Recombinant Herpetosiphon aurantiacus ATP synthase subunit alpha (atpA), partial

How can I express recombinant H. aurantiacus atpA in laboratory conditions?

Expressing recombinant H. aurantiacus atpA typically follows standard recombinant protein expression protocols with specific considerations for this protein. Based on approaches used for other ATP synthase subunits, the following methodology is recommended:

• Gene Cloning:

  • Amplify the atpA gene from H. aurantiacus genomic DNA using PCR with specific primers designed to include appropriate restriction sites

  • Clone the amplified gene into an expression vector (e.g., pET series for E. coli expression)

  • Verify the sequence to ensure no mutations were introduced during PCR

• Expression System Selection:

  • E. coli BL21(DE3) or similar strains are commonly used for recombinant ATP synthase subunit expression

  • Consider using specialized strains for membrane proteins if working with the full-length atpA

• Expression Conditions:

  • Induce expression with IPTG (0.1-1.0 mM) when cultures reach mid-log phase

  • Lower temperatures (16-25°C) often yield better results for ATP synthase subunits

  • Extended expression times (12-24 hours) may be necessary for optimal yields

• Protein Purification:

  • Use affinity tags (His-tag, GFP fusion) to facilitate purification

  • Include protease inhibitors in all buffers as ATP synthase subunits can be prone to proteolysis, as observed in similar studies

  • Consider purification under native conditions to maintain protein folding

Similar approaches have been successfully used for ATP synthase subunits from other organisms, as evidenced by studies where the atpA ORF was fused to GFP for localization and interaction studies .

What are the common challenges in purifying recombinant atpA protein?

Purification of recombinant atpA presents several challenges that researchers should anticipate:

• Proteolytic Degradation:

  • ATP synthase subunits are susceptible to proteolysis during purification

  • Evidence from studies on ValRS-ATP synthase interactions showed numerous bands corresponding to putative proteolytic products despite measures taken to avoid proteolysis

  • Solution: Use freshly prepared buffers with a cocktail of protease inhibitors, maintain cold temperatures throughout the purification process, and consider adding reducing agents

• Solubility Issues:

  • As a component of a membrane-associated complex, atpA may have hydrophobic regions that affect solubility

  • Solution: Optimize buffer conditions with appropriate detergents (e.g., DDM, CHAPS) or consider extracting the protein under native conditions that preserve its natural conformation

• Maintaining Functional Conformation:

  • The alpha subunit's functionality depends on proper folding

  • Solution: Avoid harsh purification conditions that might denature the protein; consider purifying under non-denaturing conditions

• Co-purifying Contaminants:

  • ATP synthase subunits often co-purify with other components of the complex or abundant cellular proteins

  • Studies have shown that RuBisCO is a frequent contaminant in ATP synthase purifications

  • Solution: Implement multiple purification steps (e.g., ion exchange after affinity chromatography) and validate protein purity through methods like mass spectrometry

• Low Yield:

  • Expression levels of functional recombinant ATP synthase subunits can be low

  • Solution: Optimize codon usage for the expression system, test different fusion tags, and screen multiple expression conditions to maximize yield

How can I verify the identity and purity of purified recombinant atpA?

Verifying the identity and purity of recombinant H. aurantiacus atpA should involve multiple complementary analytical techniques:

• Mass Spectrometry Analysis:

  • MALDI-TOF/MS has been effectively used to identify ATP synthase subunits in previous studies

  • Tryptic digest followed by peptide mass fingerprinting can confirm protein identity

  • Intact protein mass analysis can verify the full-length protein and detect any modifications

• Western Blotting:

  • Use antibodies specific to atpA or to affinity tags if present

  • Compare migration pattern with expected molecular weight

  • Assess the presence of degradation products or contaminating proteins

• Activity Assays:

  • While the isolated alpha subunit may not show ATP synthesis activity, ATPase activity can sometimes be measured

  • Binding assays with other ATP synthase subunits can confirm proper folding and function

• Circular Dichroism (CD) Spectroscopy:

  • Assess secondary structure content to verify proper folding

  • Compare with CD spectra of ATP synthase alpha subunits from related organisms

• Size Exclusion Chromatography:

  • Analyze oligomeric state and homogeneity of the purified protein

  • Detect potential aggregation or complex formation

• Protein Purity Assessment:

  • SDS-PAGE with Coomassie or silver staining to evaluate purity

  • Densitometric analysis to quantify purity percentage

  • Consider 2D gel electrophoresis for more detailed purity assessment

How can I investigate protein-protein interactions involving H. aurantiacus atpA?

