Recombinant Geobacillus thermodenitrificans ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its role in Geobacillus thermodenitrificans?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of ATP synthase, the enzyme complex responsible for ATP production. In Geobacillus thermodenitrificans, a thermophilic bacterium that grows optimally between 45-55°C, atpE forms part of the membrane-embedded rotary motor of ATP synthase. This protein functions within the c-ring that converts the proton motive force across the membrane into mechanical rotation, which drives ATP synthesis. The thermostable nature of this protein allows the ATP synthase complex to function efficiently at elevated temperatures, essential for the survival of this thermophilic organism .

What are the structural characteristics of Geobacillus thermodenitrificans ATP synthase subunit c?

Geobacillus thermodenitrificans ATP synthase subunit c (atpE) is a small, hydrophobic protein consisting of 70 amino acids with the sequence: MGVLAAAIAIGLAALGAGIGNGLIVSRTVEGIARQPEARGMLQTTMFIGVALVEAIPIIA VVIAFMVQGR . The protein is predominantly α-helical with two transmembrane helices connected by a polar loop region. The high proportion of alanine, glycine, and other hydrophobic residues reflects its membrane-embedded nature. The protein contains conserved residues involved in proton translocation and interaction with other subunits of the ATP synthase complex. Its compact structure and specific amino acid composition contribute to its remarkable thermostability .

How does the atpE protein from thermophilic bacteria differ from mesophilic homologs?

The atpE protein from thermophilic bacteria like G. thermodenitrificans exhibits several key differences compared to mesophilic counterparts:

These adaptations allow the protein to maintain its structure and function in the thermophilic environment where G. thermodenitrificans thrives, typically at temperatures between 45-65°C .

What are the optimal conditions for expressing recombinant G. thermodenitrificans atpE in E. coli?

Based on the available literature and experimental data, the optimal conditions for expressing recombinant G. thermodenitrificans atpE in E. coli include:

Codon optimization may be necessary due to codon usage differences between G. thermodenitrificans and E. coli. The protein has been successfully expressed in E. coli systems as described in several studies, yielding functional protein for subsequent analysis .

What purification strategies are most effective for recombinant G. thermodenitrificans atpE?

Effective purification strategies for recombinant G. thermodenitrificans atpE typically involve a multi-step approach:

• Immobilized metal affinity chromatography (IMAC): For His-tagged proteins, Ni-NTA or TALON resins can be used with imidazole gradient elution. This is particularly effective for the N-terminal His-tagged version of the protein .

• Detergent selection: Critical for maintaining protein solubility; mild detergents like DDM, LDAO, or Fos-choline-12 at concentrations just above critical micelle concentration (CMC).

• Buffer optimization: Tris or phosphate buffers at pH 7.0-8.0 with 100-300 mM NaCl and glycerol (10-20%) for stability.

• Size exclusion chromatography: As a polishing step to separate monomers from oligomers or aggregates and to exchange detergents if needed.

• Quality control: SDS-PAGE, Western blotting, and mass spectrometry to confirm purity and identity, with expected molecular weight of approximately 30 kDa .

Published protocols have achieved purification to >90% homogeneity using variations of these approaches .

How should recombinant G. thermodenitrificans atpE be stored to maintain optimal activity?

For optimal storage of recombinant G. thermodenitrificans atpE:

Short-term storage (up to one week):

• Store at 4°C in appropriate buffer

• Avoid repeated freeze-thaw cycles

Long-term storage:

• Store at -20°C or preferably -80°C

• Use buffer containing 50% glycerol or trehalose (6%) as cryoprotectants

• Aliquot to avoid repeated freeze-thaw cycles

Buffer composition:

• Tris/PBS-based buffer, pH 8.0

• Include salt (NaCl)

• Maintain appropriate detergent concentration

• Consider adding 6% trehalose for stability

Reconstitution from lyophilized form:

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Add glycerol to 5-50% final concentration for subsequent storage

Following these guidelines helps maintain structural integrity and functional activity for extended periods, which is particularly important for thermostable proteins that may be prone to aggregation upon improper handling.

What analytical methods are most appropriate for characterizing recombinant G. thermodenitrificans atpE?

