Recombinant Geobacillus sp. ATP synthase subunit c (atpE)

How is recombinant Geobacillus sp. ATP synthase subunit c (atpE) typically expressed and purified?

Recombinant expression of Geobacillus sp. ATP synthase subunit c typically employs E. coli as the heterologous host. The standard protocol includes:

• Cloning: The atpE gene is amplified by PCR using specific primers and inserted into an expression vector, typically containing an N-terminal His-tag for purification.

• Expression: Transformed E. coli cells are grown in suitable media (LB or similar) until they reach appropriate optical density, followed by induction with IPTG if using a T7-based expression system.

• Purification: Given the hydrophobic nature of subunit c, specialized protocols for membrane protein purification are employed, including:

  • Cell lysis (mechanical or detergent-based)

  • Membrane solubilization using detergents (LDAO, DDM, etc.)

  • Affinity chromatography using His-tag

  • Size exclusion chromatography for final polishing

The purified protein is typically stored in a detergent-containing buffer to maintain solubility, often with the addition of 6% trehalose to enhance stability during storage .

What are the optimal storage conditions for recombinant Geobacillus sp. ATP synthase subunit c?

The optimal storage conditions for maintaining the stability and activity of recombinant Geobacillus sp. ATP synthase subunit c include:

• Storage temperature: -20°C to -80°C for long-term storage

• Working aliquots can be maintained at 4°C for up to one week

• Buffer composition: Tris/PBS-based buffer, pH 8.0, containing 6% trehalose

• Avoidance of repeated freeze-thaw cycles that can lead to protein denaturation

• For reconstituted protein, addition of 5-50% glycerol (final concentration) before aliquoting for long-term storage

• Lyophilization can be employed for extended storage periods

How can researchers effectively incorporate recombinant Geobacillus sp. ATP synthase subunit c into proteoliposomes for functional studies?

Incorporating recombinant Geobacillus sp. ATP synthase subunit c into proteoliposomes requires precise methodology to ensure functional reconstitution. The recommended protocol involves:

Materials required:

• Purified recombinant Geobacillus sp. ATP synthase subunit c

• Lipids (typically E. coli polar lipids or synthetic phospholipids)

• Detergent (DDM, Triton X-100, or other suitable detergents)

• Bio-Beads SM-2 or similar for detergent removal

• Buffer components

Procedure:

• Prepare lipid vesicles by hydrating dried lipid film in buffer and sonication/extrusion

• Solubilize the lipid vesicles with detergent (detergent:lipid ratio typically 2:1)

• Add purified protein to the solubilized lipids (protein:lipid ratio 1:50 to 1:100)

• Remove detergent using Bio-Beads or dialysis, allowing proteoliposome formation

• Separate proteoliposomes from unincorporated protein by density gradient centrifugation

Functional validation:

• Proton pumping assays using pH-sensitive fluorescent dyes (ACMA or pyranine)

• ATP synthesis/hydrolysis assays using coupled enzyme systems

• Membrane potential measurements using voltage-sensitive dyes

For studies requiring complete F-ATP synthase, co-reconstitution with other subunits is necessary. This is typically achieved by combining individually purified components or by purifying the entire complex prior to reconstitution .

What experimental approaches can distinguish between ATP synthesis and hydrolysis activities in Geobacillus sp. ATP synthase studies?

Distinguishing between ATP synthesis and hydrolysis activities in Geobacillus sp. ATP synthase studies requires specialized experimental approaches:

ATP Synthesis Assays:

• Luciferin-Luciferase Assay: Measures ATP production in real-time through luminescence

• NADP+ Reduction Assay: Couples ATP production to NADP+ reduction via hexokinase and glucose-6-phosphate dehydrogenase

• 32P-ADP Incorporation: Measures radioactive ATP formation from labeled ADP

ATP Hydrolysis Assays:

• Coupled Enzyme Assay: Links ATP hydrolysis to NADH oxidation through pyruvate kinase and lactate dehydrogenase

• Malachite Green Assay: Quantifies released inorganic phosphate

• pH Change Monitoring: Measures proton release during ATP hydrolysis

Experimental Controls and Validation:

• Use of specific inhibitors (oligomycin, DCCD, venturicidin) to confirm ATP synthase activity

• Comparison with known mutants affecting synthetic or hydrolytic activities

• Manipulation of proton gradients to determine direction of catalysis

Table 1: Comparison of Methods for Measuring ATP Synthase Activities

Unlike mycobacterial ATP synthases which show suppressed ATP hydrolysis activity, Geobacillus enzymes typically show both synthetic and hydrolytic activities, making them valuable models for comparative studies .

