Recombinant Gracilaria tenuistipitata var. liui ATP synthase subunit c, chloroplastic (atpH)

What are the optimal growth conditions for Gracilaria tenuistipitata var. liui in laboratory settings?

Gracilaria tenuistipitata var. liui demonstrates optimal growth under controlled laboratory conditions with specific parameters for salinity, pH, temperature, and light. Research indicates that when cultivated in 500 mL cylinders, this red algal species shows significantly different growth rates at pH 6.5 and 7.0 across various salinities. For laboratory cultivation, the alga performs best when grown in aerated solutions enriched with PEM-PII medium that is replaced twice weekly. Temperature should be maintained at 20°C, and lighting should provide approximately 100 μmol photon m⁻² s⁻¹ photosynthetic photon flux (PPF) under 12-hour photoperiods. A week of acclimation is recommended before commencing experimental measurements .

For optimal results, relative growth rate (RGR) experiments demonstrate that G. tenuistipitata grown at 20°C in a salinity of 20‰ shows significantly higher growth rates compared to other salinity conditions. Interestingly, these growth rates are approximately 40% higher than those observed at 30°C, suggesting temperature-dependent growth optimization . When pH control is implemented, RGR increases significantly with increasing salinity at pH 6.5 or 7.0, with a less pronounced effect at pH 8.0.

How does the structure and function of ATP synthase subunit c differ between isoforms?

ATP synthase subunit c, also known as lipid-binding protein, forms a cylindrical c₁₀ oligomer that plays a crucial role in the proton pumping process, directly cooperating with subunit a (Atp6-equivalent) in coupling the proton gradient generated by the respiratory chain to ATP synthesis . In mammals, three isoforms of F₁F₀-ATP synthase subunit c (P1, P2, and P3) exist. These isoforms have identical mature peptides of 76 amino acids but differ in their N-terminal mitochondrial targeting peptides, which are cleaved upon import into mitochondria .

The targeting peptides have varying lengths: P1 is 61 amino acids, P2 has two alternatively spliced forms of 82 amino acids (variant a) and 123 amino acids (variant b), and P3 is 67 amino acids long. Importantly, research demonstrates that these isoforms are not functionally redundant, as silencing any individual isoform results in ATP synthesis defects. This indicates that the functional specificity of these isoforms resides in their targeting peptides, which not only mediate mitochondrial protein import but also play an essential and previously undiscovered role in respiratory chain maintenance .

What is the relationship between chloroplastic ATP synthase (atpH) and photosynthetic efficiency in Gracilaria species?

The chloroplastic ATP synthase subunit c (atpH) plays a fundamental role in energy production during photosynthesis in Gracilaria species. The synthesis and stability of ATP synthase components directly impact photosynthetic efficiency and energy conversion. Research indicates that the expression and stability of the atpH/F transcript are regulated by specific pentatricopeptide repeat (PPR) proteins, with recent studies identifying a chloroplast PPR protein called BFA2 (Biogenesis Factors required for ATP synthase 2) that binds to the atpF-atpA intergenic region in a sequence-specific manner .

The binding of BFA2 to the 3'-UTR of atpH/F has been demonstrated to be essential for stabilization of atpH/F RNA. This protective mechanism involves BFA2 acting as a site-specific barrier that blocks exoribonuclease degradation from the 3'-direction. Notably, stabilization of the atpH/F transcript requires two independent PPR proteins, PPR10 and BFA2, to protect the mRNA against exoribonucleases . These mechanisms directly influence the production of functional ATP synthase, which in turn affects photosynthetic capacity and efficiency.

What expression systems are most effective for producing recombinant atpH proteins from Gracilaria tenuistipitata var. liui?

For recombinant expression of atpH from Gracilaria tenuistipitata var. liui, bacterial expression systems, particularly E. coli, provide a practical and efficient approach. Based on methodologies applied to similar algal proteins, a combination of molecular cloning techniques with optimized expression conditions yields the best results. The procedure should begin with RNA extraction from G. tenuistipitata var. liui cultured under optimal conditions (20°C, salinity of 20-30‰, pH 7.0, and PPF of 100 μmol photon m⁻² s⁻¹) .

Following RNA extraction, RT-PCR amplification of the atpH coding sequence should be performed using gene-specific primers. The amplified sequence can then be cloned into an expression vector containing a suitable promoter (e.g., T7) and an affinity tag (e.g., His-tag) for purification. For expression, E. coli strains such as BL21(DE3) or Rosetta™ are recommended due to their enhanced production of membrane proteins. Induction of protein expression should occur when cultures reach OD₆₀₀ of 0.6-0.8, using 0.5-1.0 mM IPTG, with expression continued for 4-6 hours at 25-30°C rather than 37°C to improve protein folding.