Appropriate analytical methods for characterizing recombinant G. thermodenitrificans atpE include:

Structural characterization:

• Circular dichroism (CD) spectroscopy: For secondary structure analysis and thermal stability assessment

• NMR spectroscopy: For detailed structural information in detergent micelles

• Cryo-EM: Particularly useful if studying the entire ATP synthase complex

Functional characterization:

• Proton translocation assays: Using pH-sensitive fluorescent dyes

• ATPase activity assays: When incorporated into proteoliposomes with other ATP synthase subunits

• Membrane potential measurements: Using potential-sensitive dyes

Biophysical characterization:

• Differential scanning calorimetry (DSC): For thermostability assessment

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): For oligomeric state determination

Biochemical characterization:

• SDS-PAGE and blue native PAGE: For purity and oligomeric state analysis

• Mass spectrometry: For protein identification and post-translational modification analysis

• Limited proteolysis: To probe protein folding and domain arrangement

The combination of these methods provides comprehensive information about the structural and functional properties of the protein, essential for understanding its role in the ATP synthase complex and its potential applications in research .

How does the thermostability of G. thermodenitrificans atpE compare to other thermophilic ATP synthase components?

The thermostability of G. thermodenitrificans atpE compared to other thermophilic ATP synthase components shows interesting patterns:

Relative thermostability within ATP synthase complex:

• The c subunit (atpE) is generally one of the more thermostable components due to its highly hydrophobic nature and membrane integration

• Compared to the peripheral stalk subunits (δ, b), atpE typically exhibits greater thermostability

• The α/β catalytic hexamer shows comparable thermostability to atpE but through different mechanisms (more extensive ion-pair networks)

Temperature optima:

• G. thermodenitrificans atpE maintains functionality at temperatures up to 65-70°C

• This is consistent with the growth temperature optimum of the organism (45-55°C)

• Some hyperthermophilic archaea possess ATP synthase components with even higher thermostability (80-100°C)

Structural basis for thermostability differences:

• Membrane-embedded components like atpE rely heavily on hydrophobic interactions for stability

• Peripheral components depend more on salt bridges and hydrogen bonding networks

• The catalytic components (α/β) often incorporate additional metal binding sites for enhanced stability

Understanding these comparative thermostability profiles is crucial for reconstitution experiments and for applications requiring specific temperature ranges .

What structural features contribute to the thermostability of G. thermodenitrificans atpE?

Key structural features contributing to the thermostability of G. thermodenitrificans atpE include:

Amino acid composition:

• Increased alanine and glycine content: Provides conformational flexibility while maintaining compact packing

• Reduced number of thermolabile residues (Asn, Gln, Cys, Met) that are prone to deamidation or oxidation at high temperatures

• Higher proportion of charged residues forming salt bridges

Hydrophobic packing:

• Enhanced core hydrophobicity in transmembrane regions

• More extensive van der Waals interactions between side chains

Secondary structure stabilization:

• α-helices with optimal hydrogen bonding patterns

• Reduced flexibility in loop regions connecting transmembrane segments

Quaternary interactions:

• Specific residues at subunit interfaces that enhance c-ring stability

• Conserved motifs that facilitate proton coordination while maintaining structural integrity

These features work synergistically to maintain the functional structure of atpE under the elevated temperature conditions where G. thermodenitrificans thrives .

What is the oligomeric state of G. thermodenitrificans atpE in native conditions?

The oligomeric state of G. thermodenitrificans atpE in native conditions is characterized by:

c-ring composition:

• In ATP synthases, multiple copies of the c subunit (atpE) form a ring structure in the membrane

• For thermophilic bacteria like Geobacillus species, the c-ring typically contains 10-13 subunits

• This oligomeric arrangement is essential for the rotary mechanism of ATP synthesis

Determining factors:

• The exact number of c-subunits in the ring is species-specific and genetically determined

• This number influences the bioenergetic efficiency of ATP synthesis (H⁺/ATP ratio)

• Structural constraints imposed by interactions with other subunits (particularly a and b subunits)

Functional significance:

The oligomeric state is critical for understanding the functional properties of the ATP synthase complex in thermophilic bacteria and has implications for bioenergetic efficiency calculations .

How can recombinant G. thermodenitrificans atpE be used in bioenergetic research?