Investigating protein-protein interactions of H. aurantiacus atpA requires sophisticated approaches that can capture both stable and transient interactions:

• GFP Fusion Pull-down Assays:

  • Creating atpA-GFP fusion constructs has proven effective for studying ATP synthase interactions

  • This approach successfully identified interactions between ValRS and ATP synthase in Anabaena

  • The method involves:
    a) Generating an atpA-GFP fusion construct
    b) Expressing the fusion protein in the appropriate system
    c) Performing pull-down assays with anti-GFP antibodies
    d) Analyzing co-purifying proteins by mass spectrometry

• In vivo Crosslinking:

  • Crosslinking studies have successfully demonstrated interactions between ATP synthase subunits and other proteins

  • In Anabaena, cultures expressing AtpA-GFP were subjected to in vivo crosslinking followed by purification with anti-GFP antibodies coupled to magnetic beads

  • This approach can capture transient or weak interactions that might be lost during conventional purification

• Clear Native PAGE (CN-PAGE):

  • CN-PAGE has been used to detect ATP synthase complexes and their interactions with other proteins

  • In previous studies, ATP synthase subunits were observed in two distinct complexes in CN-PAGE, with the high molecular weight complex depending on the presence of interaction partners

  • This technique preserves native protein-protein interactions and complex integrity

• Yeast Two-Hybrid or Bacterial Two-Hybrid Systems:

  • These systems can screen for potential interaction partners

  • Consider using domain-specific constructs if the full-length protein proves challenging

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • These techniques provide quantitative measurements of binding kinetics

  • Immobilize purified recombinant atpA on a sensor chip and flow potential binding partners over the surface

How can I assess the enzymatic activity of recombinant H. aurantiacus atpA in isolation and in reconstituted systems?

Assessing the enzymatic activity of recombinant atpA requires different approaches depending on whether you're working with the isolated subunit or attempting to reconstitute functional complexes:

• Isolated Alpha Subunit Activity:

  • ATP Binding Assays:

    • Use fluorescent ATP analogs to measure binding affinity

    • Isothermal titration calorimetry (ITC) for binding thermodynamics

  • Limited ATPase Activity:

    • While the alpha subunit alone typically shows minimal ATPase activity, sensitive assays can detect basal activity

    • Use a coupled enzyme assay system (with pyruvate kinase and lactate dehydrogenase) to detect ATP hydrolysis by monitoring NADH oxidation

• Reconstituted Complex Activity:

  • Complex Assembly:

    • Combine purified alpha subunit with other essential components (β, γ, etc.)

    • Verify complex formation by native gel electrophoresis or size exclusion chromatography

  • ATP Synthesis Activity:

    • Create proteoliposomes with the reconstituted complex

    • Generate a proton gradient (pH jump or valinomycin-induced K+ diffusion potential)

    • Measure ATP production using luciferase-based assays

• Functional Complementation:

  • Express H. aurantiacus atpA in ATP synthase-deficient bacterial strains

  • Assess restoration of growth under conditions requiring ATP synthase function

  • Compare complementation efficiency with atpA from other species

• Inhibitor Studies:

  • Test sensitivity to known ATP synthase inhibitors

  • Resveratrol has been shown to bind to the ATP synthase in the F₁-domain and partially inhibit both ATP hydrolysis and ATP synthesis

  • Oligomycin A can be used as a reference inhibitor, though it has similar IC₅₀ values for bacterial and human mitochondrial ATP synthases

• Mutational Analysis:

  • Create targeted mutations in conserved catalytic residues

  • Analyze the effects on ATP binding and hydrolysis

  • Compare with equivalent mutations in well-characterized ATP synthase systems

What are the optimal conditions for expressing and purifying functional recombinant H. aurantiacus atpA?