For purification of the recombinant atpH protein, a multi-step chromatography approach is advised, beginning with immobilized metal affinity chromatography (IMAC), followed by size exclusion chromatography to increase purity. Expression yields can be further optimized by adjusting media composition, temperature, and induction parameters based on preliminary expression studies.

How can researchers effectively measure and compare ATP synthase activity between recombinant and native atpH proteins?

Comparing ATP synthase activity between recombinant and native atpH proteins requires a combination of biochemical and biophysical techniques. To establish a reliable comparison, both proteins should be analyzed under identical conditions using the following methodological approach:

• Enzymatic activity assay: Measure ATP synthesis rates using a luciferase-based ATP detection system. The reaction mixture should contain the purified ATP synthase complex (reconstituted with the respective atpH protein), ADP, inorganic phosphate, and an artificial proton gradient.

• Proton translocation measurement: Assess proton pumping efficiency using pH-sensitive fluorescent dyes (such as ACMA or pyranine) in proteoliposomes reconstituted with either recombinant or native ATP synthase complex.

• Oligomycin sensitivity: Determine the sensitivity to oligomycin, a specific inhibitor of ATP synthase, as differences in sensitivity may indicate structural variations between recombinant and native proteins.

• Thermostability analysis: Compare thermal stability profiles using differential scanning calorimetry or thermal shift assays to detect potential structural differences that might affect function.

For a comprehensive analysis, researchers should also employ structural characterization methods such as circular dichroism spectroscopy to compare secondary structure components, and native-PAGE to assess the integrity of the assembled complex. Additionally, proteomic analysis using mass spectrometry can verify post-translational modifications that might differ between recombinant and native proteins, potentially affecting function.

How do salinity and pH affect the expression and function of atpH in Gracilaria tenuistipitata var. liui?

In cultures without pH control, ambient pH increases rapidly to 9.0-9.3 within 2-3 hours after light initiation, which impacts photosynthetic efficiency and consequently ATP synthase function. This suggests that pH stabilization is critical for maintaining optimal function of photosynthetic machinery, including ATP synthase .

For experimental design, researchers should consider that optimal conditions for protein expression may differ from those for maximal growth. While growth is maximized at 20°C and 20‰ salinity, protein expression studies should examine a matrix of conditions (pH: 6.5, 7.0, 8.0; salinity: 20‰, 30‰, 39‰) to determine optimal parameters for atpH expression. RNA-seq or qPCR analysis can be used to quantify atpH transcript levels across these conditions, while western blotting with atpH-specific antibodies can assess protein abundance.

What impact do nutrient levels and light conditions have on atpH expression in laboratory cultivation?

Nutrient availability and light conditions significantly impact the expression of photosynthetic machinery components, including ATP synthase subunit c (atpH), in Gracilaria tenuistipitata var. liui. Research demonstrates that ammonium (NH₄⁺) concentration affects both growth rates and biochemical composition. For instance, the C:N ratio increases with decreasing levels of NH₄⁺, with a 78% lower ratio observed in algae grown with 2.0 mM NH₄⁺ compared to untreated specimens .

For experimental design, researchers should implement a controlled study using the following methodology:

• Culture G. tenuistipitata var. liui in media with varying NH₄⁺ concentrations (0, 0.1, 0.5, and 2.0 mM) and under different photosynthetic photon flux (PPF) levels (50, 100, and 150 μmol photon m⁻² s⁻¹).

• Monitor growth parameters including relative growth rate and biochemical composition (C:N ratio).

• Quantify atpH expression using quantitative PCR and protein levels using western blotting.

• Assess photosynthetic efficiency through chlorophyll fluorescence measurements (Fv/Fm ratio) as an indicator of photosystem II function, which cooperates with ATP synthase.

• Correlate atpH expression with photosynthetic parameters and growth rates to establish optimal conditions.

The data can be presented in a table format as follows:

This approach provides a comprehensive understanding of how nutrient and light conditions interact to influence atpH expression and function in laboratory cultivation.

What techniques are most effective for determining the oligomeric structure of recombinant atpH in isolation and within the complete ATP synthase complex?