Recombinant G. thermodenitrificans atpE offers several valuable applications in bioenergetic research:

Model system for thermophilic ATP synthesis:

• Provides insights into energy conversion mechanisms at elevated temperatures

• Allows comparative studies between mesophilic and thermophilic bioenergetic systems

Reconstitution experiments:

• Can be incorporated into liposomes to create minimal proton-translocating systems

• Mixing with components from other species allows creation of chimeric ATP synthase complexes to study compatibility and functional conservation

Structure-function studies:

• Site-directed mutagenesis to identify critical residues for proton translocation

• Investigation of the molecular basis of coupling between proton movement and rotary motion

Inhibitor development and binding studies:

• Screening for compounds that specifically interact with thermophilic ATP synthases

• Structure-based design of inhibitors targeting the c-ring

Biophysical tool development:

• Using the c-ring as a nanoscale molecular motor in bionanotechnology

• Development of biosensors based on ATP synthase activity

These applications leverage the unique properties of thermostable atpE to advance our understanding of bioenergetic processes and their applications .

What research applications benefit from using thermostable ATP synthase components like G. thermodenitrificans atpE?

Research applications benefiting from thermostable ATP synthase components like G. thermodenitrificans atpE include:

Structural biology advantages:

• Enhanced stability during crystallization attempts

• Improved behavior in cryo-EM sample preparation

• Longer shelf-life for NMR studies requiring extended data collection

Biophysical studies:

• Wider temperature range for kinetic and thermodynamic measurements

• Ability to perform experiments at elevated temperatures that better mimic certain physiological or industrial conditions

• Reduced concerns about denaturation during experimental manipulations

Biotechnological applications:

• Basis for designing ATP-regenerating systems for high-temperature enzymatic processes

• Template for engineering thermostable ATP synthases for bioenergy applications

• Development of thermostable molecular motors for nanotechnology

Evolutionary studies:

• Models for understanding molecular adaptation to extreme environments

• Probes for investigating the evolution of bioenergetic systems across thermal gradients

• Comparative analysis with mesophilic homologs to identify determinants of thermal adaptation

These diverse applications highlight the value of thermostable ATP synthase components in advancing multiple research fields, particularly those requiring robust proteins that can withstand harsh experimental conditions .

How does G. thermodenitrificans atpE perform in reconstituted membrane systems?

Performance of G. thermodenitrificans atpE in reconstituted membrane systems shows several important characteristics:

Reconstitution efficiency:

• Generally shows good incorporation into artificial liposomes or nanodiscs

• May require specific lipid compositions to maintain native-like function

• The thermostable nature allows for reconstitution protocols at higher temperatures that may improve protein insertion

Functional parameters:

• Maintains proton translocation activity when properly reconstituted

• Can participate in ATP synthesis when combined with other ATP synthase components

• Often displays higher temperature optima for activity compared to mesophilic homologs

Lipid requirements:

• May function optimally with lipids that maintain fluidity at elevated temperatures

• Could require specific lipid compositions that mimic the native membrane environment of G. thermodenitrificans

• The interaction between protein thermostability and membrane fluidity is a critical parameter

Experimental considerations:

• Detergent selection for solubilization and reconstitution significantly impacts performance

• Membrane composition affects protein orientation and oligomeric state

• Proton permeability of the reconstituted system must be carefully controlled

Understanding these performance characteristics is essential for designing effective reconstitution experiments and interpreting the resulting data .

What are common challenges in expressing and purifying functional G. thermodenitrificans atpE?

Common challenges in expressing and purifying functional G. thermodenitrificans atpE include:

Expression challenges:

• Toxicity to host cells: Membrane protein overexpression can disrupt host membrane integrity

• Inclusion body formation: Hydrophobic nature promotes aggregation rather than membrane integration

• Codon bias: Differences between thermophilic source and expression host can limit translation efficiency

• Proteolytic degradation: Improperly folded protein may be targeted by host proteases

Purification challenges:

• Detergent selection: Finding a detergent that effectively solubilizes while maintaining native structure

• Maintaining oligomeric state: Preserving the c-ring structure during extraction from membranes

• Distinguishing functional from non-functional protein: Assessing proper folding in a membrane-less environment

• Low yields: Membrane proteins typically express at lower levels than soluble proteins

Functionality assessment:

• Developing assays that work with isolated c-subunits outside the complete ATP synthase context

• Confirming proper folding of a highly hydrophobic protein with limited exposed regions

• Validating that the recombinant protein retains native-like proton translocation capability

These challenges require careful optimization of expression systems, purification protocols, and functional assays to obtain high-quality, functional protein for research applications .

