Recombinant Geobacillus kaustophilus ATP synthase subunit c (atpE)

What is the basic structure of Geobacillus kaustophilus ATP synthase subunit c (atpE)?

Geobacillus kaustophilus ATP synthase subunit c (atpE) is a 72-amino acid protein that forms the c-ring component of the Fo sector in F-type ATP synthase. The full amino acid sequence is MSLGVLAAAIAVGLGALGAGIGNGLIVSRTIEGIARQPELRPVLQTTMFIGVALVEALPIIGVVFSFIYLGR . This highly hydrophobic protein contains transmembrane domains that anchor it within the membrane portion of the ATP synthase complex. The recombinant versions typically include an N-terminal histidine tag to facilitate purification and downstream applications. The protein's compact structure is optimized for its role in the rotary mechanism of ATP synthesis.

Structurally, the c subunit contains predominantly hydrophobic residues with specific charged amino acids that are crucial for proton translocation. The protein sequence reveals a characteristic pattern of hydrophobic amino acids that enable membrane integration, with functional domains for proton binding and rotation coordination.

How does subunit c (atpE) function within the ATP synthase complex?

ATP synthase subunit c proteins form a ring-like structure in the Fo domain of ATP synthase and play a critical role in converting the proton gradient into mechanical energy. The c-ring rotates as protons flow through the Fo domain, which drives the conformational changes in the F1 domain required for ATP synthesis.

The Geobacillus kaustophilus c subunit specifically participates in this process by forming part of the proton channel. The c-ring's rotation is mechanically coupled to the central shaft (γ subunit) of the F1 domain, creating the rotary mechanism that drives ATP synthesis or hydrolysis . Unlike mycobacterial ATP synthases that show latent ATPase activity , thermophilic Geobacillus ATP synthases typically maintain both synthesis and hydrolysis capabilities, making them valuable models for studying energy conversion mechanisms.

What are the optimal conditions for reconstituting lyophilized recombinant G. kaustophilus atpE protein?

For optimal reconstitution of lyophilized G. kaustophilus atpE protein, the following protocol is recommended:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) for long-term storage

• Aliquot the reconstituted protein to avoid repeated freeze-thaw cycles

• Store working aliquots at 4°C for up to one week and long-term storage at -20°C/-80°C

The reconstitution buffer should maintain a pH of 8.0, which is optimal for protein stability. For functional studies, additional considerations may include the presence of lipids or detergents to maintain the native conformation of this membrane protein. The choice of buffer can significantly impact both structural stability and functional activity in downstream applications.

What techniques are most effective for studying the structural dynamics of the c-ring in ATP synthase?

Studying the structural dynamics of ATP synthase c-rings requires a combination of complementary techniques:

• X-ray crystallography: Provides high-resolution static structures of the c-ring, as demonstrated in studies of homologous proteins like those from Thermosynechococcus elongatus (1.98 Å resolution)

• Cryo-electron microscopy (cryo-EM): Reveals the structural arrangement of the c-ring within the intact ATP synthase complex and can capture different conformational states

• Site-directed spin labeling combined with electron paramagnetic resonance (EPR): Monitors local conformational changes during rotation

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Identifies regions with differential solvent accessibility during the functional cycle

• Molecular dynamics simulations: Models the movement of the c-ring and proton translocation through the Fo domain

• Single-molecule FRET techniques: Directly observes rotational movements in real-time

Each of these techniques provides unique insights into c-ring dynamics, and a comprehensive understanding typically requires integrating data from multiple approaches. For Geobacillus kaustophilus specifically, comparing structures with other thermophilic species like G. stearothermophilus can reveal conservation of critical functional elements.

How can sequence variations between Geobacillus species' atpE proteins be leveraged for structure-function studies?

Sequence analysis of atpE proteins from different Geobacillus species reveals both conserved and variable regions that can be targeted for structure-function studies:

Notable variations include:

• The N-terminal methionine and serine presence in G. kaustophilus and G. stearothermophilus versus only methionine in Geobacillus sp.

• A single amino acid difference between G. kaustophilus and G. stearothermophilus at position 19 (N vs. A)

• Different C-terminal regions between species that may affect functional properties

These natural sequence variations provide excellent targets for mutagenesis studies to determine how specific residues influence proton translocation, c-ring assembly, and rotational properties. Chimeric proteins constructed from different Geobacillus species can reveal which regions are critical for specific functional characteristics. Additionally, comparing these thermophilic ATP synthases with mesophilic counterparts can elucidate adaptations that enable function at elevated temperatures.

What are the implications of c subunit structure for developing novel antimicrobials targeting bacterial ATP synthases?