How can researchers employ single-molecule techniques to study the rotational dynamics of Geobacillus sp. ATP synthase?

Single-molecule techniques provide powerful insights into the rotational dynamics of ATP synthase. For Geobacillus sp. ATP synthase, the following approaches are recommended:

Single-Molecule Fluorescence Microscopy:

• Purify recombinant ATP synthase components, including the c-subunit

• Introduce site-specific cysteine mutations for fluorophore labeling

• Immobilize the α3β3 complex on a glass surface via His-tag or streptavidin-biotin linkage

• Attach a fluorescent probe (quantum dot or fluorescent bead) to the γ-subunit or c-ring

• Monitor rotation using TIRF microscopy

Analysis Parameters:

• Angular velocity: For Geobacillus-based complexes, typical rotation rates are 4.6 ± 1.0 s-1 with 300-nm beads at saturating ATP concentrations

• Step size: 120° steps corresponding to ATP binding and hydrolysis events

• Dwell times: Analysis of ATP concentration dependence to determine rate-limiting steps

A published study on Geobacillus stearothermophilus F-ATP synthase showed that when 600-nm bead duplexes were attached to the protein, the average rotational rate decreased to 1.9 ± 0.5 s-1 due to increased viscous drag .

When comparing with chimeric constructs containing Mycobacterium-specific domains, researchers observed a decrease in angular velocity during power-stroke after ATP binding, indicating that structural elements from different species can significantly impact rotational dynamics .

How does Geobacillus sp. ATP synthase subunit c differ structurally and functionally from its homologs in other bacterial species?

Geobacillus sp. ATP synthase subunit c shows important structural and functional differences when compared to homologs from other bacterial species:

Structural Comparisons:

Functional Differences:

• ATP Hydrolysis Capability: Unlike mycobacterial ATP synthases which show suppressed ATP hydrolysis, Geobacillus ATP synthases maintain both synthetic and hydrolytic functions .

• Regulatory Mechanisms: Mycobacterial ATP synthases possess unique regulatory elements, including a 36-amino acid C-terminal domain in subunit α that suppresses ATPase activity. This domain is absent in Geobacillus sp. .

• Drug Susceptibility: Mycobacterial ATP synthases are targeted by the drug bedaquiline (BDQ), which binds to subunit c. Geobacillus sp. ATP synthases show different susceptibility profiles, making them valuable for comparative inhibitor studies .

• Thermostability: As a thermophilic organism, Geobacillus sp. ATP synthase components display enhanced thermostability compared to mesophilic counterparts, with optimal activity often observed at temperatures between 55-65°C .

These differences make Geobacillus sp. ATP synthase an important comparative model for understanding the evolution and adaptation of this essential enzyme complex across bacterial species.

What role does the atpE gene play in molecular detection methods for bacterial identification?

The atpE gene, which encodes ATP synthase subunit c, has emerged as a valuable molecular target for bacterial identification and quantification, particularly for mycobacteria:

Specificity as a Molecular Target:

• In silico genomic analyses have demonstrated that the atpE gene shows high conservation within bacterial genera but sufficient variation between genera

• For mycobacteria, atpE demonstrates 80-100% similarity within the Mycobacterium genus but less than 50% similarity with closely related genera like Corynebacterium, Nocardia, and Rhodococcus

Application in Detection Methods:

• Real-time PCR Assays: Primers and probes targeting atpE have been developed for highly specific detection and quantification of bacterial species. For mycobacteria, this approach has demonstrated successful application in environmental samples including tap water and lake water .

• DNA Sequencing: Sequencing of the atpE gene provides taxonomic information useful for bacterial identification and phylogenetic studies.

• Environmental Monitoring: The stability and conservation of the atpE gene make it particularly useful for environmental monitoring applications.

Methodological Considerations:

• PCR amplification typically uses species-specific primers designed from conserved regions of the atpE gene

• For Geobacillus species identification, atpE sequencing can complement other molecular markers like 16S rRNA genes

• When using atpE for detection, cross-validation with other molecular markers is recommended to ensure specificity

This gene's utility stems from its essential role in energy metabolism, leading to high conservation within species while maintaining sufficient variation between species to enable specific detection .

How can Geobacillus sp. ATP synthase subunit c be used as a model system for studying thermophilic membrane protein adaptations?