How can researchers troubleshoot low yields of recombinant G. thermodenitrificans atpE?

Troubleshooting strategies for low yields of recombinant G. thermodenitrificans atpE:

Expression system optimization:

• Try different E. coli strains (C41/C43(DE3), BL21-AI, Rosetta) specialized for membrane protein expression

• Test inducible vs. constitutive expression systems

• Optimize codon usage for the expression host

Induction parameters:

• Reduce induction temperature (15-25°C) to slow protein synthesis and improve folding

• Decrease inducer concentration to prevent overwhelming the membrane insertion machinery

• Extend induction time to accumulate more protein gradually

• Add membrane-stabilizing compounds (glycerol, betaine) to the culture medium

Vector design improvements:

• Test different fusion tags (MBP, SUMO) known to enhance membrane protein solubility

• Include periplasmic targeting sequences to reduce cytoplasmic aggregation

• Engineer constructs with modified N/C termini to reduce proteolytic degradation

Cell disruption and extraction:

• Optimize lysis conditions to efficiently release membrane-embedded proteins

• Test different detergents and detergent concentrations for extraction

• Implement gentle extraction procedures to maintain native-like structure

Purification refinement:

• Adjust buffer components to enhance protein stability (salt concentration, pH, additives)

• Use step gradients rather than linear gradients for better separation

• Consider affinity chromatography conditions that minimize non-specific binding

Implementing these strategies systematically can help identify and overcome the specific bottlenecks limiting the yield of functional recombinant G. thermodenitrificans atpE .

How do post-translational modifications affect the function of G. thermodenitrificans atpE?

Post-translational modifications (PTMs) of G. thermodenitrificans atpE can significantly influence its function in several ways:

Known PTMs in bacterial ATP synthase c-subunits:

• N-terminal processing: Removal of initiator methionine affects membrane insertion efficiency

• Fatty acid acylation: Can occur on specific residues, enhancing membrane association

• Phosphorylation: May regulate proton translocation efficiency or c-ring assembly

Functional impacts:

• Proton coordination: Modifications near the conserved proton-binding site can alter proton affinity and translocation kinetics

• Subunit interactions: PTMs at interfaces between c-subunits or with other ATP synthase components can affect complex assembly and stability

• Membrane integration: Modifications that alter hydrophobicity profiles can influence membrane positioning

Thermophile-specific considerations:

• PTMs may contribute to thermostability by introducing additional stabilizing interactions

• Some modifications might be thermophile-specific adaptations that are absent in mesophilic homologs

• The extreme growth conditions may necessitate unique PTMs for maintaining protein function

These modifications can be critical for the proper functioning of atpE in its native thermophilic environment and may provide insights into adaptation mechanisms for extreme conditions .

What strategies can be employed to study protein-protein interactions involving G. thermodenitrificans atpE?

Strategies for studying protein-protein interactions involving G. thermodenitrificans atpE:

Biochemical approaches:

• Co-immunoprecipitation with antibodies against atpE or interaction partners

• Pull-down assays using tagged versions of atpE or potential binding partners

• Chemical cross-linking followed by mass spectrometry to identify interaction sites

• Blue native PAGE to preserve native complexes and identify interaction stoichiometry

Biophysical techniques:

• Förster Resonance Energy Transfer (FRET) between fluorescently labeled proteins

• Surface Plasmon Resonance (SPR) to measure binding kinetics

• Isothermal Titration Calorimetry (ITC) for thermodynamic characterization of interactions

• Microscale Thermophoresis (MST) to detect interactions in solution with minimal sample consumption

Structural approaches:

• Cryo-electron microscopy of assembled complexes

• X-ray crystallography of co-crystallized components

• NMR spectroscopy for detecting interaction interfaces through chemical shift perturbations

Specialized approaches for membrane proteins:

• Reconstitution in nanodiscs or liposomes to maintain native-like environment

• Site-specific labeling strategies for membrane-embedded regions

• In vivo approaches like bacterial two-hybrid or split-GFP complementation

• Lipid-protein interaction analysis through lipidomics approaches

These diverse strategies can be employed in combination to build a comprehensive understanding of how G. thermodenitrificans atpE interacts with other proteins in the ATP synthase complex and potentially with other cellular components .