Recombinant Pig ATP synthase subunit f, mitochondrial (ATP5J2)

What is the amino acid sequence and key features of pig ATP5J2?

The pig ATP synthase subunit f, mitochondrial (ATP5J2) protein consists of 87 amino acids (residues 2-88 of the mature protein). The amino acid sequence is: ASVVPLKDRRLLEVKLGELPSWILMRDFTPSGIAGAFQRGYYRYYNKYVNVKKGSVAGLS MVLAAYVVFNYCRSYKELKHERLRKYH . This sequence contains several conserved regions that are crucial for its functional interactions within the ATP synthase complex. The protein has a molecular mass of approximately 10 kDa and contains regions that facilitate its integration into the peripheral stalk of the ATP synthase complex. The sequence exhibits significant conservation across mammalian species, reflecting its important structural and functional roles in the mitochondrial ATP synthase.

What are the primary functions of the ATP5J2 subunit in mitochondrial physiology?

Recent research has revealed that the f subunit (ATP5J2) plays multiple critical roles in mitochondrial function beyond simply being a structural component of ATP synthase. Studies have demonstrated that this subunit is essential for ATP synthase dimer stability, though not for monomer stability . The f subunit significantly influences mitochondrial crista morphology, with its downregulation resulting in disruption of normal crista architecture . Additionally, it modulates the size and sensitivity of the mitochondrial permeability transition pore (mPTP), a channel involved in cell death pathways . Interestingly, these functions appear to be independent of the enzymatic activity of ATP synthase, as downregulation of the f subunit does not significantly affect the synthetic or hydrolytic activities of the complex . These discoveries highlight the multifaceted role of ATP5J2 in maintaining both structural integrity and functional regulation of mitochondria beyond its participation in ATP synthesis.

What expression systems are most effective for recombinant production of pig ATP5J2?

For successful recombinant production of pig ATP5J2, Escherichia coli expression systems have proven highly effective as demonstrated in current protocols. The recombinant protein can be efficiently expressed in E. coli with an N-terminal His tag fusion to facilitate subsequent purification steps . When designing expression constructs, researchers should include the mature protein sequence (amino acids 2-88) rather than the full precursor protein to avoid issues with mitochondrial targeting sequences that may interfere with proper expression in bacterial systems. The expression vector should contain appropriate bacterial promoters (such as T7 or tac) for high-level inducible expression. Growth conditions typically involve culture at 37°C until reaching mid-log phase, followed by induction with IPTG (isopropyl β-D-1-thiogalactopyranoside) at reduced temperatures (16-25°C) to promote proper protein folding. This approach minimizes inclusion body formation while maximizing the yield of soluble, properly folded recombinant ATP5J2 protein.

What purification strategies yield the highest purity recombinant pig ATP5J2?

Purification of recombinant pig ATP5J2 requires a multi-step approach to achieve high purity suitable for structural and functional studies. The initial purification step leverages affinity chromatography using the N-terminal His tag, with immobilized metal affinity chromatography (IMAC) on Ni-NTA or Co-NTA resins . This step should be performed under native conditions using buffers containing 20-50 mM Tris or phosphate buffer (pH 7.5-8.0), 100-300 mM NaCl, and 10-40 mM imidazole to reduce non-specific binding. After elution with 250-500 mM imidazole, a secondary purification step using size exclusion chromatography is recommended to separate monomeric ATP5J2 from aggregates and other contaminants. For applications requiring extremely high purity, an intermediate ion exchange chromatography step may be incorporated between affinity and size exclusion steps. The final purified protein should be concentrated to 0.1-1.0 mg/mL and stored in a stable buffer containing 5-50% glycerol at -20°C or -80°C to maintain long-term stability and prevent freeze-thaw damage .

How can researchers verify the proper folding and activity of purified recombinant ATP5J2?

