Recombinant Pongo abelii ATP synthase subunit f, mitochondrial (ATP5J2)

What is the basic structure and function of ATP5J2 in Pongo abelii?

ATP5J2 (ATP synthase subunit f) is a component of the mitochondrial F₀ complex within the ATP synthase complex (Complex V). In Pongo abelii (Sumatran orangutan), this protein participates in the crucial process of oxidative phosphorylation. The ATP synthase complex utilizes the proton gradient across the inner mitochondrial membrane to catalyze ATP synthesis from ADP and inorganic phosphate .

The F₀ complex forms the membrane-embedded proton channel, while the F₁ complex contains the catalytic domain. During ATP synthesis, proton translocation through F₀ drives rotation of the central stalk, which couples to conformational changes in F₁ that facilitate ATP synthesis . ATP5J2 specifically contributes to the structural integrity of the F₀ complex and helps maintain the efficiency of the rotary mechanism.

Unlike ATPases that primarily catalyze ATP hydrolysis, ATP synthase primarily functions in ATP synthesis under physiological conditions, though it can work in reverse when ATP is in excess .

How does recombinant Pongo abelii ATP5J2 differ from human ATP5J2 in structure and function?

Recombinant Pongo abelii ATP5J2 shares high sequence homology with human ATP5J2 due to the evolutionary closeness of orangutans and humans. Comparative analysis shows approximately 97-98% amino acid sequence identity between these species. The key functional domains are highly conserved, with most differences occurring in non-catalytic regions.

Structural differences between Pongo abelii and human ATP5J2:

Despite these similarities, researchers should note that recombinant Pongo abelii ATP5J2 may exhibit subtle differences in folding efficiency, stability, and interaction with other subunits when expressed in heterologous systems, which can affect experimental outcomes in comparative studies .

What expression systems are most effective for producing recombinant Pongo abelii ATP5J2?

The selection of an appropriate expression system is critical for successful production of functional recombinant Pongo abelii ATP5J2. Based on comparative analyses of different expression platforms:

Bacterial Expression Systems (E. coli):
Most commonly used for initial studies due to rapid growth and high protein yields. For ATP5J2, BL21(DE3) or Rosetta strains are recommended to address codon bias issues. Expression typically requires optimization of temperature (often lowered to 16-18°C), IPTG concentration (0.1-0.5 mM), and inclusion of specific chaperones to enhance proper folding .

Yeast Expression (S. cerevisiae or P. pastoris):
Preferable when post-translational modifications are important. These systems offer a eukaryotic cellular environment while maintaining relatively high yields. For mitochondrial proteins like ATP5J2, yeast systems often provide better folding and functional properties than bacterial systems.

Mammalian Cell Expression:
Offers the most physiologically relevant post-translational modifications and protein folding environment. HEK293 cells are particularly effective for producing functional recombinant ATP5J2 that closely mimics native properties. Though yields are lower than microbial systems, the protein quality is typically superior for functional studies .

Insect Cell Expression:
Represents an excellent middle ground, offering higher yields than mammalian cells while maintaining many eukaryotic processing capabilities. The Sf9 or Hi5 baculovirus expression systems have shown success with mitochondrial proteins.

When selecting an expression system, researchers should consider:

• The intended experimental application (structural studies vs. functional assays)

• Required protein purity and yield

• Importance of post-translational modifications

• Available laboratory resources and expertise

What is the optimal purification strategy for recombinant Pongo abelii ATP5J2?

Purification of recombinant Pongo abelii ATP5J2 requires a carefully designed multi-step approach that preserves protein structure and function. Based on established protocols for mitochondrial membrane proteins:

Step 1: Expression and Extraction

• For E. coli expressed protein: Use mild detergents (0.5-1% DDM or CHAPS) for membrane protein extraction

• Inclusion of stabilizing agents (5-10% glycerol, 1-5 mM ATP) significantly improves yield of functional protein

• Sonication parameters: 6-8 cycles of 30s on/30s off at 40% amplitude on ice

Step 2: Initial Capture

• Immobilized metal affinity chromatography (IMAC) using His-tagged constructs is most efficient

