituted systems.

How should researchers design experiments to investigate the function of atpE1 in membrane proton translocation?

Investigating the function of atpE1 in membrane proton translocation requires careful experimental design. A comprehensive methodological approach includes:

• Reconstitution in Liposomes:

  • Prepare liposomes with defined lipid composition

  • Incorporate purified recombinant atpE1 into liposomes

  • Create a proton gradient across the membrane

  • Measure proton translocation using pH-sensitive fluorescent dyes

• Site-Directed Mutagenesis:

  • Identify conserved residues through sequence alignment with other ATP synthase c subunits

  • Generate point mutations of key residues potentially involved in proton translocation

  • Assess the impact of mutations on proton channel function

• Biophysical Characterization:

  • Circular dichroism to assess secondary structure

  • NMR or X-ray crystallography for detailed structural information

  • Molecular dynamics simulations to model proton movement

• Controls and Validations:

  • Use well-characterized ATP synthase inhibitors

  • Compare with other subunit c variants (e.g., atpE2)

  • Include negative controls (liposomes without protein)

Data from these experiments should be organized systematically, as shown in this example table format:

This approach enables comprehensive characterization of atpE1's role in proton translocation and ATP synthesis.

How does atpE1 contribute to the unique metabolic capabilities of P. carbinolicus compared to related Geobacter species?

The contribution of atpE1 to P. carbinolicus metabolism must be understood in the context of the organism's unique ecological niche and metabolic capabilities. Unlike its acetate-oxidizing Geobacter relatives, P. carbinolicus has adapted to grow by fermentation, syntrophic hydrogen/formate transfer, or electron transfer to sulfur from short-chain alcohols .

Methodological approach to investigating this question:

• Comparative Genomics Analysis:

  • Compare the atpE1 sequence and genomic context with related species

  • Identify unique features that might contribute to differential regulation or function

  • Examine synteny and gene neighborhoods

• Metabolic Flux Analysis:

  • Trace ATP production in wildtype vs. atpE1-modified strains

  • Measure ATP/ADP ratios under different growth conditions

  • Quantify hydrogen and formate production in cocultures with methanogens

• Proteomics Integration:

  • Perform 2D-PAGE analysis of P. carbinolicus grown on different substrates

  • Identify differential expression of atpE1 and other ATP synthase components

  • Correlate atpE1 expression with specific metabolic pathways

Research has shown that P. carbinolicus expresses multiple isozymes of ATP synthase that are differentiated and assigned roles according to their structural properties and genomic contexts . These isozymes likely contribute to the organism's ability to adapt to different energy sources and environmental conditions.

The unique features of atpE1 may enable P. carbinolicus to maintain ATP synthesis under low-energy conditions, such as during syntrophic growth with methanogens, where energetic constraints are severe . Studies have shown that during growth on ethanol in coculture with methanogens, P. carbinolicus expresses specific ATP synthase components, suggesting a specialized role in energy conservation during syntrophic metabolism .

What is the role of tungsten in regulating ATP synthase expression and function in P. carbinolicus?

The role of tungsten in regulating ATP synthase in P. carbinolicus represents a complex interaction between metal availability and energy metabolism. Research has revealed:

• Tungsten-Dependent Regulation:

  • Comparative 2D-PAGE of ethanol-grown P. carbinolicus revealed enhanced expression of tungsten-dependent enzymes including components affecting ATP synthase function

  • Tungsten limitation results in slower growth and expression of molybdenum-dependent isoenzymes

• Methodological Investigation Approach:

  • Metal-Limited Cultures: Cultivate P. carbinolicus in defined media with controlled tungsten/molybdenum concentrations

  • Proteome Analysis: Perform quantitative proteomics to track ATP synthase subunit expression under varying metal conditions

  • Enzymatic Assays: Measure ATP synthase activity in membrane preparations from cells grown with different metal availabilities

  • Gene Expression Analysis: Quantify atpE1 transcription under varying metal conditions

• Integrative Model:

  • Tungsten availability appears to influence the expression pattern of ATP synthase components, potentially including differential regulation of atpE1 and atpE2

  • This regulation is likely coordinated with expression of tungsten-dependent acetaldehyde:ferredoxin oxidoreductases and formate dehydrogenase

  • The metabolic implications include adaptation to different electron acceptor conditions

A comprehensive experimental approach would include controlled growth experiments with the following design:

How can researchers resolve contradictory findings about the function of atpE1 across different experimental systems?