Determining the oligomeric structure of recombinant atpH requires a multi-technique approach for both isolated subunits and the assembled ATP synthase complex. The following methodological workflow is recommended:

• For isolated atpH analysis:

  • Analytical ultracentrifugation (AUC) to determine the oligomeric state in solution

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to measure molecular weight and oligomeric distribution

  • Chemical cross-linking followed by mass spectrometry (CLMS) to identify interaction interfaces

  • Transmission electron microscopy (TEM) with negative staining to visualize oligomeric structures

• For assembled ATP synthase complex analysis:

  • Blue native polyacrylamide gel electrophoresis (BN-PAGE) to preserve native protein interactions and determine complex integrity

  • Cryo-electron microscopy (cryo-EM) for high-resolution structural analysis

  • Atomic force microscopy (AFM) to visualize membrane-embedded complexes

Based on research with ATP synthase subunit c from various organisms, recombinant atpH likely forms cylindrical c-ring oligomers. In mammals, subunit c assembles into a cylindrical c₁₀ oligomer that is critical for the proton pumping process, directly cooperating with subunit a in coupling the proton gradient to ATP synthesis . Analysis of targeting peptides may also reveal their role in complex assembly, as research shows that subunit c isoforms with different targeting peptides have non-redundant functions in respiratory chain maintenance .

For comprehensive characterization, researchers should compare the oligomeric structure of recombinant atpH with native protein from Gracilaria tenuistipitata var. liui using identical analytical techniques. This comparison will validate the structural integrity of the recombinant protein and identify any differences that might impact functional studies.

How can researchers investigate the interaction between atpH and RNA stability factors in chloroplasts?

Investigating the interaction between atpH and RNA stability factors in chloroplasts requires a combination of molecular biology and biochemical techniques focused on RNA-protein interactions. Research has identified the importance of proteins like BFA2 (Biogenesis Factors required for ATP synthase 2), a chloroplast PPR protein that binds to the atpF-atpA intergenic region and is essential for stabilizing atpH/F RNA . The following methodological approach is recommended:

• RNA-protein interaction analysis:

  • RNA Electrophoretic Mobility Shift Assay (EMSA) to detect binding of PPR proteins like BFA2 to the atpH/F RNA

  • RNA immunoprecipitation (RIP) to identify proteins associated with atpH transcripts in vivo

  • UV crosslinking and immunoprecipitation (CLIP) to map precise RNA-protein interaction sites

• Functional validation:

  • Generate knockout or knockdown lines of candidate RNA stability factors using CRISPR-Cas9 or RNAi

  • Quantify atpH/F transcript levels using northern blotting or qRT-PCR

  • Perform RNA degradation assays to assess stability of atpH/F transcripts in the presence or absence of specific factors

• Structural characterization:

  • Determine the binding motif in the 3'-UTR of atpH/F using truncated RNA constructs in binding assays

  • Employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of PPR proteins that interact with RNA

Based on current research, BFA2 binds to the atpF-atpA intergenic region in a sequence-specific manner, protecting atpH/F RNA from degradation by exoribonucleases. Stabilization of the atpH/F transcript requires two independent PPR proteins, PPR10 and BFA2, to protect the mRNA against exoribonucleases . These proteins act as site-specific barriers, blocking exoribonuclease degradation from the 3'-direction. This protection mechanism is essential for maintaining adequate levels of ATP synthase components and ensuring proper photosynthetic function.

How can recombinant atpH be utilized in bioenergetic research and potential biotechnological applications?

Recombinant atpH from Gracilaria tenuistipitata var. liui presents significant opportunities for both fundamental bioenergetic research and biotechnological applications. The following methodological approaches can be implemented:

• Bioenergetic research applications:

  • Model system for studying proton translocation: Recombinant atpH can be reconstituted into liposomes to create a minimal system for studying proton translocation mechanisms across membranes.

  • Structure-function relationship studies: Site-directed mutagenesis of recombinant atpH can identify critical residues involved in proton translocation and c-ring assembly.

  • Comparative studies with other species: Recombinant atpH can be used in comparative analyses with homologous proteins from different organisms to understand evolutionary adaptations in ATP synthases.

• Biotechnological applications:

  • Biomimetic energy conversion systems: The c-ring structure formed by atpH can inspire the development of synthetic nanomotors or molecular machines for energy conversion applications.

  • Biosensor development: Engineered variants of atpH could be developed as biosensors for proton gradients or membrane potential in various systems.

  • Drug screening platform: Reconstituted ATP synthase complexes containing recombinant atpH can serve as targets for screening compounds that modulate energy metabolism.

The unique properties of Gracilaria tenuistipitata var. liui atpH, particularly its adaptation to variable salinity and pH conditions , make it an especially valuable model for understanding how ATP synthases function under environmental stress. This knowledge could lead to the development of stress-resistant bioenergetic systems for biotechnological applications or enhanced understanding of algal adaptation mechanisms.

For researchers pursuing these applications, it is essential to optimize expression systems to produce sufficient quantities of functional protein. The combined acid and enzymatic hydrolysis approaches developed for other Gracilaria proteins may provide valuable methodological insights for processing recombinant atpH.

What methodologies are most effective for studying the role of atpH targeting peptides in respiratory chain maintenance?

