Recombinant Pongo abelii ATP synthase lipid-binding protein, mitochondrial (ATP5G2)

What is Recombinant Pongo abelii ATP synthase lipid-binding protein, mitochondrial (ATP5G2)?

Recombinant Pongo abelii ATP synthase lipid-binding protein (ATP5G2) is a full-length mitochondrial protein derived from orangutan (Pongo abelii) that functions as part of the C-subunit of mitochondrial ATP synthase complex. The commercially available recombinant form typically includes amino acids 67-141 of the mature protein, fused to an N-terminal His tag, and is expressed in E. coli expression systems . ATP5G2 belongs to a family of proteins that includes ATP5G1 and ATP5G3, all of which constitute the C-subunit of mitochondrial ATP synthases despite being regulated distinctly . The protein is encoded by the ATP5G2 gene (also known as ATP5MC2) and its amino acid sequence is: DIDTAAKFIGAGAATVGVAGSGAGIGTVFGSLIIGYARNPSLKQQLFSYAILGFALSEAMGLFCLMVAFLILFAM .

How does ATP5G2 differ functionally from other ATP synthase subunits?

ATP5G2 functions as one of three C-subunit isoforms (along with ATP5G1 and ATP5G3) required for complete ATP synthase functionality. While the mature protein sequences of these isoforms are identical after proteolytic processing, they cannot substitute for one another, suggesting unique regulatory roles in ATP synthase assembly or function . Research indicates that ATP5G2 typically shows higher expression levels than ATP5G1 in mammalian neural progenitor cells, though the relative abundance varies across tissue types and species . Unlike ATP5I (subunit e) which has been shown to interact with certain pharmacological agents like biguanides and influence the stability of respiratory complexes I and IV, ATP5G2's primary role appears to be structural within the c-ring of ATP synthase . The protein's lipid-binding property is critical for maintaining proper membrane integration and rotation of the c-ring during ATP synthesis.

What expression systems are most effective for producing ATP5G2?

E. coli expression systems have proven most effective for producing recombinant ATP5G2 with high purity and yield. The procedure typically involves fusing the mature protein sequence (amino acids 67-141) with an N-terminal His tag to facilitate purification . When expressing highly hydrophobic membrane proteins like ATP5G2, using specialized E. coli strains optimized for membrane protein expression (such as C41(DE3) or C43(DE3)) can improve yield. For optimal expression, induction conditions should be carefully controlled with lower IPTG concentrations (0.1-0.5 mM) and reduced temperatures (16-25°C). Purification is typically performed using immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography to achieve >90% purity . For functional studies, reconstitution into liposomes or nanodiscs may be necessary to maintain the native conformation of this lipid-binding protein.

How can ATP5G2 be used to study mitochondrial membrane dynamics?

ATP5G2 offers a valuable tool for investigating mitochondrial membrane dynamics due to its integral role in the ATP synthase complex. To study membrane dynamics, researchers can employ fluorescently tagged ATP5G2 constructs (using GFP or RFP fusions at the C-terminus to avoid interfering with mitochondrial targeting) for live-cell imaging of ATP synthase distribution during various cellular states. In comparative studies with ATP5G1, researchers have observed that modifications to ATP synthase subunits can significantly affect mitochondrial morphology, with some variants reducing mitochondrial fragmentation during stress conditions . For advanced applications, super-resolution microscopy techniques (STORM or PALM) combined with ATP5G2 labeling can reveal nanoscale organization of ATP synthase within cristae membranes. Additionally, reconstituting purified ATP5G2 into artificial membrane systems allows for controlled biophysical studies of lipid-protein interactions that influence membrane curvature and organization. These approaches provide insights into how ATP synthase arrangement contributes to cristae formation and mitochondrial membrane integrity.

What methodologies can be employed to investigate ATP5G2's role in cellular resilience to metabolic stress?

Investigating ATP5G2's role in metabolic stress resilience requires a multi-faceted approach. First, CRISPR/Cas9-mediated gene editing can be employed to knock out endogenous ATP5G2 while introducing wild-type or mutant versions to assess functional consequences . This should be followed by metabolic stress challenges including exposure to hypoxia (1% O₂), hypothermia (31°C), or mitochondrial toxins like rotenone (20 μM) to evaluate cell survival using flow cytometry-based viability assays with propidium iodide staining . Researchers should quantify mitochondrial respiratory parameters using Seahorse XF analyzers to determine oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and particularly spare respiratory capacity, which has been linked to stress resilience in studies of related ATP5G isoforms . Mitochondrial network morphology should be assessed via confocal microscopy with appropriate mitochondrial markers (e.g., MitoTracker or antibodies against TOMM20) to quantify fragmentation patterns before and after stress exposure . Additionally, measurement of NAD⁺/NADH ratios provides insight into metabolic adaptations, as restoration of NAD⁺ concentrations has been associated with improved stress responses in ATP synthase subunit studies .

What are the evolutionary implications of ATP5G2 sequence variations across primate species?