When researchers encounter contradictory findings about atpE1 function, a systematic approach to resolving these contradictions is essential:

• Identify Sources of Experimental Variation:

  • Expression systems used (E. coli, yeast, mammalian cells)

  • Buffer compositions and pH conditions

  • Membrane reconstitution methods

  • Presence of associated proteins or subunits

• Statistical Analysis and Reproducibility Assessment:

  • Use meta-analysis techniques to integrate findings across studies

  • Apply statistical tests appropriate for the data type (parametric vs. non-parametric)

  • Assess power calculations to determine if sample sizes were adequate

  • Evaluate reproducibility across independent laboratories

• Controlled Variable Experiments:

  • Design experiments that systematically vary one parameter at a time

  • Include appropriate controls for each variable

  • Document all experimental conditions meticulously

• Multi-method Validation:

  • Use complementary techniques to verify findings (e.g., both in vitro and in vivo approaches)

  • Apply both structural and functional assays

  • Validate with both recombinant and native proteins

When analyzing contradictions in findings about atpE1, consider that the protein may have context-dependent functions. For example, its behavior in isolation may differ from its function within the complete ATP synthase complex . Additionally, P. carbinolicus contains multiple ATP synthase isozymes with potentially overlapping or complementary functions .

A data comparison table can be particularly useful in resolving contradictions:

This approach helps identify patterns and sources of variation that might explain apparently contradictory results.

What methodological approaches should be used to differentiate between the functions of atpE1 and atpE2 in P. carbinolicus?

Differentiating between the functions of atpE1 and atpE2 requires sophisticated methodological approaches that can delineate their specific roles:

• Gene Knockout and Complementation Studies:

  • Generate single and double knockout mutants (ΔatpE1, ΔatpE2, ΔatpE1/ΔatpE2)

  • Complement knockouts with wild-type and chimeric constructs

  • Assess growth phenotypes under various metabolic conditions (fermentation, syntrophic growth, sulfur reduction)

• Condition-Specific Expression Analysis:

  • Use quantitative RT-PCR to measure relative expression of atpE1 and atpE2 under different growth conditions

  • Perform ribosome profiling to assess translation rates

  • Use reporter gene fusions to visualize expression patterns

• Structural-Functional Analysis:

  • Create chimeric proteins with domains swapped between atpE1 and atpE2

  • Perform site-directed mutagenesis of non-conserved residues

  • Use molecular dynamics simulations to predict functional differences

• Protein-Protein Interaction Studies:

  • Identify differential interaction partners using pull-down assays or yeast two-hybrid screens

  • Characterize the composition of ATP synthase complexes containing atpE1 versus atpE2

  • Use crosslinking approaches to capture transient interactions

Data from these studies should be integrated into a comprehensive comparison table:

This integrated approach would provide a comprehensive understanding of the functional differentiation between these highly similar proteins and their contributions to P. carbinolicus metabolism .

What are the best practices for purifying recombinant P. carbinolicus atpE1 while maintaining its native conformation?

Purifying membrane proteins like atpE1 while preserving native conformation requires careful methodology:

• Expression System Selection and Optimization:

  • For structural studies, E. coli expression systems with specialized strains (C41(DE3) or C43(DE3)) designed for membrane proteins are recommended

  • For functional studies requiring post-translational modifications, consider eukaryotic expression systems

  • Use inducible promoters with gentle induction conditions (lower temperature, reduced inducer concentration)

• Membrane Extraction Protocol:

  • Harvest cells in mid-log phase

  • Use gentle lysis methods (osmotic shock or enzymatic methods rather than sonication)

  • Extract membranes with carefully selected detergents:

    • Initial screening of multiple detergents (DDM, LMNG, CHAPS)

    • Optimize detergent concentration to maintain protein-lipid interactions

    • Consider nanodiscs or amphipols for long-term stability

• Purification Strategy:

  • Use affinity chromatography based on the tag selected during cloning

  • Include a size exclusion chromatography step to ensure homogeneity

  • Maintain detergent above critical micelle concentration throughout purification

  • Consider on-column detergent exchange to more stable detergents

• Quality Control Assessments:

  • Circular dichroism to confirm secondary structure

  • Fluorescence spectroscopy to assess tertiary structure

  • Thermal stability assays to optimize buffer conditions

  • Negative-stain electron microscopy to verify protein integrity

Storage recommendations for purified atpE1:

• Store in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for long-term storage

• Avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week

• Consider lyophilization for very long-term storage

What experimental design is most appropriate for studying the interaction between atpE1 and other components of the ATP synthase complex?