Geobacillus sp. ATP synthase subunit c provides an excellent model system for studying adaptations of membrane proteins to thermophilic conditions:

Research Applications:

• Thermostability Studies:

  • Comparative analysis of amino acid composition with mesophilic counterparts reveals adaptations that confer thermostability

  • Identification of specific stabilizing interactions (ionic bonds, hydrophobic interactions, hydrogen bonding networks)

  • Investigation of membrane lipid interactions that contribute to thermal stability

• Structure-Function Relationships:

  • Analysis of how thermophilic adaptations impact proton translocation efficiency

  • Studies on the relationship between c-ring size/stoichiometry and thermophilic adaptation

  • Investigation of subunit interface interactions at elevated temperatures

• Protein Engineering Applications:

  • Use as a scaffold for engineering thermostable membrane proteins

  • Identification of thermostabilizing motifs that can be transferred to other proteins

  • Development of robust membrane protein expression systems

Experimental Approaches:

• Comparative Mutagenesis:

  • Generation of chimeric proteins with regions from mesophilic and thermophilic sources

  • Site-directed mutagenesis to identify key residues contributing to thermostability

  • Functional assays at varying temperatures to correlate structural features with thermal tolerance

• Advanced Biophysical Techniques:

  • Circular dichroism (CD) spectroscopy to monitor secondary structure stability at different temperatures

  • Differential scanning calorimetry to determine melting temperatures and thermodynamic parameters

  • Molecular dynamics simulations to model protein behavior at elevated temperatures

• Reconstitution Studies:

  • Incorporation into liposomes with different lipid compositions to study lipid-protein interactions

  • Investigation of functional properties in native-like membrane environments

  • Comparison of proton translocation efficiency at different temperatures

These studies contribute significantly to our understanding of protein thermostability and provide insights that can be applied to protein engineering for biotechnological applications .

What are the major challenges in expressing and purifying functional Geobacillus sp. ATP synthase subunit c, and how can they be overcome?

Expression and purification of functional Geobacillus sp. ATP synthase subunit c present several technical challenges that researchers must address:

Major Challenges and Solutions:

• Protein Toxicity during Expression:

  • Challenge: The hydrophobic nature of subunit c can disrupt host cell membranes

  • Solution: Use tightly controlled expression systems (e.g., pET with T7 lac promoter)

  • Solution: Employ reduced temperature expression (16-25°C) to slow production rate

  • Solution: Consider specialized E. coli strains like C41(DE3) or C43(DE3) designed for membrane protein expression

• Protein Solubilization:

  • Challenge: Extracting membrane-embedded subunit c without denaturation

  • Solution: Screen multiple detergents (DDM, LDAO, FC-12) for optimal extraction

  • Solution: Use detergent:protein ratios of 4:1 to 10:1 for effective solubilization

  • Solution: Consider addition of lipids during solubilization to stabilize native structure

• Maintaining Functionality:

  • Challenge: Preserving native conformation during purification

  • Solution: Include lipids or lipid-like molecules in purification buffers

  • Solution: Minimize exposure to harsh conditions (extreme pH, high salt)

  • Solution: Use size exclusion chromatography as a final step to ensure homogeneity

• Assembly into Functional c-rings:

  • Challenge: Ensuring proper oligomerization into functional c-rings

  • Solution: Develop reconstitution protocols with appropriate lipid compositions

  • Solution: Monitor c-ring formation using native PAGE or analytical ultracentrifugation

  • Solution: Consider co-expression with other F0 subunits to facilitate proper assembly

• Thermostability Considerations:

  • Challenge: Maintaining protein stability during manipulation at non-physiological temperatures

  • Solution: Include stabilizing agents (glycerol, trehalose) in storage buffers

  • Solution: Perform purification steps at elevated temperatures (30-45°C) when possible

  • Solution: Consider heat activation steps to promote proper folding

Optimized Purification Protocol:

Based on published methodologies and the specific characteristics of Geobacillus sp. ATP synthase subunit c, the following optimized protocol is recommended:

• Express with N-terminal His-tag in E. coli C41(DE3) at 25°C for 18 hours

• Harvest cells and lyse in buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl

• Solubilize membranes with 1% DDM for 1 hour at 40°C

• Purify using Ni-NTA affinity chromatography with 0.05% DDM in all buffers

• Apply size exclusion chromatography for final purification

• Store in Tris/PBS-based buffer with 6% trehalose at pH 8.0

How can researchers employ cutting-edge structural biology techniques to elucidate the high-resolution structure of Geobacillus sp. ATP synthase subunit c?