Verification of proper folding and functional integrity of purified recombinant pig ATP5J2 requires multiple complementary approaches. Structural integrity can be initially assessed using circular dichroism (CD) spectroscopy to analyze secondary structure elements, comparing the spectrum with predicted patterns based on the known structure of ATP synthase subunit f. Thermal shift assays provide information about protein stability and can be used to optimize buffer conditions for maximal stability. Functional verification presents a greater challenge since ATP5J2 functions as part of a multi-subunit complex. Researchers can employ reconstitution assays where purified recombinant ATP5J2 is incorporated into ATP synthase subcomplexes depleted of the native f subunit, followed by assessment of complex assembly using blue native PAGE or analytical ultracentrifugation. Alternatively, interaction studies using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can verify binding to known protein partners within the ATP synthase complex. For advanced functional studies, reconstitution of the protein into liposomes or nanodiscs followed by analysis of its effects on proton conductance or ATP synthase dimer formation can provide valuable insights into its native activity.

How does the f subunit contribute to ATP synthase dimer stability and what methods can detect this function?

The f subunit plays a crucial role in maintaining ATP synthase dimer stability but not monomer stability, as demonstrated by recent research . To investigate this function, researchers can employ several methodological approaches. Blue native polyacrylamide gel electrophoresis (BN-PAGE) represents a primary technique for analyzing ATP synthase oligomeric states, allowing visualization of the relative abundance of monomers versus dimers in samples with normal or depleted ATP5J2 levels. Crosslinking studies using chemical crosslinkers with various spacer lengths can identify specific interaction partners of the f subunit within the dimer interface. Cryo-electron microscopy (cryo-EM) provides high-resolution structural information about the positioning of the f subunit within ATP synthase dimers and can reveal conformational changes resulting from its absence. Complementary approaches include fluorescence resonance energy transfer (FRET) analysis using fluorescently labeled subunits to measure proximity relationships in intact mitochondria, and analytical ultracentrifugation to determine the sedimentation properties of ATP synthase complexes with and without the f subunit. These methods collectively provide comprehensive insights into how ATP5J2 contributes to dimer formation and stability.

What experimental approaches can best assess the role of ATP5J2 in mitochondrial crista morphology?

Investigating the relationship between ATP5J2 and mitochondrial crista morphology requires sophisticated imaging and biochemical techniques. Transmission electron microscopy (TEM) serves as the gold standard for visualizing crista ultrastructure in cells with normal or depleted ATP5J2 levels, providing nanometer-resolution images of mitochondrial membrane architecture . Live-cell imaging using mitochondria-targeted fluorescent proteins combined with super-resolution microscopy techniques (such as STED or PALM/STORM) allows dynamic analysis of crista structure in living cells. Immunogold labeling of ATP5J2 coupled with electron microscopy can precisely localize the protein relative to crista junctions and other architectural features. Tomographic reconstruction from electron microscopy data generates three-dimensional models of crista morphology, revealing subtle structural changes not apparent in two-dimensional projections. Biochemical fractionation methods can separate inner membrane, outer membrane, and crista membrane components to analyze the distribution of ATP5J2 and other ATP synthase subunits between these compartments. Researchers should employ these complementary approaches to establish causal relationships between ATP5J2 function and crista morphology maintenance.

How can researchers accurately measure the impact of ATP5J2 modifications on mitochondrial permeability transition?

The mitochondrial permeability transition pore (mPTP) represents a critical pathway in cell death mechanisms, and ATP5J2 has been implicated in modulating both the size and sensitivity of this pore . To investigate this function, researchers can utilize multiple experimental approaches. Calcium retention capacity (CRC) assays measure mitochondrial ability to sequester calcium before undergoing permeability transition, providing a quantitative metric for mPTP sensitivity in mitochondria with modified ATP5J2. Mitochondrial swelling assays, monitored by changes in light scattering, can detect the kinetics of permeability transition in response to various inducers. Patch-clamp electrophysiology of mitochondrial membranes allows direct measurement of pore conductance and size characteristics. Live-cell imaging with membrane potential-sensitive dyes (such as TMRM or JC-1) can monitor permeability transition events in intact cells. In conjunction with these techniques, genetic approaches using CRISPR/Cas9 to generate specific ATP5J2 mutations or site-directed mutagenesis of recombinant protein can establish structure-function relationships. These methodologies together provide comprehensive understanding of how ATP5J2 regulates this crucial aspect of mitochondrial physiology.

