Recombinant Geobacter sp. ATP synthase subunit c (atpE)

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

ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex in Geobacter species. This protein forms a cylindrical oligomeric ring (c-ring) embedded in the membrane that functions as a proton channel. The rotation of this c-ring, driven by proton translocation across an electrochemical gradient, is mechanically coupled to ATP synthesis in the F1 region of the complex .

The specific function of subunit c involves:

• Formation of the membrane-embedded proton channel (F0 sector)

• Participation in the mechanical rotation that drives ATP synthesis

• Coupling proton movement across membranes to the catalysis of ATP formation

ATP synthase subunit c is also known by several alternative names: ATP synthase F(0) sector subunit c, F-type ATPase subunit c (F-ATPase subunit c), and lipid-binding protein .

How does the expression system affect the properties of recombinant Geobacter sp. atpE protein?

The expression system significantly impacts the properties, yield, and functionality of recombinant Geobacter sp. atpE protein. The choice of host organism, expression vector, and culture conditions all contribute to the final protein characteristics.

When expressed in E. coli, as commonly done, the recombinant protein may be fused with affinity tags (such as His-tag) to facilitate purification . The expression system must be optimized to ensure:

• Proper folding of the protein

• Adequate yield for experimental purposes

• Retention of native-like structure and function

• Minimization of inclusion body formation

For optimal expression in E. coli systems, codon optimization of the synthetic gene may be necessary to accommodate codon usage preferences, as has been reported for other ATP synthase subunits . Additionally, the expression of membrane proteins like atpE often requires specialized strains and careful optimization of induction conditions to avoid toxicity to the host cells.

What are the optimal purification strategies for recombinant Geobacter sp. atpE protein?

Purification of recombinant Geobacter sp. atpE requires a specialized approach due to its hydrophobic nature and membrane association. Based on established protocols for similar proteins, the following methodological approach is recommended:

Step-by-step purification protocol:

• Cell lysis and membrane fraction isolation:

  • Harvest cells and resuspend in lysis buffer (typically 20 mM Tris-HCl pH 8.0 with protease inhibitors)

  • Add lysozyme (1 mg/mL) and incubate at 4°C for approximately 1.5 hours

  • Sonicate at 50-75 W to complete cell disruption

  • Isolate membrane fractions by ultracentrifugation

• Detergent solubilization:

  • Solubilize membrane fractions using appropriate detergents (e.g., n-dodecyl β-D-maltoside)

  • Optimize detergent concentration to maximize protein extraction while maintaining protein structure

• Affinity chromatography:

  • For His-tagged constructs, use immobilized metal affinity chromatography (IMAC)

  • Apply solubilized protein to Ni-NTA or similar resin

  • Wash with buffers containing low imidazole concentrations

  • Elute with buffer containing higher imidazole concentration (typically 250-500 mM)

• Size exclusion chromatography:

  • Further purify by gel filtration to separate monomeric protein from aggregates

  • Analyze fractions by SDS-PAGE to confirm purity

• Quality control:

  • Verify protein purity (>90%) by SDS-PAGE

  • Confirm identity via immunoblotting using anti-ATP synthase subunit c antibodies

  • Assess secondary structure by circular dichroism spectroscopy to confirm α-helical content

The purified protein should be stored according to established guidelines, typically in a Tris-based buffer with 50% glycerol at -20°C or -80°C to maintain stability .

How can researchers assess the functional activity of purified recombinant Geobacter sp. atpE?