Optimizing expression and purification of functional recombinant H. aurantiacus atpA requires careful consideration of multiple factors:

• Expression System Selection:

  • E. coli BL21(DE3) remains the most common system for recombinant ATP synthase subunit expression

  • Consider C41(DE3) or C43(DE3) strains specifically designed for membrane protein expression

  • For challenging expressions, alternative systems such as Bacillus subtilis or cell-free expression systems may be evaluated

• Vector and Fusion Tag Optimization:

  • N-terminal vs. C-terminal tags can significantly affect expression and solubility

  • GFP fusions have been successfully used for ATP synthase subunits and provide visual confirmation of expression

  • Consider a cleavable tag system (TEV or PreScission protease) to obtain native protein after purification

• Expression Condition Optimization:

  • Temperature: Lower temperatures (16-20°C) often yield more properly folded protein

  • Inducer concentration: Typical IPTG concentrations range from 0.1-0.5 mM, but lower concentrations may improve folding

  • Media composition: Enriched media (TB, 2YT) typically yield higher biomass but defined minimal media may improve folding

  • Test expression matrix:

  Temperature	IPTG Concentration	Induction Time	Media Type
  16°C	0.1 mM	16-24 hours	TB
  25°C	0.25 mM	6-8 hours	2YT
  30°C	0.5 mM	3-4 hours	LB
  37°C	1.0 mM	2-3 hours	M9

• Purification Buffer Optimization:

  • pH range: Typically 7.0-8.0 for ATP synthase subunits

  • Salt concentration: 150-300 mM NaCl is standard, but higher concentrations may improve stability

  • Additives to consider:

    • 5-10% glycerol to prevent aggregation

    • 1-5 mM MgCl₂ (important for nucleotide binding proteins)

    • ATP or non-hydrolyzable ATP analogs (0.1-1 mM) to stabilize conformation

    • Reducing agents (1-5 mM DTT or 0.5-2 mM TCEP)

• Multi-step Purification Strategy:

  • Initial capture: Affinity chromatography based on fusion tag (IMAC for His-tag, etc.)

  • Intermediate purification: Ion exchange chromatography to remove similarly charged contaminants

  • Polishing: Size exclusion chromatography to separate oligomeric states and remove aggregates

  • Consider specific precautions to avoid proteolysis, which has been observed during purification of ATP synthase components

• Quality Control Checkpoints:

  • After each purification step, assess:

    • Purity by SDS-PAGE

    • Identity by Western blot or mass spectrometry

    • Oligomeric state by native PAGE or size exclusion chromatography

    • Activity using appropriate functional assays

How can I investigate the potential therapeutic applications of targeting H. aurantiacus atpA?

While the search results don't specifically address therapeutic applications of H. aurantiacus atpA, research on ATP synthase from other organisms provides a framework for such investigations:

• Antimicrobial Development Based on ATP Synthase Inhibition:

  • ATP synthase inhibition sensitizes S. aureus to antimicrobial peptides and neutrophil killing

  • Design experiments to test if:

    • H. aurantiacus atpA can complement ATP synthase function in pathogenic bacteria

    • Structural differences between H. aurantiacus atpA and human ATP synthase could be exploited for selective inhibitor design

    • Recombinant H. aurantiacus atpA can be used in high-throughput screening for novel inhibitors

• Cancer Research Applications:

  • ATP synthase α-subunit has been identified as a potential therapeutic target for breast cancer

  • Expression is upregulated in highly metastatic cells compared to low metastatic cells

  • Studies have shown ATP synthase α-subunit expression in 94.6% of breast cancer specimens compared to only 21.2% in normal breast tissues

  • Using recombinant H. aurantiacus atpA, researchers could:

    • Develop molecular probes to study ATP synthase structure and function

    • Create tools for comparative studies between bacterial and human ATP synthases

    • Identify structural elements that could inform development of selective inhibitors

• Experimental Design for Therapeutic Targeting:

  • Structure-Based Drug Design:

    • Use recombinant H. aurantiacus atpA to generate structural data

    • Perform in silico screening for potential binding molecules

    • Verify binding with biophysical techniques (SPR, ITC, etc.)