The c subunit structure presents several promising characteristics for antimicrobial development:

• Essential function: The c subunit is critical for bacterial energy metabolism, making it an attractive drug target

• Structural uniqueness: Bacterial c subunits differ significantly from mammalian counterparts, potentially allowing selective targeting

• Accessible binding sites: Specific inhibitors can bind to the c-ring interface or proton-binding sites

• Precedent: The mycobacterial ATP synthase inhibitor bedaquiline targets the c subunit, demonstrating clinical efficacy

Research on Geobacillus kaustophilus atpE provides valuable insights for antimicrobial development due to its thermostability and structural similarity to pathogenic bacterial c subunits. Understanding the detailed structure-function relationship in G. kaustophilus atpE can facilitate the design of inhibitors that disrupt c-ring rotation or assembly, potentially creating new classes of antibiotics with novel mechanisms of action.

Studies comparing G. kaustophilus atpE with mycobacterial counterparts (which exhibit latent ATPase activity ) could reveal structural determinants that might be exploited for selective inhibition of pathogenic bacteria while minimizing effects on human ATP synthases.

How can researchers overcome the challenges of expressing and purifying functional atpE protein?

Expression and purification of functional atpE protein present several challenges due to its hydrophobic nature and membrane association. A systematic approach includes:

• Expression system optimization:

  • Use specialized E. coli strains designed for membrane protein expression (C41(DE3), C43(DE3))

  • Control expression levels through tunable promoters to prevent aggregation

  • Consider low-temperature induction (16-20°C) to improve folding

• Solubilization strategies:

  • Test multiple detergents (DDM, LDAO, Triton X-100) for optimal extraction

  • Consider membrane scaffold proteins for nanodisc formation

  • Evaluate amphipols for stabilizing the native conformation

• Purification refinement:

  • Utilize the N-terminal His-tag for initial IMAC purification

  • Implement size-exclusion chromatography to separate monomers from oligomers

  • Consider lipid supplementation during purification to maintain function

• Functional verification:

  • Assess c-ring assembly using native PAGE

  • Verify proper folding through circular dichroism spectroscopy

  • Evaluate proton-binding capability through pH-dependent tryptophan fluorescence

For recombinant Geobacillus kaustophilus atpE specifically, E. coli expression systems have demonstrated success as documented in the product specifications . Maintaining protein stability post-purification requires careful buffer optimization and storage conditions that prevent protein aggregation while preserving the native conformation.

What methods can resolve contradictory data regarding the oligomeric state of the c-ring in different experimental conditions?

Contradictory data on c-ring oligomeric states can be resolved through multiple complementary approaches:

• Native mass spectrometry:

  • Directly measures the intact complex mass under near-physiological conditions

  • Can distinguish between different oligomeric states (c9-c15 rings reported across species)

• Cross-linking coupled with mass spectrometry:

  • Captures the spatial relationships between subunits

  • Identifies interaction interfaces that define the ring structure

• Atomic force microscopy:

  • Provides direct visualization of c-ring dimensions and subunit arrangement

  • Can be performed in lipid bilayers to mimic native environment

• Blue native PAGE with quantitative Western blotting:

  • Compares migration patterns across different conditions

  • Measures stoichiometry through calibrated antibody binding

• Analytical ultracentrifugation:

  • Determines molecular weight of complexes in solution

  • Distinguishes between different assembly states

When examining contradictory results, researchers should consider that experimental conditions (detergents, lipids, pH, salt concentration) can dramatically affect the observed oligomeric state. For thermophilic species like G. kaustophilus, temperature-dependent oligomerization may be particularly relevant. Additionally, ensuring that the N-terminal His-tag doesn't interfere with assembly is critical for obtaining physiologically relevant results.

How do the structural and functional properties of G. kaustophilus atpE compare with other thermophilic ATP synthase c subunits?

Comparison of G. kaustophilus atpE with other thermophilic ATP synthase c subunits reveals important structural and functional insights:

The G. kaustophilus atpE protein (UniProt ID: Q5KUI8) shares extremely high sequence similarity with G. stearothermophilus atpE (UniProt ID: P42011), differing in only one amino acid position (N vs. A at position 19) . Both proteins maintain similar structural features that contribute to thermostability, including optimized hydrophobic interactions and salt bridges.

Unlike mesophilic counterparts, these thermophilic c subunits typically exhibit enhanced structural rigidity while maintaining the functional flexibility needed for rotary catalysis. The amino acid composition favors residues that contribute to thermal stability, such as increased alanine content and reduced thermolabile residues.