Investigating the role of atpH targeting peptides in respiratory chain maintenance requires a comprehensive approach that integrates molecular, biochemical, and cell biological techniques. Based on research demonstrating that ATP synthase subunit c isoforms have non-redundant functions due to their targeting peptides , the following methodological framework is recommended:

• Targeting peptide analysis and engineering:

  • Generate chimeric constructs with targeting peptides fused to reporter proteins (e.g., GFP)

  • Create variant constructs with systematic mutations or deletions in the targeting peptide sequence

  • Express these constructs in appropriate model systems (algal cells or heterologous systems)

• Functional assessment:

  • Measure ATP synthesis rates in cells expressing different targeting peptide variants

  • Assess respiratory chain assembly and function using Blue Native PAGE and activity assays

  • Quantify the abundance and stability of respiratory chain complexes using western blotting and pulse-chase experiments

• Interaction studies:

  • Identify proteins that interact with the targeting peptides using pull-down assays and mass spectrometry

  • Validate interactions using techniques such as co-immunoprecipitation and FRET (Fluorescence Resonance Energy Transfer)

  • Map interaction domains through truncation analysis and peptide competition assays

Research with mammalian ATP synthase subunit c has shown that targeting peptides play roles beyond protein import, contributing to respiratory chain maintenance . Expression of targeting peptides fused to GFP variants rescued ATP synthesis and respiratory chain defects in silenced cells, demonstrating their functional significance. This suggests that atpH targeting peptides may have similar dual roles in chloroplasts, potentially interacting with components involved in thylakoid membrane organization or photosynthetic complex assembly.

For comprehensive analysis, researchers should compare effects across different environmental conditions, as the function of targeting peptides may be influenced by factors such as pH, salinity, or light intensity that affect membrane properties and protein interactions in Gracilaria tenuistipitata var. liui .

What are common challenges in recombinant expression of atpH and strategies to overcome them?

Recombinant expression of membrane proteins like ATP synthase subunit c (atpH) presents several challenges due to their hydrophobic nature and complex assembly requirements. The following table outlines common challenges and corresponding optimization strategies:

Based on research with similar proteins, a promising approach is to use a combination of moderate temperature (20°C) and low inducer concentration for extended periods (16-24 hours) to favor proper folding and membrane insertion. Additionally, extraction protocols should be carefully optimized, as the choice of detergent significantly impacts both yield and functional integrity of membrane proteins like atpH.

For validation of properly expressed and folded recombinant atpH, researchers should employ multiple analytical techniques including circular dichroism spectroscopy to assess secondary structure content, size exclusion chromatography to evaluate oligomeric state, and functional reconstitution assays to confirm proton translocation activity.

How can researchers optimize protocols for analyzing the interaction between atpH and RNA stability factors in different environmental conditions?

Optimizing protocols for analyzing atpH-RNA stability factor interactions across environmental conditions requires careful consideration of experimental parameters. The following methodological framework addresses key optimization points:

• RNA-protein interaction analysis under variable conditions:

  • Modify EMSA buffer conditions to mimic different pH and salt concentrations

  • Perform binding assays at various temperatures to assess thermostability of interactions

  • Include competitive binding assays with RNA variants to determine specificity under different conditions

• In vivo interaction validation:

  • Develop dual-reporter systems to monitor RNA-protein interactions in real-time

  • Implement RNA immunoprecipitation followed by sequencing (RIP-seq) under different growth conditions

  • Utilize proximity-based labeling techniques (BioID or APEX) to capture transient interactions

• Troubleshooting common challenges:

  a. Low signal-to-noise ratio:

  • Increase stringency of washes in immunoprecipitation procedures

  • Optimize crosslinking parameters for specific RNA-protein pairs

  • Use background reduction techniques such as competitive elution

  b. Inconsistent results across conditions:

  • Standardize RNA and protein preparation methods

  • Implement internal controls for normalization

  • Design factorial experiments to identify interaction effects between variables

  c. Difficulty in detecting low-abundance interactions:

  • Employ signal amplification techniques

  • Consider enrichment strategies before analysis

  • Use more sensitive detection methods (e.g., digital PCR, single-molecule imaging)

Research has demonstrated that PPR proteins like BFA2 bind to specific regions of chloroplast transcripts, including the atpF-atpA intergenic region, protecting RNA from exoribonuclease degradation . Under different environmental conditions, the efficiency of this protection mechanism may vary, potentially affecting ATP synthase production and function. By systematically analyzing these interactions across a range of conditions relevant to Gracilaria tenuistipitata var. liui ecology (such as pH 6.5-8.0 and salinities of 20-39‰) , researchers can gain insights into how RNA stability mechanisms adapt to environmental changes.