• Reconstitution into liposomes:

  • Incorporate purified atpE into artificial liposomes

  • Assess proton translocation capability using pH-sensitive fluorescent dyes

  • Monitor proton gradient formation across the liposome membrane

• Assembly with other ATP synthase subunits:

  • Combine purified atpE with additional purified subunits to reconstitute partial or complete ATP synthase complexes

  • Evaluate the assembly of c-ring structures using analytical ultracentrifugation or native gel electrophoresis

  • Attempt reconstitution of functional ATP synthase activity by combining all necessary subunits

• Structural characterization:

  • Use circular dichroism spectroscopy to confirm proper α-helical secondary structure

  • Employ analytical techniques such as size exclusion chromatography or dynamic light scattering to assess oligomerization state

  • Apply advanced structural biology methods (X-ray crystallography, cryo-EM) to determine the structural arrangement of assembled c-rings

• Binding assays:

  • Evaluate binding to known interaction partners such as subunit a or the F1 sector components

  • Use surface plasmon resonance or isothermal titration calorimetry to quantify binding affinities

These methodological approaches provide complementary information about the structural integrity and functional potential of the purified recombinant atpE protein.

What factors influence the assembly of the c-ring structure in ATP synthase, and how can this be studied using recombinant Geobacter sp. atpE?

The assembly of the c-ring structure in ATP synthase is influenced by multiple factors that can be systematically investigated using recombinant Geobacter sp. atpE. This represents a fundamental question in understanding ATP synthase stoichiometry and function.

Key factors influencing c-ring assembly:

Research methodology to study c-ring assembly:

• In vitro reconstitution experiments:

  • Purify recombinant atpE protein and systematically vary conditions (detergents, lipids, pH, salts)

  • Monitor assembly using analytical ultracentrifugation, native gel electrophoresis, or electron microscopy

  • Quantify c-ring stoichiometry using mass spectrometry or structural methods

• Cross-linking approaches:

  • Employ chemical cross-linking to stabilize c-ring assemblies

  • Analyze cross-linked products by SDS-PAGE and mass spectrometry

  • Identify specific residues involved in subunit-subunit interactions

• Comparative studies:

  • Compare assembly properties of atpE from different Geobacter species or other organisms

  • Correlate differences in sequence with variations in c-ring stoichiometry

  • Create chimeric proteins to identify domains responsible for specific assembly characteristics

The variable stoichiometry of c-rings across species (c10 to c15) remains a fascinating biological question , and recombinant expression systems provide powerful tools to investigate the underlying determinants of this variation.

How should expression conditions be optimized for maximum yield of functional Geobacter sp. atpE protein?

Optimization of expression conditions is critical for obtaining high yields of functional Geobacter sp. atpE protein. A systematic approach should address multiple parameters:

Expression strain selection:

• BL21(DE3) and derivatives are commonly used for membrane protein expression

• C41(DE3) and C43(DE3) strains, specifically designed for toxic membrane proteins, may improve yields

• Consider Lemo21(DE3) for tunable expression level control

Vector design optimization:

• Incorporate appropriate affinity tags (His-tag commonly used)

• Consider fusion partners (MBP, SUMO) to enhance solubility

• Optimize codon usage for E. coli expression

• Include tightly controlled promoters (T7, araBAD) to prevent leaky expression

Induction protocol optimization matrix:

Methodological approach for optimization:

• Perform small-scale expression tests varying the above parameters

• Analyze protein expression by SDS-PAGE and Western blotting

• Assess protein solubility through fractionation experiments

• Determine protein functionality using structural or functional assays

• Scale up production using optimized conditions

For Geobacter sp. atpE specifically, lower temperatures (16-25°C) after induction and extended expression periods may prove beneficial for proper membrane protein folding. The addition of specific lipids or membrane-stabilizing agents to the culture medium might also enhance functional expression.

What mutagenesis strategies can elucidate structure-function relationships in Geobacter sp. atpE?