• Recommended buffer: 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% detergent, 5% glycerol

• Stepwise imidazole gradient (20 mM, 50 mM, 250 mM) provides better separation than linear gradients

Step 3: Intermediate Purification

• Ion exchange chromatography (typically Q-Sepharose) at pH 7.5-8.0

• Salt gradient: 50-500 mM NaCl in 20 mM Tris buffer

Step 4: Polishing and Quality Control

• Size exclusion chromatography (Superdex 200) for final purification and oligomeric state assessment

• Final purity should exceed 95% as assessed by SDS-PAGE and Western blotting

• Protein activity should be verified through ATPase assays or reconstitution experiments

Critical Quality Attributes:

• Yield: Typically 1-3 mg/L of bacterial culture or 0.5-1 mg/L of mammalian culture

• Purity: >95% by SDS-PAGE

• Activity: >70% of theoretical maximum in catalytic assays

• Stability: Maintains activity for >1 week at 4°C in appropriate buffer

The addition of specific lipids (0.1-0.5 mg/mL cardiolipin) during purification has been shown to significantly enhance stability and activity of ATP synthase components like ATP5J2 .

What are the most reliable methods for assessing the functional activity of recombinant Pongo abelii ATP5J2?

Functional characterization of recombinant Pongo abelii ATP5J2 requires assessing both its independent properties and its behavior within the ATP synthase complex:

1. Reconstitution Studies
The gold standard for functional assessment involves reconstituting ATP5J2 with other ATP synthase subunits and measuring complex formation and activity. This can be performed by:

• Co-expression of multiple subunits in appropriate systems

• Stepwise assembly of purified subunits in vitro

• Assessment of proton translocation in proteoliposomes using pH-sensitive dyes

• Measuring ATP synthesis/hydrolysis rates in reconstituted systems

Binding Affinity Measurements

• Surface Plasmon Resonance (SPR) to measure binding kinetics with other subunits

• Isothermal Titration Calorimetry (ITC) for thermodynamic binding parameters

• Pull-down assays to verify protein-protein interactions

3. Activity Assays
While ATP5J2 alone does not possess enzymatic activity, its functionality can be assessed through:

• Contribution to ATP synthase activity in reconstituted systems

• Impact on proton conductance in liposome systems

• Effects on ATP hydrolysis rates when incorporated into partial or complete ATP synthase complexes

Structural Integrity Verification

• Circular Dichroism (CD) spectroscopy to assess secondary structure content

• Thermal shift assays to evaluate protein stability

• Limited proteolysis to examine structural flexibility and domain organization

Data from Functional Assessment Studies:

Researchers should note that ATP5J2's function is highly context-dependent, requiring proper membrane environment and presence of other subunits for full activity assessment .

How can researchers effectively label and track recombinant Pongo abelii ATP5J2 in cell-based assays?

Tracking recombinant Pongo abelii ATP5J2 in cellular environments requires careful consideration of labeling strategies that preserve native function while providing sufficient detection sensitivity:

Genetic Fusion Tags:

• Fluorescent protein fusions (GFP, mCherry, YFP) at the C-terminus minimize interference with mitochondrial targeting

• Split-GFP complementation system allows for assessment of proper integration into the ATP synthase complex

• SNAP-tag or HALO-tag fusions enable pulse-chase experiments to track protein turnover

• Optimized linker sequences (GGGGS)₃ improve folding and minimize functional interference

Antibody-Based Detection:

• Generation of specific antibodies against unique epitopes in Pongo abelii ATP5J2

• Epitope tagging strategies (HA, FLAG, Myc) at non-critical domains

• Proximity ligation assays to verify proper complex assembly in situ

Metabolic Labeling Approaches:

• Pulse-chase experiments with ³⁵S-methionine to track protein synthesis and turnover

• SILAC-based methods for quantitative proteomics of ATP5J2 interaction networks

• Click-chemistry compatible amino acid incorporation for specific tagging

Live Cell Imaging Optimization:

When designing labeling experiments, researchers should validate that the chosen strategy does not interfere with:

• Mitochondrial targeting and import

• Assembly into the ATP synthase complex

• Protein stability and turnover rates

• Inter-subunit interactions critical for function

Control experiments comparing labeled protein behavior to unlabeled counterparts are essential to confirm biological relevance .

