Recombinant Thunnus obesus ATP synthase subunit a (mt-atp6)

What is the basic structure and function of ATP synthase subunit a in Thunnus obesus?

ATP synthase subunit a (mt-atp6) in Thunnus obesus is a key component of the mitochondrial ATP synthase complex. The complete protein consists of 133 amino acids as indicated by the full sequence analysis. The primary function of this subunit involves facilitating proton translocation through the F₀ subcomplex, which is coupled to ATP synthesis/hydrolysis in the F₁ subcomplex through a unique rotary mechanism. The proton gradient established across the inner mitochondrial membrane drives this process, converting electrochemical energy into mechanical energy through subunit rotation, which is ultimately transformed into chemical energy in the form of ATP .

How does the primary sequence of Thunnus obesus mt-atp6 compare with other species?

The Thunnus obesus mt-atp6 protein sequence (LALTLPWVLFPTPTSRWLNNRLLTLQNWFIGRFGHELFTPVNLPGHKWAVLLTSLMLFLI SLNMLGLLPYTFTPTTQLSLNMGLAFPLWLATVIIGMRNQPTEALGHLLPEGTPTLLIPV LIVIETISLFIRP) demonstrates conservation of key functional domains while exhibiting species-specific variations. Comparative analysis with other species can be performed using standard alignment tools. In particular, the transmembrane regions and the proton channel components show higher conservation compared to other regions of the protein. This evolutionary conservation reflects the critical functional importance of these domains in the ATP synthesis mechanism .

What expression systems are most effective for producing recombinant Thunnus obesus mt-atp6?

Based on current research protocols, E. coli expression systems have been successfully employed for the recombinant production of Thunnus obesus mt-atp6. The protein can be effectively expressed as a fusion construct with an N-terminal His-tag to facilitate purification. E. coli provides several advantages including rapid growth, high protein yields, and established protocols for membrane protein expression. The expression construct typically includes the full-length sequence (amino acids 1-133) of the mature mt-atp6 protein. Following expression, the recombinant protein requires careful handling during purification and storage to maintain structural integrity and functionality .

What are the optimal storage and reconstitution conditions for recombinant Thunnus obesus mt-atp6?

Recombinant Thunnus obesus mt-atp6 protein is typically supplied as a lyophilized powder and requires specific handling for optimal stability and functionality. The recommended storage protocol involves maintaining the protein at -20°C to -80°C upon receipt, with aliquoting necessary for multiple uses to avoid repeated freeze-thaw cycles. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance long-term stability, addition of 5-50% glycerol (with 50% being the standard final concentration) is recommended. Once reconstituted, working aliquots can be stored at 4°C for up to one week, but extended storage should be at -20°C/-80°C. The storage buffer typically consists of Tris/PBS-based buffer with 6% trehalose at pH 8.0 .

How can researchers verify the functional activity of recombinant Thunnus obesus mt-atp6?

Verifying the functional activity of recombinant mt-atp6 requires several complementary approaches. First, structural integrity can be assessed using SDS-PAGE, which should show a single band corresponding to the expected molecular weight. For functional analysis, researchers can employ reconstitution into liposomes followed by proton translocation assays. This involves measuring pH changes using pH-sensitive fluorescent dyes or direct proton flux measurements. ATP hydrolysis coupling assays can also be performed by reconstituting the recombinant mt-atp6 with complementary ATP synthase subunits and measuring ATP hydrolysis rates in the presence of a proton gradient. Additionally, binding assays with known interacting partners from the ATP synthase complex can provide indirect evidence of proper folding and functional capacity .

What methodologies are recommended for studying mt-atp6 mutations and their effects on ATP synthase function?