Systematic mutagenesis approaches provide powerful tools to investigate structure-function relationships in Geobacter sp. atpE. The following methodological strategies can be employed:

1. Alanine-scanning mutagenesis:

• Systematically replace individual amino acids with alanine

• Focus on the conserved regions and transmembrane segments

• Analyze effects on assembly, stability, and function

• Create a comprehensive functional map of the protein

2. Targeted mutagenesis of key functional residues:

• Identify highly conserved residues across species through sequence alignment

• Focus on the essential proton-binding glutamate/aspartate residue in the middle of the second transmembrane helix

• Investigate residues at subunit interfaces that may influence c-ring stoichiometry

• Explore the role of lipid-binding regions

3. Chimeric protein construction:

• Create fusion proteins between Geobacter sp. atpE and corresponding subunits from organisms with different c-ring stoichiometries

• Swap specific domains to identify regions responsible for assembly determinants

• Analyze how sequence differences correlate with functional variations

4. Cross-species comparative mutagenesis:

• Compare atpE sequences from different Geobacter species and other organisms

• Identify natural sequence variations

• Introduce these variations into the recombinant protein to assess functional impacts

5. Analysis methods for mutant proteins:

• Expression and purification using standardized protocols

• Structural assessment via circular dichroism, native gel electrophoresis

• Assembly assays to determine effects on c-ring formation

• Functional reconstitution to evaluate proton translocation capability

By systematically applying these mutagenesis strategies, researchers can develop a comprehensive understanding of how specific residues and regions contribute to the structure, assembly, and function of Geobacter sp. atpE in the context of ATP synthase.

How can researchers overcome protein aggregation and inclusion body formation when expressing Geobacter sp. atpE?

Membrane proteins like Geobacter sp. atpE are prone to aggregation and inclusion body formation during recombinant expression. The following methodological approaches can help overcome these challenges:

Prevention strategies:

• Expression system modifications:

  • Reduce expression rate through lower inducer concentrations (e.g., 0.1-0.5 mM IPTG instead of 1.0 mM)

  • Lower cultivation temperature (16-25°C) after induction

  • Use specialized E. coli strains designed for membrane protein expression

  • Employ weaker promoters or tunable expression systems

• Fusion partners and solubility enhancers:

  • Express atpE as a fusion with solubility-enhancing partners (MBP, SUMO, thioredoxin)

  • Include molecular chaperones by co-expression (GroEL/GroES, DnaK/DnaJ)

  • Add specific lipids or detergents to culture medium

• Buffer optimization:

  • Include glycerol (5-10%) in lysis buffer

  • Add mild detergents during cell disruption

  • Use protease inhibitors to prevent degradation

Recovery strategies for inclusion bodies:

• Solubilization protocol:

  • Isolate inclusion bodies by centrifugation

  • Wash with detergents and low concentrations of denaturants

  • Solubilize using appropriate detergents or chaotropic agents (urea, guanidinium HCl)

• Refolding methodology:

  • Remove denaturant by dialysis or dilution

  • Refold in the presence of appropriate lipids and detergents

  • Use pulsed refolding with gradual denaturant removal

  • Include redox pairs to facilitate disulfide bond formation if applicable

• Verification of refolded protein:

  • Analyze secondary structure by circular dichroism

  • Assess oligomeric state by size exclusion chromatography

  • Compare with natively purified protein where possible

When working with recombinant Geobacter sp. atpE, researchers should be particularly attentive to the detergent selection, as this can dramatically impact the solubility and native-like folding of the protein. A systematic screen of different detergent types and concentrations is often necessary to identify optimal conditions.

What approaches can resolve difficulties in obtaining sufficient purity and yield of Geobacter sp. atpE for structural studies?

Structural studies require high purity (>95%) and substantial yields of Geobacter sp. atpE, which presents specific challenges. The following methodological approaches address these difficulties:

Enhanced purification strategies:

• Affinity tag optimization:

  • Compare N-terminal versus C-terminal His-tag placement

  • Test alternative affinity tags (Strep-tag II, FLAG-tag)

  • Consider dual affinity tags with protease cleavage sites

  • Optimize tag removal protocols if tags interfere with structural studies

• Multi-step chromatography approach:

  • Implement sequential purification steps:

    • Immobilized metal affinity chromatography (IMAC)

    • Ion exchange chromatography

    • Size exclusion chromatography

  • Develop custom purification schemes based on protein properties

• Detergent screening and optimization:

  • Systematically evaluate different detergents for:

    • Extraction efficiency

    • Protein stability

    • Compatibility with structural techniques

  • Consider detergent exchange during purification

Yield enhancement approaches:

• Scale-up strategies:

  • Transition to high-density fermentation

  • Implement fed-batch cultivation

  • Optimize media composition for membrane protein expression

• Expression system alternatives:

  • Evaluate insect cell expression systems

  • Consider cell-free expression systems for membrane proteins

  • Test eukaryotic hosts for specific applications

• Stabilization approaches:

  • Identify and add specific lipids that enhance stability

  • Screen additives (glycerol, specific salts, osmolytes)

  • Optimize buffer composition through systematic testing

Quality control methodology:

By implementing these methodological approaches, researchers can overcome challenges in obtaining sufficient quantities of pure, homogeneous Geobacter sp. atpE suitable for structural and functional studies.

How can recombinant Geobacter sp. atpE be utilized in bioenergetic research and comparative studies of ATP synthases?

Recombinant Geobacter sp. atpE offers unique opportunities for advancing bioenergetic research and comparative studies of ATP synthases. The following methodological approaches highlight its research applications:

Comparative bioenergetics research:

• c-ring stoichiometry studies:

  • Investigate the determinants of c-ring size in Geobacter sp.

  • Compare with other organisms having different c-ring stoichiometries (c10-c15)

  • Correlate ring size with bioenergetic efficiency and environmental adaptation

  • Analyze the relationship between c-ring stoichiometry and H+/ATP ratio

• Evolutionary analysis:

  • Compare atpE sequences and structures across diverse species

  • Investigate evolutionary conservation patterns

  • Identify adaptations in extremophiles versus mesophiles

  • Study co-evolution with other ATP synthase subunits

Structural biology applications:

• Cryo-EM and crystallographic studies:

  • Use purified recombinant atpE to reconstitute c-rings for structural determination

  • Map the proton translocation pathway

  • Investigate the structural basis of c-ring assembly

  • Analyze lipid-protein interactions in the c-ring

• Hybrid approaches:

  • Combine recombinant Geobacter sp. atpE with subunits from other organisms

  • Create chimeric ATP synthases with novel properties

  • Investigate the structural compatibility between components from different species

Biotechnological applications:

• Bioenergy systems:

  • Explore the potential of modified ATP synthases in bioenergy applications

  • Investigate ATP production efficiency in engineered systems

  • Study the role of c-ring stoichiometry in determining energy conversion efficiency

• Nanomotor development:

  • Utilize the rotary mechanism of ATP synthase for nanomotor applications

  • Engineer modified c-rings with altered rotational properties

  • Develop hybrid biological-synthetic nanomachines

By pursuing these research directions, investigators can leverage recombinant Geobacter sp. atpE to advance our understanding of bioenergetic principles, evolutionary adaptations in energy-converting systems, and potential biotechnological applications of ATP synthases.

What insights can be gained from studying the role of targeting peptides in ATP synthase assembly using recombinant Geobacter sp. atpE?

The study of targeting peptides in ATP synthase assembly represents an advanced research direction with significant implications for understanding cellular bioenergetics. While the search results specifically mention targeting peptides in the context of mammalian ATP synthase subunit c isoforms , similar principles can be investigated using recombinant Geobacter sp. atpE through the following methodological approaches:

Experimental strategies for targeting peptide research:

• Comparative analysis of targeting sequences:

  • Identify and characterize native targeting sequences in Geobacter sp. atpE

  • Compare with targeting sequences from other organisms

  • Analyze the evolutionary conservation of targeting mechanisms

  • Investigate species-specific adaptations in targeting systems

• Fusion protein experiments:

  • Create fusion constructs with different targeting peptides

  • Assess localization and assembly efficiency

  • Investigate the impact on protein stability and function

  • Determine whether targeting peptides have additional roles beyond localization

• Structure-function analysis of targeting domains:

  • Perform systematic mutagenesis of targeting sequences

  • Identify critical residues for proper localization

  • Investigate potential interactions with chaperones or assembly factors

  • Determine the structural basis for targeting specificity

Research questions addressable through this approach:

• Do targeting peptides in ATP synthase subunits serve functions beyond membrane targeting?