What are the key structural and functional differences between pig ATP5J2 and human ATP5MF?

Comparative analysis of pig ATP5J2 and human ATP5MF (formerly known as ATP5J2) reveals both conservation and species-specific adaptations in this important ATP synthase subunit. The human ATP5MF gene encodes the ATP synthase subunit f, which is functionally homologous to the pig ATP5J2 protein . Sequence alignment shows substantial conservation between these proteins, reflecting their shared evolutionary origin and conserved function in the ATP synthase complex. Both proteins participate in similar structural roles within the F₀ complex of mitochondrial ATP synthase, contributing to proton channel formation and ATP synthesis coupling. The human ATP5MF gene is located on chromosome 7 and can produce alternatively spliced transcript variants encoding different isoforms . This may represent a mechanism for fine-tuning ATP synthase function in different human tissues that is potentially less developed in pigs. Functional studies suggest conservation of the role in dimer stability and crista morphology maintenance between species, though subtle differences in interaction partners or regulatory mechanisms may exist. These comparative insights are valuable for translating findings from porcine models to human mitochondrial biology and disease.

What experimental approaches can determine species-specific functional differences in ATP synthase subunit f?

Investigating species-specific functional differences in ATP synthase subunit f requires sophisticated comparative experimental approaches. Heterologous complementation studies represent a powerful method, where the endogenous f subunit in a model system (yeast, cultured mammalian cells) is replaced with orthologs from different species to assess functional rescue. This approach can reveal subtle functional differences that have evolved between species. Cross-species reconstitution experiments using purified components can directly test whether pig ATP5J2 can assemble correctly with human ATP synthase subcomplexes, and vice versa. Structural biology approaches, including cryo-EM and X-ray crystallography, can capture species-specific conformational differences in the assembled complex. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can identify regions with different solvent accessibility or conformational dynamics between species. Comparative proteomic analysis using proximity labeling (BioID or APEX) with the f subunit as bait can map species-specific interaction networks. Mitochondrial assays comparing oxygen consumption, ATP production, and membrane potential maintenance in systems with swapped f subunits can detect functional consequences of species-specific variations. These approaches collectively provide a comprehensive assessment of how evolutionary changes in this protein may contribute to species-specific adaptations in bioenergetics.

How can structure-based drug design target ATP synthase subunit f for therapeutic development?

ATP synthase represents a promising target for therapeutic development, with the f subunit offering unique opportunities for structure-based drug design. The strategic position of subunit f at the base of the peripheral stalk makes it an attractive target for developing compounds that could modulate specific functions of ATP synthase . Researchers can approach this by first obtaining high-resolution structural data of the f subunit within the complete ATP synthase complex using cryo-EM or X-ray crystallography. Virtual screening of compound libraries against identified binding pockets can identify potential ligands with specificity for the f subunit. Molecular dynamics simulations can predict how these compounds might affect the conformational dynamics of ATP synthase, particularly regarding dimer stability and interaction with the permeability transition pore. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can experimentally validate binding of candidate compounds to recombinant ATP5J2. Functional assays measuring ATP synthesis, hydrolysis, or permeability transition in isolated mitochondria treated with these compounds can assess their biological effects. This integrated approach could lead to the development of compounds that selectively modulate specific functions of ATP synthase without disrupting its essential catalytic activity, potentially offering therapeutic avenues for mitochondrial disorders.

What role does ATP5J2 play in the formation and regulation of the mitochondrial permeability transition pore?