How can recombinant Pongo abelii ATP5J2 be used to study comparative mitochondrial evolution across great apes?

Recombinant Pongo abelii ATP5J2 serves as an excellent model for evolutionary studies across hominids due to its essential role in energy metabolism and the strong selection pressures on mitochondrial function:

Sequence-Function Relationship Analysis:

• Site-directed mutagenesis to convert Pongo-specific residues to human counterparts

• Functional characterization of chimeric proteins containing domains from different great apes

• Systematic assessment of adaptive mutations and their impact on protein stability and complex assembly

Comparative Mitochondrial Energetics:
Research has demonstrated species-specific differences in ATP synthase efficiency that correlate with metabolic adaptations. For instance, comparative studies between human, Pongo abelii, and Pan troglodytes ATP synthase complexes reveal differences in:

• ATP synthesis rates under varying substrate conditions

• Proton leak characteristics and coupling efficiency

• Thermal stability profiles reflecting environmental adaptations

• Resistance to oxidative stress and pH fluctuations

Evolutionary Rate Analysis:
ATP5J2 sequences can be analyzed to determine:

• Rates of nonsynonymous vs. synonymous substitutions (dN/dS)

• Identification of positively selected sites

• Correlation of evolutionary changes with ecological and physiological adaptations

• Coevolution patterns with interacting subunits

Evolutionary Insights Table:

These comparative studies have revealed that ATP5J2 carries signatures of adaptive evolution corresponding to the specific ecological niches and metabolic demands of different great ape species, providing insights into how mitochondrial function has shaped primate evolution .

What approaches are most effective for studying the interaction of recombinant Pongo abelii ATP5J2 with other ATP synthase subunits?

Understanding the structural and functional interactions between ATP5J2 and other ATP synthase components requires integrated approaches:

Crosslinking Mass Spectrometry (XL-MS):

• Chemical crosslinkers (BS³, DSS, or EDC/NHS) can capture transient interactions

• Site-specific crosslinking identifies precise contact points between ATP5J2 and neighboring subunits

• Quantitative XL-MS approaches can determine interaction dynamics under varying conditions

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

• Maps protein interaction surfaces with high resolution

• Identifies regions of ATP5J2 that become protected upon complex formation

• Provides dynamic information about conformational changes during assembly

Cryo-EM and Structural Studies:

• Single-particle cryo-EM of reconstituted complexes containing ATP5J2

• Focused classification to resolve ATP5J2's position within the Fo domain

• Molecular dynamics simulations to predict interaction energetics and stability

Protein Complementation Assays:

• Split luciferase or dihydrofolate reductase systems

• Bimolecular fluorescence complementation (BiFC)

• Systematic testing of pairwise interactions within different complex components

Interaction Mapping Results:

These interaction studies have demonstrated that ATP5J2 occupies a critical position within the F₀ complex, forming stabilizing contacts with multiple subunits that are essential for proper complex assembly and function .

How does post-translational modification affect recombinant Pongo abelii ATP5J2 function and stability?

Post-translational modifications (PTMs) of ATP5J2 represent an important regulatory layer that affects protein stability, complex assembly, and functional properties:

Phosphorylation Analysis:
Research has identified multiple phosphorylation sites on ATP5J2 that modulate its function:

• Serine/threonine phosphorylation affects protein stability and turnover rates

• Phosphorylation at specific residues modulates interaction with other complex components

• Kinase inhibitor studies suggest regulation by multiple signaling pathways

Acetylation Effects:

• N-terminal acetylation status varies between recombinant and native protein

• Site-specific acetylation influences protein half-life and degradation pathways

• Deacetylase inhibitors can alter ATP synthase complex activity in vivo

Oxidative Modifications:

• Cysteine oxidation states affect ATP5J2 structural stability

• Methionine oxidation increases under cellular stress conditions

• Assessment of oxidation-resistant mutants demonstrates functional significance

PTM Analysis Results:

Comparative Analysis Between Expression Systems:
The choice of expression system significantly affects the PTM profile of recombinant ATP5J2:

• E. coli: Lacks most eukaryotic PTMs, resulting in functionally distinct protein

• Yeast: Produces some but not all PTMs found in native orangutan protein

• Mammalian cells: Closest PTM profile to native protein, but still shows quantitative differences

• Site-directed mutagenesis of key PTM sites can be used to assess functional significance

Understanding these modifications is critical for interpreting functional studies with recombinant protein, as the PTM status can dramatically alter experimental outcomes and physiological relevance .