When studying mt-atp6 mutations, researchers should employ a multi-faceted approach. Site-directed mutagenesis can be used to introduce specific mutations into the recombinant protein expression construct. Following expression and purification, biophysical characterization using circular dichroism spectroscopy helps assess potential structural changes. Functional assays should include proton translocation measurements and ATP synthesis/hydrolysis rates when the mutant protein is reconstituted with other ATP synthase components. For in vivo effects, introducing equivalent mutations in model organisms and measuring mitochondrial function provides physiological context. When analyzing disease-associated mutations, complementation studies in cellular models with mt-atp6 deficiencies can determine if the mutant protein restores function. Molecular dynamics simulations complement experimental data by predicting how specific mutations might alter protein dynamics and interactions .

How is mt-atp6 used in phylogenetic analyses of tuna species and other marine organisms?

The mt-atp6 gene, often analyzed alongside mt-atp8, serves as a valuable genetic marker for phylogenetic studies of tuna species and other marine organisms. Researchers typically amplify the complete mitochondrial ATP synthase subunits 6 and 8 regions using primers such as ATP 8.2L (5'AAAGCRTYRGCCTTTTAAGC3') and COIII.2H (5'GTTAGTGGTCAKGGGCTTGGRTC3'), which yield fragments approximately 950 base pairs in length. For more specific analyses, internal primers can be designed to produce smaller fragments (around 540 bp) suitable for techniques like temperature gradient gel electrophoresis (TGGE). These genetic sequences provide resolution at both the species and population levels, revealing evolutionary relationships and genetic diversity patterns. The relatively high mutation rate of mitochondrial genes, combined with their maternal inheritance pattern, makes mt-atp6 particularly useful for tracing evolutionary lineages and identifying cryptic species within marine ecosystems .

What differences exist between Thunnus obesus mt-atp6 and homologous proteins in other organisms?

The Thunnus obesus mt-atp6 protein exhibits notable structural and functional differences compared to homologous proteins in other organisms. While the core functional domains involved in proton translocation are generally conserved, sequence variations reflect adaptations to different physiological demands and evolutionary histories. Compared to mammalian counterparts, the tuna mt-atp6 shows adaptations potentially related to the high metabolic demands of these active pelagic fish. The protein's adaptation to different thermal environments is particularly relevant given the unique physiology of tuna species, which maintain elevated body temperatures. When compared to bacterial ATP synthase subunits, the mitochondrial mt-atp6 displays more extensive differences, reflecting the long evolutionary divergence between prokaryotic and eukaryotic systems. These comparative analyses provide insights into both the essential conserved features of ATP synthase and the adaptable elements that respond to different selective pressures .

How do researchers use mt-atp6 sequences for stock identification in fisheries management?

For fisheries management applications, mt-atp6 sequences provide valuable genetic markers for stock identification and population structure analysis. Researchers typically collect tissue samples (muscle, fin clips, or blood) from multiple individuals across different geographic regions, followed by DNA extraction using standard protocols or salt extraction methods. The mt-atp6 gene region is then amplified using specific primers and sequenced. Analysis of sequence polymorphisms can reveal distinct genetic stocks, which may represent demographically independent units requiring separate management strategies. Population genetic metrics such as haplotype diversity, nucleotide diversity, and F-statistics help quantify genetic differentiation between putative stocks. Recent studies have demonstrated the utility of mt-atp6 sequences in revealing previously undetected stock structures in tuna populations, informing more effective conservation and fisheries management practices. This approach is often complemented by additional genetic markers for comprehensive stock assessment .

What role does the mt-atp6 subunit play in bioenergetic adaptation of deep-sea fish like Thunnus obesus?

The mt-atp6 subunit plays a crucial role in the bioenergetic adaptation of deep-sea fish like Thunnus obesus (Bigeye tuna), which experiences variable temperatures and oxygen levels during vertical migrations. Research suggests that specific amino acid substitutions in the mt-atp6 protein may influence proton translocation efficiency and thereby ATP production under different environmental conditions. These adaptations potentially optimize ATP synthase performance across the temperature and pressure gradients encountered by these fish. The structural elements of mt-atp6 that form part of the proton channel are particularly important for maintaining function under varying conditions. Advanced research approaches include comparative analysis of mt-atp6 sequences from species with different depth distributions, functional studies of recombinant proteins under simulated deep-sea conditions, and measurement of ATP synthesis efficiency at different temperatures and pressures. Understanding these adaptations provides insights into both evolutionary biology and potential biomimetic applications for energy conversion technologies .