The mitochondrial permeability transition pore (mPTP) represents a critical pathway in cell death mechanisms, and recent research has implicated ATP5J2 in its regulation. Studies have demonstrated that downregulation of the f subunit modulates both the size and sensitivity of the permeability transition pore without affecting ATP synthase enzymatic activity . This finding positions ATP5J2 as a potential regulatory component of the mPTP complex. Several models have been proposed for how ATP5J2 might influence mPTP formation: it may stabilize a conformation of ATP synthase that resists pore formation, it could interact with other proteins that directly regulate the pore, or it might affect lipid organization at potential pore-forming regions. Experimental approaches to investigate these possibilities include site-directed mutagenesis of ATP5J2 to identify specific residues involved in mPTP regulation, reconstitution of purified components in liposomes to recapitulate pore formation in vitro, and high-resolution structural studies of ATP synthase under conditions favoring or inhibiting mPTP formation. The relationship between ATP5J2, ATP synthase dimers, and the mPTP represents a frontier in understanding mitochondrial membrane permeabilization during stress conditions and cell death pathways.

How can recombinant ATP5J2 be used in studies of ATP synthase assembly and oligomerization?

Recombinant ATP5J2 provides a valuable tool for detailed investigation of ATP synthase assembly and oligomerization processes. Researchers can utilize fluorescently labeled recombinant ATP5J2 for real-time visualization of incorporation into ATP synthase complexes in reconstitution systems or permeabilized cells. Site-directed mutagenesis of recombinant ATP5J2 allows systematic mapping of residues critical for dimer formation by introducing point mutations and assessing their effects on oligomerization . In vitro assembly assays using purified ATP synthase components with wild-type or modified recombinant ATP5J2 can reconstruct the assembly pathway under controlled conditions. Cross-linking studies combined with mass spectrometry using recombinant ATP5J2 with incorporated photo-activatable amino acids can identify precise interaction interfaces with neighboring subunits. Cryo-electron tomography of mitochondrial membranes reconstituted with tagged recombinant ATP5J2 can visualize its position in the context of ATP synthase arrays along cristae. Single-molecule techniques tracking labeled recombinant ATP5J2 can provide insights into the dynamics of association and dissociation from ATP synthase complexes. These advanced applications of recombinant ATP5J2 continue to expand our understanding of the intricate molecular architecture and assembly of this crucial bioenergetic machinery in mitochondria.

What are the optimal storage and handling conditions for recombinant pig ATP5J2?

Maintaining the stability and activity of recombinant pig ATP5J2 requires careful attention to storage and handling conditions. The purified protein is typically supplied as a lyophilized powder, which should be briefly centrifuged prior to opening to ensure all material collects at the bottom of the vial . Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol as a cryoprotectant for long-term storage . The optimal storage buffer typically consists of a Tris/PBS-based system at pH 8.0 with 6% trehalose to maintain protein stability . After reconstitution, the protein should be stored at -20°C/-80°C in small working aliquots to avoid repeated freeze-thaw cycles, which can severely compromise protein integrity. For short-term use, working aliquots can be maintained at 4°C for up to one week . When handling the protein during experiments, researchers should maintain temperature conditions above 0°C but below room temperature whenever possible to preserve native structure while preventing precipitation. These careful handling procedures ensure maximal retention of structural integrity and functional activity for experimental applications.

What methods can effectively assess the purity and integrity of recombinant ATP5J2 preparations?

Comprehensive quality assessment of recombinant ATP5J2 preparations requires multiple analytical techniques to evaluate purity, integrity, and structural properties. SDS-PAGE represents the primary method for purity assessment, with high-quality preparations showing a single dominant band at approximately 10 kDa corresponding to the ATP5J2 protein (plus any tag modifications) . Western blotting using specific antibodies against ATP5J2 or detection tags provides additional confirmation of protein identity. Mass spectrometry analysis, particularly MALDI-TOF or ESI-MS, can verify the exact molecular weight and detect any unexpected modifications or degradation products. Size-exclusion chromatography can assess aggregation state and homogeneity of the preparation. Circular dichroism spectroscopy provides information about secondary structure content, which should match predictions based on known structural data for the f subunit. For advanced applications, thermal shift assays can determine protein stability under various buffer conditions, while dynamic light scattering measures size distribution and potential aggregation. Nuclear magnetic resonance (NMR) spectroscopy can provide atomic-level information about protein folding for smaller constructs or domains. These complementary approaches together ensure that recombinant ATP5J2 preparations meet the stringent quality requirements for structural and functional studies.