• How do targeting sequences influence the assembly of the complete ATP synthase complex?

• Are there species-specific adaptations in targeting mechanisms related to different ecological niches?

• Can targeting peptides from different organisms be interchanged while maintaining functionality?

Similar to findings in mammalian systems, where ATP synthase subunit c isoforms with different targeting peptides were found to be non-redundant despite identical mature peptides , investigation of targeting sequences in bacterial systems like Geobacter sp. could reveal unexpected functional roles. The methodological approaches outlined above provide a framework for exploring these questions systematically.

How can studies of Geobacter sp. atpE contribute to understanding microbial adaptation to different energy environments?

Research on Geobacter sp. atpE can provide critical insights into microbial adaptation mechanisms across different energy environments. The following methodological approaches facilitate integration of atpE studies with broader ecological and bioenergetic contexts:

Ecological adaptation research:

• Comparative analysis across Geobacter species:

  • Analyze atpE sequence variations among Geobacter species from different environments

  • Correlate sequence differences with habitat-specific energy limitations

  • Investigate adaptations in species specialized for different electron acceptors

  • Compare ATP synthase efficiency across species with different metabolic strategies

• Environmental response studies:

  • Examine how atpE expression and modification respond to:

    • Varying energy availability

    • Different electron acceptors

    • Environmental stressors (pH, temperature, toxicants)

  • Monitor ATP synthase activity as an indicator of metabolic state during bioremediation

Integration with metabolic network studies:

• Systems biology approaches:

  • Correlate ATP synthase activity with expression of other metabolic enzymes

  • Analyze the relationship between citrate synthase levels (a metabolic marker) and ATP synthase function

  • Develop metabolic flux models incorporating ATP synthesis efficiency

  • Investigate energy conservation strategies in different growth conditions

• Environmental transcriptomics and proteomics:

  • Monitor atpE expression in field samples during bioremediation

  • Correlate expression patterns with environmental parameters

  • Identify co-expressed genes that may participate in coordinated energy regulation

  • Develop biomarkers for monitoring Geobacter metabolic state in environmental applications

By pursuing these research directions, investigators can bridge the gap between molecular-level understanding of ATP synthase function and ecosystem-level understanding of microbial energy management strategies. This integrated approach enables insights into how fundamental bioenergetic systems like ATP synthase contribute to microbial adaptation and ecological function.

What methodological approaches can connect recombinant protein studies of atpE with in situ investigations of Geobacter energy metabolism?

Bridging laboratory studies of recombinant Geobacter sp. atpE with in situ investigations presents methodological challenges and opportunities. The following approaches can establish these connections:

Translational research strategies:

• Development of activity-based protein profiling:

  • Create activity-based probes targeting ATP synthase

  • Apply these probes to environmental samples

  • Quantify ATP synthase activity in situ

  • Correlate with environmental parameters and metabolic rates

• Antibody-based approaches:

  • Develop antibodies against recombinant Geobacter sp. atpE

  • Use these for immunodetection in environmental samples

  • Quantify protein abundance in field samples

  • Compare with laboratory cultures under defined conditions

• Genetic reporter systems:

  • Create reporter constructs linking atpE expression to fluorescent proteins

  • Introduce these into Geobacter for in situ monitoring

  • Track expression dynamics during environmental transitions

  • Correlate with other metabolic indicators like citrate synthase expression

Integrated methodological framework:

By implementing these methodological approaches, researchers can establish meaningful connections between detailed molecular studies of recombinant atpE and the complex in situ function of ATP synthase in environmental settings. This integrated strategy enables a more comprehensive understanding of how ATP synthase contributes to Geobacter's remarkable metabolic capabilities in diverse environments.