How can researchers investigate the interaction between recombinant mt-atp6 and other ATP synthase subunits?

Investigating interactions between recombinant mt-atp6 and other ATP synthase subunits requires sophisticated biophysical and biochemical approaches. Surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) provide quantitative measurements of binding affinities between mt-atp6 and potential interaction partners. Crosslinking studies using bifunctional reagents followed by mass spectrometry analysis can identify specific interaction sites. For structural characterization, cryo-electron microscopy of reconstituted complexes offers insights into the spatial arrangement of subunits within the assembled complex. Fluorescence resonance energy transfer (FRET) using labeled subunits enables monitoring of dynamic interactions in real-time. Computational approaches like molecular docking and molecular dynamics simulations complement experimental data by predicting interaction interfaces and conformational changes. Finally, functional reconstitution assays measure how specific subunit interactions affect ATP synthesis rates and proton translocation efficiency, linking structural insights to functional outcomes .

What are the implications of mt-atp6 mutations for mitochondrial diseases, and how does research on Thunnus obesus inform human disease studies?

Research on mt-atp6 mutations has significant implications for understanding mitochondrial diseases, as exemplified by conditions like Leigh syndrome. While studies on Thunnus obesus mt-atp6 may seem taxonomically distant from human applications, the highly conserved nature of ATP synthase mechanics makes comparative research valuable. Specific mutations, such as the m.9176T>G mutation in human mt-ATP6, have been associated with severe neurodegenerative conditions. Comparative analysis of mt-atp6 across species helps identify conserved residues where mutations are likely to be pathogenic. Research methodologies developed for studying recombinant fish proteins can be adapted for investigating human disease mutations. For instance, high-resolution melt (HRM) analysis and DNA sequencing techniques used to detect heteroplasmic mutations in tuna mt-atp6 are directly applicable to human diagnostics. Furthermore, understanding how certain species tolerate specific variations in mt-atp6 may provide insights into potential compensatory mechanisms that could be therapeutically relevant for human mitochondrial disorders .

What purification strategies yield the highest purity and activity for recombinant Thunnus obesus mt-atp6?

Optimal purification of recombinant Thunnus obesus mt-atp6 requires a strategic multi-step approach. Initial purification typically employs immobilized metal affinity chromatography (IMAC) using Ni-NTA resins to capture the His-tagged protein. For membrane proteins like mt-atp6, inclusion of appropriate detergents (such as n-dodecyl-β-D-maltoside or digitonin) throughout the purification process is critical to maintain protein solubility and native conformation. Following IMAC, size exclusion chromatography helps remove protein aggregates and further increases purity. Ion exchange chromatography may be employed as an additional purification step depending on the isoelectric point of the specific construct. Throughout the purification process, maintaining a controlled temperature (typically 4°C) and including protease inhibitors prevents degradation. Quality assessment by SDS-PAGE should demonstrate purity greater than 90%, as observed with current recombinant preparations. For functional studies, detergent exchange or reconstitution into liposomes may be necessary following purification to restore a lipid environment conducive to proper protein function .

How should researchers optimize PCR conditions for amplifying the mt-atp6 gene from Thunnus obesus samples?