What insights can be gained from comparing G. kaustophilus atpE with mycobacterial ATP synthase components?

Comparison between G. kaustophilus atpE and mycobacterial ATP synthase components reveals critical differences in regulatory mechanisms and potential therapeutic targets:

• Regulatory mechanisms:

  • Mycobacterial ATP synthases exhibit latent ATPase activity, largely regulated by the C-terminal extension of subunit α

  • G. kaustophilus ATP synthase lacks these specialized regulatory features, functioning bidirectionally as both synthase and ATPase

  • These differences suggest evolutionary adaptations to different ecological niches and metabolic requirements

• Structural adaptations:

  • Mycobacterial ATP synthases contain unique structural elements including an extended C-terminus in subunit α and an extra 14-amino-acid γ-loop

  • G. kaustophilus lacks these features, with its c subunit optimized for thermostability rather than regulatory control

  • The c-ring stoichiometry may differ between species, affecting the bioenergetic efficiency

• Therapeutic implications:

  • The structural differences provide opportunities for selective targeting of mycobacterial ATP synthases

  • Understanding G. kaustophilus atpE structure as a thermostable model can inform rational design of inhibitors

  • Comparative studies can identify conserved functional sites versus species-specific regions

This comparison highlights how ATP synthase components have evolved different regulatory mechanisms to adapt to specific environmental and metabolic challenges. While mycobacterial ATP synthases have developed complex regulatory mechanisms to prevent wasteful ATP hydrolysis , thermophilic Geobacillus species have optimized their components for stable function at high temperatures.

What emerging technologies might revolutionize our understanding of c subunit function in ATP synthase?

Several cutting-edge technologies are poised to transform our understanding of c subunit function:

• Time-resolved cryo-EM:

  • Captures structural intermediates during the rotational cycle

  • Provides dynamic snapshots of conformational changes at near-atomic resolution

  • Could reveal transient states previously inaccessible to structural determination

• Advanced computational methods:

  • Quantum mechanical/molecular mechanical (QM/MM) simulations to model proton transfer with electronic precision

  • Machine learning approaches to predict functional consequences of mutations

  • Extended molecular dynamics simulations that reach biologically relevant timescales

• Single-molecule biophysics:

  • High-speed AFM to directly visualize c-ring rotation in real-time

  • Magnetic tweezers to measure torque generation during proton translocation

  • Correlative microscopy combining fluorescence and structural data

• Advanced genetic and synthetic biology tools:

  • CRISPR-based approaches for precise mutagenesis of atpE in native contexts

  • Expanded genetic code incorporation for site-specific labeling with novel probes

  • Synthetic c-rings with non-natural amino acids to probe functional mechanisms

• Nanotechnology applications:

  • Biomimetic nanomotors based on c-ring design principles

  • Integration of functional c-rings into synthetic membranes for energy harvesting

  • Development of c-ring-based biosensors for proton gradient detection

These technologies, when applied to G. kaustophilus atpE, will likely reveal unprecedented details about the coupling between proton movement and rotary mechanics, potentially inspiring new biomimetic applications in nanotechnology.

How might G. kaustophilus atpE serve as a model for bioenergy applications and synthetic biology?

G. kaustophilus atpE offers several advantages as a model for bioenergy applications and synthetic biology:

• Thermostable molecular machinery:

  • Functions efficiently at elevated temperatures (50-70°C)

  • Resists denaturation under harsh industrial conditions

  • Could be engineered into hybrid energy-generating systems

• Biofuel cell development:

  • Integration into electrode surfaces to create ATP from electrical potentials

  • Coupling with photosynthetic proteins for light-driven ATP production

  • Construction of artificial vesicles with oriented ATP synthases for energy storage

• Biosensor applications:

  • Development of pH gradient sensors based on c-ring conformational changes

  • Creation of ATP biosensors utilizing the coupling between proton flow and ATP synthesis

  • Integration into microfluidic devices for energy metabolism studies

• Synthetic biology platforms:

  • Chassis for designing modified ATP synthases with altered ion specificities

  • Template for creating nanomotors with controllable rotational properties

  • Component of minimal cells designed for specialized energy production

The availability of recombinant G. kaustophilus atpE facilitates these applications by providing a stable, well-characterized protein component that can be modified and integrated into various synthetic systems. Its thermostability is particularly valuable for industrial applications where operational conditions might denature mesophilic proteins.

Future research will likely focus on engineering G. kaustophilus atpE and related components to create tailored energy systems with improved efficiency, alternative fuel sources, or novel regulatory mechanisms inspired by natural ATP synthase diversity.