  • Functional Screening:

    • Develop high-throughput assays using recombinant protein

    • Screen compound libraries for inhibitors or modulators

    • Validate hits with secondary assays and selectivity testing

• Antibody Development:

  • Generate antibodies against recombinant H. aurantiacus atpA

  • Test cross-reactivity with human ATP synthase

  • Evaluate potential for diagnostic or therapeutic applications

  • Previous studies have shown antibodies against ATP synthase α-subunit can inhibit proliferation, migration and invasion in breast cancer cells

• Host-Pathogen Interaction Studies:

  • Investigate if H. aurantiacus atpA or its derivatives interact with human immune components

  • Test if structural elements from bacterial ATP synthase could be used to enhance AMP activity

  • Develop peptide mimetics based on bacterial ATP synthase structure that could potentiate antimicrobial activity

What imaging techniques can be used to study the localization and dynamics of atpA in bacterial cells?

Advanced imaging techniques are essential for understanding the localization and dynamics of ATP synthase components in bacterial cells:

• Fluorescent Protein Fusion Microscopy:

  • GFP-tagged ATP synthase subunits have been successfully used to study localization patterns

  • In Anabaena, AtpA-GFP showed thylakoidal localization in vegetative cells and enrichment at the cell poles in heterocysts

  • Methodology:

    • Create C-terminal or N-terminal GFP fusions of atpA

    • Express in appropriate bacterial system

    • Image using confocal or wide-field fluorescence microscopy

    • For optimal results, use photo-stable GFP variants (e.g., mNeonGreen, mEGFP)

• Super-Resolution Microscopy:

  • Techniques like STORM, PALM, or STED provide nanoscale resolution beyond the diffraction limit

  • These approaches can resolve individual ATP synthase complexes and their organization

  • Implementation:

    • Use photoactivatable or photoswitchable fluorophores for PALM/STORM

    • Optimize sample preparation to minimize background fluorescence

    • Consider dual-color imaging to study co-localization with other proteins

• Single-Particle Tracking:

  • Tracks the movement of individual ATP synthase complexes in live cells

  • Reveals dynamics, diffusion rates, and potential confinement zones

  • Approach:

    • Use photoactivatable fluorophores or quantum dots for labeling

    • Acquire time-lapse images at high frame rates

    • Analyze trajectories to determine diffusion coefficients and confinement

• Fluorescence Recovery After Photobleaching (FRAP):

  • Measures protein mobility within membranes

  • Can reveal if ATP synthase complexes are freely diffusing or restricted

  • Protocol:

    • Express atpA-GFP fusion in bacteria

    • Photobleach a defined region of interest

    • Monitor fluorescence recovery over time

    • Calculate diffusion coefficients from recovery curves

• Förster Resonance Energy Transfer (FRET):

  • Detects interactions between ATP synthase subunits or with other proteins

  • Provides information on conformational changes during function

  • Implementation:

    • Create donor-acceptor fluorophore pairs on different components

    • Measure energy transfer efficiency

    • Correlate with functional states of the enzyme

• Cryo-Electron Tomography:

  • Visualizes ATP synthase complexes in their native cellular environment

  • Provides structural information in the cellular context

  • Approach:

    • Vitrify bacterial cells expressing tagged atpA

    • Collect tilt series for tomographic reconstruction

    • Identify ATP synthase complexes through sub-tomogram averaging

How can I characterize the interaction between H. aurantiacus atpA and potential inhibitors for drug development?