Studying protein-protein interactions within the ATP synthase complex requires a multi-faceted experimental design:

• Crosslinking and Co-immunoprecipitation:

  • Use chemical crosslinkers of varying arm lengths to capture direct interactions

  • Apply site-specific photo-crosslinking to map interaction interfaces

  • Perform co-immunoprecipitation with antibodies against atpE1 or other complex components

  • Analyze precipitated complexes with mass spectrometry for comprehensive interaction mapping

• Reconstitution Studies:

  • Purify individual ATP synthase components

  • Perform stepwise reconstitution experiments adding components sequentially

  • Measure ATP synthesis activity at each stage of reconstitution

  • Use fluorescently labeled components to track complex assembly

• Structural Biology Approaches:

  • Apply cryo-electron microscopy to visualize the intact complex

  • Use NMR to study interactions between smaller subcomplexes

  • Employ hydrogen-deuterium exchange mass spectrometry to identify interaction interfaces

  • Develop computational models of the complete ATP synthase structure

• Functional Assay Design:

  • Develop assays that can detect ATP synthesis activity

  • Measure proton translocation in reconstituted systems

  • Assess complex stability under varying conditions

  • Include appropriate controls with known ATP synthase inhibitors

An example experimental design table for interaction studies:

This comprehensive approach would elucidate the structural and functional relationships between atpE1 and other components of the ATP synthase complex in P. carbinolicus .

How might atpE1 be utilized in synthetic biology applications for bioenergy production?

The application of atpE1 in synthetic biology for bioenergy production represents an exciting frontier of research. A methodological framework includes:

This research direction could leverage the unique properties of P. carbinolicus atpE1, which has evolved to function efficiently under anaerobic conditions and in syntrophic relationships , potentially providing advantages for certain bioenergy applications.

What emerging technologies will advance our understanding of ATP synthase subunit interactions in anaerobic bacteria?

Emerging technologies are poised to revolutionize our understanding of ATP synthase subunit interactions in anaerobic bacteria like P. carbinolicus:

• Advanced Structural Biology Techniques:

  • Cryo-electron tomography for visualizing ATP synthases in their native membrane environment

  • Micro-electron diffraction (MicroED) for determining structures of membrane protein microcrystals

  • Time-resolved X-ray free-electron laser (XFEL) crystallography for capturing dynamic states during ATP synthesis

  • Integrative structural biology approaches combining multiple data types for complete complex modeling

• Single-Molecule Technologies:

  • Single-molecule FRET to observe dynamic conformational changes during ATP synthesis

  • Magnetic tweezers to directly measure torque generation in ATP synthase

  • High-speed atomic force microscopy to visualize rotation of ATP synthase in real-time

  • Nanopore-based approaches for studying proton translocation at the single-molecule level

• Genetic and Genomic Tools:

  • CRISPR interference (CRISPRi) for precise control of gene expression in anaerobic bacteria

  • Ribosome profiling to assess translation efficiency of ATP synthase components

  • Single-cell transcriptomics to explore cell-to-cell variability in ATP synthase expression

  • Genomic integration of reporter constructs for in vivo monitoring of protein expression and interaction

• Computational and AI-based Methods:

  • AlphaFold2 and similar tools for accurate protein structure prediction

  • Molecular dynamics simulations with enhanced sampling techniques

  • Machine learning approaches for predicting protein-protein interactions

  • Systems biology modeling of complete ATP synthase function in cellular context

These technologies will enable researchers to address fundamental questions about atpE1 function that were previously inaccessible, including:

• How does the c-ring assemble and maintain stability in the membrane?

• What are the precise dynamics of proton translocation through the Fo complex?

• How do environmental conditions affect ATP synthase complex formation and activity?

• What is the evolutionary trajectory of ATP synthase diversification in anaerobic bacteria?

By integrating these technologies into comprehensive research programs, scientists will develop a more complete understanding of ATP synthase function in P. carbinolicus and related anaerobic bacteria.