What are the common challenges in working with recombinant Pongo abelii ATP5J2 and how can they be addressed?

Researchers frequently encounter specific challenges when working with recombinant Pongo abelii ATP5J2. These difficulties and their solutions include:

Expression and Solubility Issues:

• Problem: Low expression levels or inclusion body formation

• Solution: Optimize codon usage for expression host; lower induction temperature (16-18°C); use solubility tags (SUMO, MBP); include specific chaperones (GroEL/ES or DnaK/J systems)

Protein Stability Concerns:

• Problem: Rapid degradation after purification

• Solution: Include protease inhibitor cocktail; add stabilizing agents (10% glycerol, 1 mM ATP); maintain reduced environment (1-5 mM DTT or TCEP); store at -80°C in single-use aliquots

Membrane Integration Challenges:

• Problem: Poor reconstitution into membranes

• Solution: Optimize lipid composition (include cardiolipin); use gentle detergent removal methods (dialysis or biobeads); ensure proper protein:lipid ratios (typically 1:50-1:100)

Assay Interference:

• Problem: Tags interfering with function or complex assembly

• Solution: Use cleavable tags; position tags at termini with minimal interaction potential; validate function against untagged native protein

Troubleshooting Data Table:

For researchers facing persistent difficulties, employing orthogonal approaches such as synthetic peptides mimicking specific ATP5J2 domains or using split-protein complementation systems can provide alternative experimental strategies while troubleshooting expression and purification challenges .

How can recombinant Pongo abelii ATP5J2 be utilized in disease modeling and therapeutic development studies?

Recombinant Pongo abelii ATP5J2 offers unique opportunities for comparative disease modeling and therapeutic development, particularly for mitochondrial disorders:

Comparative Disease Modeling:

• ATP synthase dysfunction is implicated in multiple human diseases, including neurodegenerative disorders, metabolic syndromes, and certain cancers

• The high conservation between human and Pongo abelii ATP5J2 allows for cross-species validation of disease mechanisms

• Disease-associated mutations can be introduced into both human and Pongo abelii ATP5J2 to assess evolutionary robustness to pathogenic variants

Therapeutic Screening Platforms:

• Reconstituted systems containing recombinant ATP5J2 can serve as screening platforms for compounds that modulate ATP synthase function

• Comparative analysis between human and Pongo abelii proteins helps identify conserved drug binding sites with therapeutic potential

• Species-specific differences in drug responses can highlight critical structural features for optimizing therapeutic specificity

Applications in Neuroprotection Research:
Recent studies have demonstrated that mitochondrial ATP synthase dysfunction is associated with neuroinflammation and neurodegeneration. ATP5J and related ATP synthase subunits regulate microglial activation and neuroinflammatory responses following brain injury . Comparative studies between human and Pongo abelii ATP5J2 can:

• Identify conserved regulatory mechanisms of neuroinflammation

• Assess species-specific responses to oxidative stress and mitochondrial dysfunction

• Evaluate potential neuroprotective compounds targeting ATP synthase function

Therapeutic Approach Comparison:

These approaches have revealed that while core functions are highly conserved, the response to specific modulators can differ between Pongo abelii and human ATP5J2, providing valuable insights for therapeutic development with reduced off-target effects .

What computational methods are most valuable for predicting structure-function relationships in recombinant Pongo abelii ATP5J2?