Optimization of PCR conditions for amplifying the mt-atp6 gene from Thunnus obesus samples requires careful consideration of several parameters. DNA extraction should employ either standard commercial kits or modified salt extraction methods optimized for fish tissues. The primer selection is critical, with established primers such as ATP 8.2L (5'AAAGCRTYRGCCTTTTAAGC3') and COIII.2H (5'GTTAGTGGTCAKGGGCTTGGRTC3') targeting the entire mt-ATP synthase subunits 6 and 8 region. For studies requiring shorter fragments, custom internal primers can be designed to produce ~540 bp fragments suitable for techniques like temperature gradient gel electrophoresis (TGGE). The PCR reaction mixture typically includes: 10-50 ng template DNA, 0.2-0.4 μM of each primer, 0.2 mM dNTPs, 1.5-2.5 mM MgCl₂, and 1-1.5 units of high-fidelity DNA polymerase in appropriate buffer. Thermal cycling conditions should include an initial denaturation (95°C, 3-5 minutes), followed by 30-35 cycles of denaturation (95°C, 30 seconds), annealing (50-58°C, 30-45 seconds), and extension (72°C, 1 minute), with a final extension at 72°C for 7-10 minutes. Touchdown PCR approaches may improve specificity when working with samples of variable quality .

What analytical techniques are most informative for characterizing structural properties of recombinant mt-atp6?

Comprehensive structural characterization of recombinant mt-atp6 requires a combination of complementary analytical techniques. Circular dichroism (CD) spectroscopy provides valuable insights into secondary structure content, particularly the predominantly α-helical nature of the protein. For tertiary structure analysis, intrinsic tryptophan fluorescence spectroscopy can report on the local environment of aromatic residues, while thermal denaturation studies using differential scanning calorimetry (DSC) reveal thermodynamic stability parameters. Given the membrane protein nature of mt-atp6, techniques like attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) are particularly useful for analyzing secondary structure in membrane-mimetic environments. For higher-resolution structural information, solution NMR with isotopically labeled protein can provide residue-specific information, though this is challenging for full-length membrane proteins. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers insights into protein dynamics and solvent accessibility. Ultimately, cryo-electron microscopy represents the most promising approach for determining high-resolution structures, especially when mt-atp6 is assembled within the complete ATP synthase complex .

How can recombinant Thunnus obesus mt-atp6 be used to develop specific antibodies for research applications?

Recombinant Thunnus obesus mt-atp6 provides an excellent antigen source for developing specific antibodies with multiple research applications. The purified recombinant protein (>90% purity by SDS-PAGE) can be used for immunization following standard protocols. For polyclonal antibody production, rabbits or other suitable host animals are immunized with the purified protein, typically 100-200 μg per injection, using appropriate adjuvants. Multiple boost injections at 2-3 week intervals enhance antibody titer and specificity. For monoclonal antibody development, mice are immunized similarly, followed by hybridoma generation and screening. To ensure specificity, antibodies should be validated using Western blotting against both the recombinant protein and native protein from Thunnus obesus mitochondrial preparations. The resulting antibodies can be used for immunolocalization studies to investigate the distribution and assembly of ATP synthase complexes in tissue samples, for immunoprecipitation to study protein-protein interactions, and for functional studies such as inhibition assays. These antibodies also facilitate research on ATP synthase assembly, regulation, and potential roles in cellular processes beyond energy production .

What role does the mt-atp6 gene play in population genetics and conservation of tuna species?

The mt-atp6 gene serves as a valuable genetic marker in population genetics and conservation studies of tuna species. As part of the mitochondrial genome, it undergoes maternal inheritance without recombination, making it particularly useful for tracing evolutionary lineages and population histories. Research protocols typically involve sampling from multiple individuals across different geographic regions, followed by DNA extraction, PCR amplification using specific primers, and sequence analysis. The resulting data can reveal population structure, genetic diversity, and historical demographic events. Analysis of mt-atp6 sequences has identified distinct genetic stocks in tuna populations, information critical for establishing appropriate management units for conservation. The relatively high mutation rate of this gene provides resolution at both species and population levels. Furthermore, comparison of contemporary samples with historical specimens (from museum collections or archaeological remains) using mt-atp6 sequences can document temporal changes in genetic diversity, potentially revealing impacts of fishing pressure or environmental changes. This approach can be particularly valuable for monitoring the genetic health of commercially exploited tuna populations and informing sustainable management practices .