Characterizing interactions between H. aurantiacus atpA and potential inhibitors requires a systematic approach combining biophysical, structural, and functional techniques:

• Binding Affinity and Kinetics Determination:

  • Surface Plasmon Resonance (SPR):

    • Immobilize purified recombinant atpA on a sensor chip

    • Flow inhibitor solutions at varying concentrations

    • Calculate association (k₁) and dissociation (k₋₁) rate constants

    • Determine equilibrium dissociation constant (KD)

  • Isothermal Titration Calorimetry (ITC):

    • Measures binding thermodynamics (ΔH, ΔS, ΔG)

    • Provides stoichiometry of binding

    • Works with inhibitors of diverse solubility properties

  • Microscale Thermophoresis (MST):

    • Requires small amounts of protein

    • Works well for membrane proteins and challenging targets

    • Detects binding in near-native conditions

• Structural Characterization of Binding:

  • X-ray Crystallography:

    • Co-crystallize atpA with inhibitors to determine binding site

    • Provides atomic-level details of interaction interfaces

    • Identifies key residues involved in binding

  • Nuclear Magnetic Resonance (NMR):

    • Chemical shift perturbation to map binding interface

    • Dynamics of protein-inhibitor complex

    • Works well for weak or transient interactions

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

    • Maps regions protected from exchange upon inhibitor binding

    • Does not require crystallization

    • Provides information on conformational changes

• Functional Impact Assessment:

  • Enzyme Activity Assays:

    • Measure IC₅₀ and Ki values using ATP hydrolysis assays

    • Determine mechanism of inhibition (competitive, non-competitive, uncompetitive)

    • Assess reversibility of inhibition

  • Thermal Shift Assays:

    • Monitor protein stability changes upon inhibitor binding

    • High-throughput screening capability

    • Can provide indirect evidence of binding

• Comparative Studies with Known Inhibitors:

  • Resveratrol binds to the F₁-domain of ATP synthase and partially inhibits both ATP hydrolysis and ATP synthesis

  • Oligomycin A is a reference inhibitor but has similar IC₅₀ values between bacterial and human mitochondrial ATP synthases

  • Structure-activity relationship studies comparing novel compounds with established inhibitors

• Selectivity Profiling:

  • Counter-screening against human ATP synthase

  • Testing against panel of other nucleotide-binding proteins

  • Assessing off-target effects in cellular systems

How should I interpret contradictory results when studying atpA function across different experimental systems?

When encountering contradictory results in atpA research across different experimental systems, a systematic approach to data interpretation is essential:

• Experimental System Differences Assessment:

  • Expression System Variables:

    • Different host organisms may process or fold the recombinant protein differently

    • Post-translational modifications may vary between systems

    • Example: Studies of ATP synthase in different organisms have shown system-specific interactions, such as the ValRS-ATP synthase interaction in Anabaena

  • Assay Condition Variations:

    • Buffer components, pH, ionic strength, and temperature can significantly impact protein function

    • ATP synthase activity is particularly sensitive to membrane environment and proton gradient

    • Inhibitor effectiveness may vary with experimental conditions (e.g., resveratrol's inhibition of ATP synthase depends on specific binding conditions)

• Protein Construct Considerations:

  • Fusion Tags Impact:

    • Different tags (His, GFP, etc.) may affect protein folding, activity, or interactions

    • In studies of ATP synthase, GFP fusions have been successfully used but may influence certain interactions

  • Truncation Effects:

    • Full-length versus partial constructs may exhibit different properties

    • Domain-specific functions may be lost in truncated constructs

    • Studies have shown that deletion of specific domains (like CAAD in ValRS) can affect interactions with ATP synthase

• Physiological Context Differences:

  • In Vivo vs. In Vitro Discrepancies:

    • Cellular environment provides cofactors, interacting partners, and appropriate membrane context

    • Reconstituted systems may lack critical components

    • Example: ATP synthase mutants show different phenotypes in growth conditions versus nitrogen-depleted conditions

  • Cell Type-Specific Effects:

    • ATP synthase function may differ between cell types or developmental stages

    • In Anabaena, ATP synthase shows different localization patterns in vegetative cells versus heterocysts

• Systematic Troubleshooting Approach:

  • Control Experiment Matrix:

    • Design experiments with positive and negative controls for each variable

    • Include system-specific controls to validate assay performance

  • Parameter Isolation:

    • Systematically vary one parameter at a time to identify critical variables

    • Test protein activity across a range of conditions to establish optimal parameters

  • Data Integration Framework:

    • Develop a model that accommodates apparently contradictory results

    • Consider that different experimental systems may reveal different aspects of atpA function

What are the critical quality control checkpoints for ensuring reliable results when working with recombinant H. aurantiacus atpA?