Elucidating the high-resolution structure of Geobacillus sp. ATP synthase subunit c requires employing cutting-edge structural biology techniques:

Cryo-Electron Microscopy (Cryo-EM):

• Sample Preparation: Purify intact c-rings or complete ATP synthase complexes

• Grid Preparation: Apply 3-4 μL of sample (2-5 mg/mL) to glow-discharged grids

• Data Collection: Collect data on a high-end microscope (e.g., Titan Krios) with direct electron detector

• Processing: Use software like RELION or cryoSPARC for 3D reconstruction

• Resolution: Can achieve 2.5-4 Å resolution for membrane protein complexes

• Advantages: Visualizes the protein in a near-native environment; can resolve entire ATP synthase complex

X-ray Crystallography:

• Crystallization: Screen detergent:lipid:protein ratios using vapor diffusion methods

• Crystal Optimization: Focus on lipid cubic phase (LCP) crystallization for membrane proteins

• Data Collection: Use synchrotron radiation with microfocus beamlines for small crystals

• Processing: Process diffraction data and solve structure using molecular replacement

• Resolution: Can achieve 1.5-3 Å resolution with well-diffracting crystals

• Challenges: Obtaining well-diffracting crystals is particularly difficult for membrane proteins

Solution NMR Spectroscopy:

• Sample Preparation: Express isotopically labeled protein (15N, 13C, 2H)

• Micelle Selection: Screen detergent micelles for optimal NMR spectra

• Experiments: Collect 2D/3D heteronuclear NMR spectra for backbone and side-chain assignments

• Structure Calculation: Use distance restraints from NOEs and dihedral angle restraints

• Resolution: Provides atomic-level information on structure and dynamics

• Limitations: Size limitations may require study of individual transmembrane helices rather than entire c-ring

Integrative Structural Biology Approach:

• Combine low-resolution data (Cryo-EM, SAXS) with high-resolution techniques

• Use computational methods to integrate diverse structural data

• Perform molecular dynamics simulations to model behavior in membranes

• Validate structural models with functional assays and mutagenesis

By integrating these approaches, researchers can obtain comprehensive structural insights into the Geobacillus sp. ATP synthase subunit c, particularly in the context of the complete c-ring or the entire F-ATP synthase complex .

What are the potential applications of Geobacillus sp. ATP synthase subunit c in the development of novel antimicrobial compounds?

The unique properties of Geobacillus sp. ATP synthase subunit c present several opportunities for development of novel antimicrobial compounds:

Antimicrobial Drug Development Potential:

• Comparative Analysis for Selectivity:

  • Structural comparison between bacterial (including Geobacillus sp.) and human ATP synthase c-subunits reveals distinct differences that can be exploited for selective targeting

  • Analysis of binding sites for known inhibitors like bedaquiline provides insights for structure-based drug design

  • Thermophilic properties of Geobacillus proteins offer unique structural features that might influence inhibitor binding

• Model System for Inhibitor Screening:

  • Recombinant expression systems for Geobacillus sp. ATP synthase subunit c enable high-throughput screening assays

  • Thermostability allows for screening at elevated temperatures, which may reduce false positives from compounds that denature at higher temperatures

  • Comparative screening against human ATP synthase can identify selective inhibitors with reduced toxicity

• In Silico Approaches:

  • Molecular docking studies with the 3D structure of Geobacillus sp. ATP synthase c-subunit can identify potential binding sites

  • Virtual compound libraries can be screened against these targets

  • Quantum mechanical calculations can predict binding energies and selectivity

Experimental Design for Drug Screening:

• Biochemical Assays:

  • ATP synthesis/hydrolysis assays in reconstituted systems

  • Proton pumping assays using pH-sensitive dyes

  • Competitive binding assays with known inhibitors

• Structural Studies:

  • Co-crystallization with potential inhibitors

  • NMR studies to identify binding interfaces

  • Hydrogen-deuterium exchange mass spectrometry to map binding sites

• Whole-Cell Approaches:

  • Construction of chimeric ATP synthases with Geobacillus sp. components in model organisms

  • Minimum inhibitory concentration (MIC) determination

  • Cytotoxicity assessment against mammalian cell lines

Research Findings:

Studies with mycobacterial ATP synthase have already demonstrated the potential of this approach. For example, TMC207 (bedaquiline) effectively inhibits mycobacterial ATP synthase by binding to subunit c. The atpE gene, encoding subunit c, has been identified as the target for mutations conferring resistance to this drug .