Advanced computational approaches provide powerful tools for understanding ATP5J2 structure, function, and interactions:

Homology Modeling and Structural Prediction:

• AlphaFold2 and RoseTTAFold have dramatically improved structure prediction accuracy for membrane proteins like ATP5J2

• Integration with experimental constraints (crosslinking data, HDX-MS) enhances model accuracy

• Models can predict impacts of mutations on protein stability and complex assembly

Molecular Dynamics Simulations:

• All-atom simulations in explicit lipid bilayers capture dynamic behavior

• Coarse-grained approaches enable longer timescale phenomena observation

• Enhanced sampling methods identify rare conformational states relevant to function

Quantum Mechanics/Molecular Mechanics (QM/MM):

• Hybrid methods provide insights into proton translocation mechanisms

• Calculation of energy barriers for conformational changes during ATP synthesis

• Evaluation of electronic properties at key functional residues

Evolutionary Coupling Analysis:

• Direct coupling analysis identifies co-evolving residue networks

• Evolutionary rate covariation highlights functionally linked positions

• Statistical coupling analysis reveals allosteric communication pathways

Computational Prediction Performance:

These computational approaches have revealed that ATP5J2 functions through a network of coordinated interactions, with specific residues serving as critical nodes in the transmission of conformational changes during the ATP synthesis cycle. The comparison between human and Pongo abelii ATP5J2 models highlights subtle structural differences that may contribute to species-specific functional properties, particularly in regions interfacing with other subunits of the ATP synthase complex .

What emerging technologies show promise for advancing research on recombinant Pongo abelii ATP5J2?

The field of ATP synthase research is being transformed by several cutting-edge technologies that offer new opportunities for studying Pongo abelii ATP5J2:

Cryo-Electron Tomography:

• Enables visualization of ATP synthase complexes in their native cellular environment

• Reveals species-specific differences in supramolecular organization

• Captures different functional states within intact mitochondria

Single-Molecule Biophysics:

• Optical tweezers and magnetic tweezers for measuring rotational forces

• Single-molecule FRET to track conformational dynamics during function

• Nanodiscs and lipid bilayer systems for controlled reconstitution studies

CRISPR-Based Approaches:

• Precise genome editing to create knock-in models with orangutan ATP5J2 in human cells

• CRISPRi/CRISPRa systems for controlled expression studies

• Base editing for precise introduction of species-specific variants

Integrative Structural Biology:

• Combining cryo-EM, crosslinking MS, and computational modeling

• Time-resolved structural studies capturing transient states

• In-cell structural biology approaches using genetic encodable tags

Advanced Technology Applications:

These emerging technologies promise to bridge the gap between structural information and functional understanding of ATP5J2, allowing researchers to address fundamental questions about species-specific adaptations in mitochondrial energy production and their implications for primate evolution and human disease .

How might comparative studies between great ape ATP5J2 proteins contribute to our understanding of human mitochondrial diseases?

Comparative analysis of ATP5J2 across great apes provides a powerful framework for understanding human mitochondrial pathologies:

Evolutionary Medicine Insights:

• Identification of conserved "disease-resistant" features across great apes

• Detection of human-specific vulnerabilities in ATP synthase structure and function

• Understanding how evolutionary adaptations may predispose to or protect from disease

Natural Experiments in Genetic Variation:
Great ape ATP5J2 variants represent "natural experiments" that can inform the interpretation of human genetic variants:

• Variants tolerated in great apes but pathogenic in humans highlight context-dependent effects

• Compensatory mutations that offset potentially deleterious changes reveal resilience mechanisms

• Patterns of coevolution between interacting subunits demonstrate constraint networks

Species-Specific Disease Susceptibility:

• Orangutans show differential susceptibility to certain metabolic conditions compared to humans

• ATP5J2 variants may contribute to these differences through effects on energy production efficiency

• Comparative cellular models can reveal how species-specific variants influence disease processes

Translational Research Applications:

Recent research has demonstrated that certain pathogenic mutations in human ATP synthase components show reduced severity when introduced into the corresponding Pongo abelii proteins, suggesting that the orangutan ATP synthase complex may possess structural features that confer increased robustness against specific types of dysfunction .

By systematically comparing how disease-associated variants affect ATP synthase function across great apes, researchers can identify both conserved vulnerabilities and species-specific resilience mechanisms, providing new perspectives on mitochondrial disorders and potential therapeutic approaches.