Ensuring reliable results with recombinant H. aurantiacus atpA requires rigorous quality control at multiple stages of the experimental workflow:

• Gene and Construct Verification:

  • Sequence Confirmation:

    • Complete DNA sequencing of the expression construct

    • Verification of reading frame and absence of mutations

    • Confirmation of regulatory elements and fusion tag sequences

  • Expression Vector Stability:

    • Plasmid stability testing in the expression host

    • Verification of selection marker functionality

    • Assessment of copy number consistency

• Protein Expression Quality Control:

  • Expression Level Monitoring:

    • Time-course analysis of expression

    • Comparison of soluble versus insoluble fractions

    • Optimization of induction conditions to maximize functional protein

  • Protein Solubility Assessment:

    • Differentiate between truly soluble protein and solubilized aggregates

    • Analyze oligomeric state by native PAGE or size exclusion chromatography

    • ATP synthase subunits can form aggregates or incorrect assemblies if expression conditions are suboptimal

• Purification Quality Metrics:

  • Purity Assessment:

    • SDS-PAGE with densitometric analysis

    • Mass spectrometry to identify contaminants

    • ATP synthase purifications often contain contaminants like RuBisCO

  • Integrity Verification:

    • Western blot to confirm full-length protein

    • Mass spectrometry to detect proteolytic degradation

    • Studies have shown that ATP synthase subunits can undergo proteolysis during purification despite preventive measures

  • Homogeneity Evaluation:

    • Dynamic light scattering to assess polydispersity

    • Analytical ultracentrifugation for detailed characterization

    • Size exclusion chromatography to detect aggregates

• Functional Validation:

  • Activity Benchmarking:

    • Comparison with established standards or reference preparations

    • Dose-response relationships with known activators/inhibitors

    • ATP synthase activity can be measured through ATP synthesis/hydrolysis assays

  • Binding Properties:

    • Verification of nucleotide binding

    • Assessment of interaction with other ATP synthase subunits

    • Confirmation of inhibitor binding if relevant

• Stability Monitoring:

  • Storage Stability:

    • Activity retention during storage at different temperatures

    • Monitoring of degradation or aggregation over time

    • Optimization of buffer conditions for long-term stability

  • Freeze-Thaw Stability:

    • Quantification of activity loss after freeze-thaw cycles

    • Development of aliquoting strategy to minimize freeze-thaw events

    • Assessment of cryoprotectant effectiveness

What emerging technologies could advance our understanding of H. aurantiacus atpA structure-function relationships?

Several cutting-edge technologies are poised to revolutionize our understanding of ATP synthase subunit structure-function relationships:

• Cryo-Electron Microscopy (Cryo-EM) Advancements:

  • Single-particle cryo-EM now routinely achieves near-atomic resolution

  • Capable of resolving conformational heterogeneity in complex systems

  • Can capture different functional states of ATP synthase

  • Application to H. aurantiacus atpA:

    • Determine structure in different nucleotide-bound states

    • Visualize interactions with other ATP synthase subunits

    • Identify conformational changes during catalytic cycle

• Integrative Structural Biology Approaches:

  • Combining multiple structural techniques:

    • X-ray crystallography for high-resolution static structures

    • Cryo-EM for conformational ensembles

    • NMR for dynamics information

    • SAXS for solution conformation

  • Computational integration of diverse structural data

  • Benefits for atpA research:

    • Comprehensive view of structure-function relationships

    • Identification of allosteric networks

    • Improved models for inhibitor design

• Single-Molecule Techniques:

  • Single-molecule FRET:

    • Monitors conformational dynamics in real-time

    • Can observe rare or transient states

    • Correlates structural changes with function

  • Magnetic tweezers or optical traps:

    • Measure mechanical forces during ATP synthase operation

    • Directly observe rotational movements

  • Application to atpA research:

    • Monitor conformational changes during catalysis

    • Correlate ATP binding/hydrolysis with structural transitions

    • Investigate effects of inhibitors on molecular dynamics

• Advanced Computational Methods:

  • Molecular Dynamics Simulations:

    • Atomistic simulations now reaching millisecond timescales

    • Enhanced sampling techniques for rare event observation

    • Integration with experimental data via hybrid approaches

  • Machine Learning Applications:

    • Prediction of protein-protein interaction interfaces

    • Virtual screening for novel inhibitors

    • Extraction of patterns from large experimental datasets

  • Benefits for H. aurantiacus atpA:

    • Prediction of functional effects of mutations

    • Design of improved inhibitors

    • Understanding species-specific functional differences

• In-cell Structural Biology:

  • Cryo-electron tomography of intact cells

  • In-cell NMR spectroscopy

  • Proximity labeling methods (APEX, BioID)

  • Application to ATP synthase research:

    • Visualization of ATP synthase in native membrane environment

    • Mapping of protein-protein interactions in cellular context

    • Understanding of spatial organization and dynamics in vivo

• Synthetic Biology and Protein Engineering:

  • Creation of minimal functional units

  • Domain swapping between species

  • Incorporation of non-canonical amino acids for site-specific probes

  • Potential for atpA research:

    • Engineering chimeric proteins to identify functional domains

    • Introduction of biophysical probes at specific sites

    • Creation of optogenetically controlled variants

How might studies of H. aurantiacus atpA inform our understanding of ATP synthase evolution and specialization across species?

Comparative studies of H. aurantiacus atpA can provide valuable insights into ATP synthase evolution and specialization:

• Evolutionary Conservation and Divergence:

  • Sequence and Structural Homology Analysis:

    • Alignment of atpA sequences across diverse species

    • Identification of universally conserved versus lineage-specific features

    • Structure-based alignments to identify functional conservation despite sequence divergence

  • Phylogenetic Analysis:

    • Construction of robust phylogenetic trees

    • Correlation of ATP synthase evolution with ecological niches

    • Identification of horizontal gene transfer events

• Functional Adaptations in Different Environments:

  • Thermodynamic and Kinetic Adaptations:

    • Comparison of ATP synthesis/hydrolysis efficiency across species

    • Adaptation to different energy demands and metabolic contexts

    • Studies in other organisms have shown specialization of ATP synthase function in different cell types, such as heterocysts in Anabaena

  • Membrane Environment Adaptations:

    • Lipid interactions and requirements

    • Adaptation to different membrane compositions

    • Integration with other membrane protein complexes

• Novel Functions and Moonlighting Roles:

  • Non-canonical Functions:

    • ATP synthase components have been identified as cell-surface receptors for apparently unrelated ligands

    • In breast cancer, ATP synthase α-subunit is detected on the cell surface and may be involved in cancer progression

    • In bacteria like S. aureus, ATP synthase affects tolerance to antimicrobial peptides

  • Species-Specific Interactions:

    • Identification of lineage-specific binding partners

    • Studies in Anabaena revealed a specific interaction between ATP synthase and ValRS that may not be universal

    • Mapping interaction networks across species

• Structural Specializations:

  • Subunit Composition Variations:

    • Differences in subunit number and arrangement

    • Alternative isoforms in different species or tissues

    • Presence or absence of regulatory subunits

  • Catalytic Site Adaptations:

    • Variations in nucleotide binding affinity

    • Adaptations to different ATP/ADP ratios or pH environments

    • Species-specific inhibitor sensitivity

• Research Approaches and Methodologies:

  • Comparative Biochemistry:

    • Side-by-side functional assays of atpA from different species

    • Reciprocal complementation experiments

    • Chimeric proteins with domains from different species

  • Structural Comparisons:

    • Superposition of ATP synthase structures from diverse species

    • Identification of conformational differences in equivalent functional states

    • Analysis of species-specific structural elements

  • Systems Biology Approaches:

    • Integration of atpA function with metabolic networks

    • Comparison of regulatory mechanisms across species

    • Modeling of ATP synthase contribution to cellular energetics in different organisms