By analyzing how structural variations in Geobacillus sp. ATP synthase subunit c affect inhibitor binding compared to pathogenic bacteria, researchers can develop more selective antimicrobial compounds with reduced side effects and resistance potential. This approach represents a promising direction for addressing the growing challenge of antimicrobial resistance .

How might genetic engineering of Geobacillus sp. ATP synthase subunit c contribute to the development of bioenergy applications?

Genetic engineering of Geobacillus sp. ATP synthase subunit c offers promising avenues for bioenergy applications, leveraging the thermostability and efficiency of this component:

Bioenergy Applications:

• Enhanced ATP Production Systems:

  • Engineer c-subunits with modified proton binding sites to optimize the proton:ATP ratio

  • Develop hybrid systems with increased efficiency at elevated temperatures

  • Create robust ATP production systems for bioreactor applications

• Artificial Photosynthesis Integration:

  • Couple engineered ATP synthase complexes with light-harvesting systems

  • Develop light-driven ATP regeneration systems for biocatalysis

  • Create semi-synthetic energy conversion platforms combining biological and artificial components

• Bioelectrochemical Systems:

  • Immobilize engineered ATP synthase on electrode surfaces

  • Develop ATP-producing bioelectronic devices driven by electrical potential

  • Create bioelectrochemical cells with enhanced stability at various operating conditions

Engineering Approaches:

• Rational Design Strategies:

  • Modify c-ring stoichiometry to adjust the ATP:proton ratio

  • Engineer interfaces between subunits to enhance stability

  • Introduce non-natural amino acids at key positions to optimize function

• Directed Evolution:

  • Develop high-throughput screening systems for ATP synthase function

  • Apply error-prone PCR to generate variant libraries

  • Select for variants with enhanced performance under defined conditions

• Chimeric Enzyme Development:

  • Combine thermostable Geobacillus components with functional elements from other species

  • Create hybrid systems with tailored properties for specific applications

  • Engineer interface regions to ensure compatibility between components from different sources

Research Challenges and Solutions:

These engineering approaches leverage the natural thermostability and efficiency of Geobacillus sp. ATP synthase components while enhancing their utility for bioenergy applications, potentially contributing to sustainable energy solutions .

What research gaps remain in understanding the evolution and adaptation of ATP synthase subunit c across thermophilic bacterial species?

Despite significant progress in understanding ATP synthase structure and function, several important research gaps remain regarding the evolution and adaptation of subunit c in thermophilic bacteria such as Geobacillus species:

Current Research Gaps:

• Evolutionary Trajectory:

  • Limited understanding of the evolutionary processes that led to thermophilic adaptations in ATP synthase subunit c

  • Incomplete knowledge of how c-ring stoichiometry evolved across species and its relationship to environmental adaptation

  • Unclear evolutionary relationship between ATP synthase components and environmental niche specialization

• Structure-Function Relationships:

  • Incomplete characterization of specific amino acid residues that confer thermostability

  • Limited understanding of how thermophilic adaptations affect proton translocation efficiency

  • Unclear relationship between c-ring size and optimal operating temperature across species

• Comparative Genomics:

  • Need for expanded analysis of ATP synthase gene clusters across diverse thermophilic species

  • Limited understanding of how horizontal gene transfer may have influenced ATP synthase evolution

  • Incomplete knowledge of regulatory elements controlling ATP synthase expression in thermophiles

Proposed Research Directions:

• Comprehensive Phylogenetic Analysis:

  • Construct detailed phylogenies of ATP synthase components across thermophilic and mesophilic species

  • Apply molecular clock analyses to estimate when thermophilic adaptations arose

  • Correlate evolutionary changes with environmental transitions

• Ancestral Sequence Reconstruction:

  • Computationally reconstruct ancestral sequences of ATP synthase subunit c

  • Express and characterize these reconstructed proteins

  • Identify key mutations that enabled thermophilic adaptation

• Comparative Functional Studies:

  • Develop standardized assays to compare ATP synthase performance across species

  • Evaluate proton translocation efficiency at different temperatures

  • Measure ATP synthesis rates and coupling efficiency across evolutionary variants

• Integrated Structural Biology:

  • Obtain high-resolution structures of ATP synthase from diverse thermophilic species

  • Compare structural features across the temperature adaptation spectrum

  • Identify conserved and variable regions associated with thermostability

These research directions would significantly enhance our understanding of how ATP synthase subunit c has evolved and adapted across thermophilic bacterial species, providing insights into both fundamental evolutionary processes and potential applications in biotechnology and synthetic biology .