Evolutionary analysis of ATP5G2 across primate lineages reveals important adaptive patterns in mitochondrial energy production. To study these implications, researchers should perform comprehensive phylogenetic analyses comparing ATP5G2 sequences from diverse primate species, with particular attention to the Pongo genus compared to other great apes. Key metrics to calculate include BLOSUM62 scores and Jensen-Shannon Divergence (JSD) scores for amino acid substitutions, which can identify functionally significant variations . Researchers should focus on amino acid substitutions in both the mature protein sequence and the mitochondrial targeting sequence, as the latter shows considerable variability despite conservation of the mature protein . Functional validation of identified variants requires site-directed mutagenesis to introduce primate-specific substitutions into human or mouse ATP5G2, followed by assessment of effects on mitochondrial function, stress resilience, and protein-protein interactions. Comparative assays measuring Complex V enzymatic activity with and without specific inhibitors (e.g., oligomycin at 1 μM) can reveal functional consequences of these evolutionary changes . This approach has revealed significant functional effects of specific amino acid substitutions in the related ATP5G1, suggesting similar evolutionary adaptations may occur in ATP5G2.

What are the optimal storage and handling conditions for recombinant ATP5G2 protein?

Optimal storage and handling of recombinant ATP5G2 requires careful attention to its hydrophobic membrane protein characteristics. The lyophilized protein should be stored at -20°C/-80°C upon receipt . Before opening, the vial should be briefly centrifuged to bring contents to the bottom. For reconstitution, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . To enhance stability, add glycerol to a final concentration of 30-50% (with 50% being optimal for many applications) . After reconstitution, the solution should be divided into small working aliquots to avoid repeated freeze-thaw cycles, which significantly reduce protein activity. Working aliquots can be stored at 4°C for up to one week, while long-term storage requires -20°C/-80°C conditions . For experimental use, the buffer composition may need adjustment depending on the application; a Tris/PBS-based buffer at pH 8.0 containing 6% trehalose has been shown to maintain protein stability . When handling the protein, avoid vigorous vortexing which can cause denaturation; instead, mix by gentle pipetting or flicking of the tube.

How can researchers effectively measure ATP5G2 incorporation into functional ATP synthase complexes?

Measuring ATP5G2 incorporation into functional ATP synthase complexes requires a combination of biochemical and biophysical techniques. Begin with isolation of intact mitochondria using differential centrifugation, followed by gentle solubilization of membrane proteins using digitonin (0.5-1%) or other mild detergents to preserve protein-protein interactions . Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) should be performed to separate intact ATP synthase complexes, followed by Western blotting with antibodies against ATP5G or other ATP synthase subunits . For functional assessment, researchers should measure ATP synthase (Complex V) activity using spectrophotometric assays that couple ATP hydrolysis to NADH oxidation, with activity normalized to protein content and expressed relative to a control sample . Oligomycin sensitivity serves as an important control to confirm specific ATP synthase activity . For more detailed structural analysis, cryo-electron microscopy of purified ATP synthase complexes can determine if ATP5G2 is properly positioned within the c-ring. Additionally, proteomic approaches involving crosslinking mass spectrometry can identify interaction partners of ATP5G2 within the complex to confirm proper assembly. Pulse-chase experiments using isotope-labeled amino acids can track the kinetics of ATP5G2 incorporation into assembled complexes.

What experimental approaches can be used to investigate the specific lipid-binding properties of ATP5G2?

Investigating ATP5G2's lipid-binding properties requires specialized biophysical techniques. Surface plasmon resonance (SPR) represents an effective method to characterize binding interactions, though researchers must consider the size difference between ATP5G2 and potential lipid binding partners when designing experimental setups . For optimal results, immobilize the smaller molecule on the gold surface and add increasing concentrations of the protein to determine affinity constants (KD) . Isothermal titration calorimetry (ITC) provides complementary thermodynamic data on lipid-protein interactions, revealing both binding affinity and stoichiometry. To investigate specific lipid interactions in membrane environments, reconstitution studies using liposomes of defined composition allow researchers to assess how different lipids affect ATP5G2 function. Fluorescence-based approaches using environment-sensitive probes (attached either to the protein or to lipids) can detect conformational changes upon binding. For determining the specific lipid-binding regions within ATP5G2, hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify protected regions that likely interact with lipids. Additionally, molecular dynamics simulations based on the known structure of ATP synthase c-subunits can predict lipid interaction sites for targeted mutagenesis studies to confirm their functional importance.

How can researchers address common challenges in ATP5G2 expression and purification?

Researchers encountering difficulties with ATP5G2 expression and purification should implement systematic troubleshooting approaches. For poor expression yields, optimize codon usage for E. coli by synthesizing a codon-optimized gene, as the Pongo abelii sequence may contain rare codons . Consider using lower induction temperatures (16-18°C) and extended expression times (16-24 hours) to allow proper folding of this membrane protein. For proteins forming inclusion bodies, add mild solubilizing agents like 0.5-1% sarkosyl during lysis, followed by dilution before affinity purification. If protein aggregation occurs during purification, increase the concentration of stabilizing agents in buffers (trehalose can be increased from 6% to 10-15%) . For difficulties with His-tag detection or binding to Ni-NTA resin, verify protein expression by Western blot using antibodies against the ATP5G2 protein itself rather than the tag. Consider alternative purification approaches such as ion exchange chromatography if affinity purification proves challenging. For reconstitution difficulties, optimize the lipid:protein ratio (typically starting with 100:1 molar ratio) and try different lipid compositions that better mimic the mitochondrial inner membrane. Adding cardiolipin (10-20% of total lipids) often improves stability and functionality of mitochondrial membrane proteins in reconstituted systems.

How should researchers interpret contradictory data when comparing ATP5G2 with other ATP synthase subunits?

When interpreting contradictory data comparing ATP5G2 with other ATP synthase subunits, researchers should implement a systematic analytical framework. First, examine the experimental context carefully, as different cell types, species origins, and experimental conditions can significantly impact results—for instance, studies comparing mouse and Arctic Ground Squirrel (AGS) neural progenitor cells revealed different expression patterns of ATP5G isoforms . Consider the precise genetic constructs used, as even small variations in protein sequences or tags can alter function; for example, specific amino acid substitutions in ATP5G1 (L32P) showed dramatic effects on cellular resilience to metabolic stress . When comparing knockout phenotypes, distinguish between acute (siRNA/shRNA) versus chronic (CRISPR/Cas9) depletion methods, as compensatory mechanisms may develop in the latter . For seeming contradictions in protein-protein interactions, evaluate the experimental methods (co-IP, crosslinking, BN-PAGE) for differences in stringency that might affect detection sensitivity. Compare protein expression levels relative to physiological abundance, as overexpression artifacts can confound interpretation. When analyzing functional data, normalize to appropriate controls and consider that ATP5G2 may have both canonical (ATP synthesis) and non-canonical functions. For integrating conflicting findings, employ computational models incorporating data from multiple experimental approaches to identify parameters that might reconcile apparent contradictions.

How does ATP5G2 functionality compare with ATP5G1 and ATP5G3 in mitochondrial ATP synthesis?

What insights can comparison between ATP5G2 and ATP5I provide for mitochondrial research?

Comparing ATP5G2 and ATP5I offers valuable insights into the multifaceted roles of ATP synthase subunits beyond their structural functions. While ATP5G2 forms part of the c-ring (subunit c) essential for proton translocation and rotary motion of ATP synthase , ATP5I (subunit e) occupies a peripheral position in the membrane domain and appears to have additional regulatory functions . Research on ATP5I has revealed its capability to interact with pharmacological agents such as biguanides with relatively high affinity (KD ≈ 11.10 μM), whereas similar specific interactions have not been demonstrated for ATP5G2 . ATP5I knockout studies have shown that this subunit influences the stability of respiratory complexes I and IV, with its absence leading to decreased expression of NDUFB8 (complex I) and COX II (complex IV) proteins, a regulatory role not observed with ATP5G2 . Additionally, ATP5I appears to influence mitochondrial morphology and network organization, with its reintroduction in knockout cells restoring filamentous mitochondrial networks . While ATP5G2's primary role relates to the core catalytic function of ATP synthase, ATP5I exemplifies how peripheral subunits can serve as regulatory nodes connecting ATP synthase to other respiratory complexes and cellular processes. This comparison highlights the value of studying seemingly secondary ATP synthase subunits for understanding integrated mitochondrial function and identifying novel pharmacological targets.

How do species-specific variations in ATP5G2 correlate with metabolic adaptations across primates?

Species-specific variations in ATP5G2 across primates reveal important evolutionary adaptations in mitochondrial energy metabolism. While the mature ATP5G2 protein sequence shows high conservation across species, the mitochondrial targeting sequence exhibits considerable variability, suggesting adaptation to species-specific import machinery or regulatory mechanisms . This pattern parallels findings with ATP5G1, where specific amino acid substitutions in the Arctic Ground Squirrel variant (particularly L32P) confer significant cytoprotection against metabolic stressors including hypoxia and hypothermia . In Pongo abelii, the ATP5G2 protein maintains core functionality while potentially harboring adaptations to the orangutan's unique metabolic demands and habitat. Comparative analyses examining BLOSUM62 scores and Jensen-Shannon Divergence (JSD) scores can identify amino acid substitutions with high probability of functional consequence . Similar analytical approaches might reveal whether Pongo abelii ATP5G2 contains adaptations comparable to the protective L32P substitution found in AGS ATP5G1. These evolutionary modifications likely reflect adaptations to environmental pressures, dietary patterns, and metabolic requirements across primate lineages. The study of such variations provides insights into both the fundamental conservation of mitochondrial energy production mechanisms and the species-specific adaptations that allow primates to thrive in diverse ecological niches.