Recombinant Thalassiosira pseudonana ATP synthase subunit c, chloroplastic (atpH)

What is the function of ATP synthase subunit c (atpH) in Thalassiosira pseudonana?

ATP synthase subunit c (atpH) is a critical component of the F0 sector of the ATP synthase complex in the chloroplast of Thalassiosira pseudonana. This subunit forms the proton channel within the membrane domain of ATP synthase, allowing protons to flow across the membrane, which drives the rotation of the complex and subsequently ATP synthesis. In diatoms like T. pseudonana, this process is particularly important for harnessing energy from photosynthesis, as these organisms have unique photosynthetic pathways that differ from green algae and plants. T. pseudonana exhibits a typical C3-type photosynthetic carbon fixation pathway, where ATP synthase plays a crucial role in energy conversion during photosynthesis .

How does the amino acid sequence of T. pseudonana atpH compare to other organisms?

The amino acid sequence of T. pseudonana ATP synthase subunit c shows conservation with other photosynthetic organisms but contains unique features specific to diatoms. While the search results don't provide the exact sequence for atpH, we can compare it to the related ATP synthase subunit b' (atpG) from T. pseudonana, which has a sequence of 156 amino acids: "MINLSILISSSEVSGPGGLFDINATLPLVAIQFILLMVTLNIILYSPLLTIIEERKEYVLSHLAQASEKLAQAKELTTQYEQDLETARKEAQLEIANSQNIHKEILDIELDISQKYIDNLLETISSDLLNKKKTALDSLDTIVTSLCTEVETKLSI" .

The genome-scale metabolic model of T. pseudonana (iThaps987) has revealed that this organism has 183 unique enzymes compared to another model diatom (Phaeodactylum tricornutum), indicating distinct metabolic features that influence the structure and function of its energy-generating complexes like ATP synthase .

What are the key structural characteristics of recombinant ATP synthase subunits in diatoms?

Recombinant ATP synthase subunits from T. pseudonana, including atpH, typically exhibit structural features adapted to the unique cellular architecture of diatoms. These proteins often contain chloroplast targeting sequences when expressed in their native context. When expressed as recombinant proteins, they can be produced with fusion tags to aid in purification and detection. For example, the related ATP synthase subunit b' (atpG) from T. pseudonana has been successfully expressed with His-tags, which allows for efficient purification using metal affinity chromatography .

The full-length recombinant atpG protein (156 amino acids) has been produced and characterized, suggesting that similar approaches could be applied to atpH . The structural integrity of these recombinant proteins is critical for functional studies and can be verified through methods such as SDS-PAGE and Western blotting to confirm purity and molecular weight.

How do expression conditions affect the yield and activity of recombinant T. pseudonana atpH protein?

Expression conditions significantly impact the yield and activity of recombinant T. pseudonana atpH. Based on similar recombinant protein expression studies with T. pseudonana proteins, optimal expression typically occurs when E. coli cultures reach an OD600 of approximately 0.6 before induction with IPTG (1 mM). Post-induction cultivation is generally performed at 37°C for 3 hours, followed by cell harvesting and processing .

For challenging membrane proteins like ATP synthase subunits, expression parameters can be adjusted in the following ways:

The lysis and extraction procedures should be carefully optimized, potentially using detergents or specialized buffers to solubilize membrane-associated proteins like atpH. The preparation typically involves cell resuspension in a buffer containing sucrose (25% w/v), EDTA, and DTT, followed by treatment with lysozyme, DNase I, and MgCl2 to facilitate cell disruption .

What are the challenges in distinguishing between native and recombinant ATP synthase activity in experimental setups?

Distinguishing between native and recombinant ATP synthase activity presents several challenges in experimental setups. When studying T. pseudonana atpH, researchers must account for:

• Oligomeric assembly requirements: ATP synthase subunits function within a multi-subunit complex. Recombinant atpH must integrate properly with other subunits to exhibit native-like activity.

• Post-translational modifications: Native atpH may undergo specific modifications in T. pseudonana that are absent in recombinant systems like E. coli.

• Membrane environment: As a membrane protein, atpH activity is highly dependent on the lipid environment, which differs between diatom chloroplasts and recombinant expression systems.

To address these challenges, researchers can employ the following strategies:

• Use tag-specific antibodies to differentiate between native and recombinant proteins in activity assays

• Perform complementation studies in ATP synthase-deficient mutants

• Develop in vitro reconstitution systems with purified components

• Utilize isotope labeling to track the recombinant protein in complex mixtures

How does the electron transport chain in T. pseudonana chloroplasts interact with ATP synthase compared to other photosynthetic organisms?

The electron transport chain in T. pseudonana chloroplasts exhibits distinct interactions with ATP synthase compared to other photosynthetic organisms. According to metabolic modeling studies, T. pseudonana primarily utilizes linear electron flow (LEF) under normal phototrophic conditions, while cyclic electron flow (CEF) appears to be inactive—a notable difference from green algae and plants .

This has important implications for ATP synthase function:

• The proton motive force that drives ATP synthase is generated primarily through LEF in T. pseudonana under normal conditions.

• The ATP/NADPH ratio produced through photosynthesis likely differs from that in organisms that utilize both LEF and CEF.

• The regulation of ATP synthase activity may be tailored to these electron flow characteristics.

These differences reflect the unique evolutionary history of diatoms, which acquired chloroplasts through secondary endosymbiosis. The genome-scale metabolic model (iThaps987) validates these observations about electron flow patterns in T. pseudonana, providing a framework for understanding how ATP synthase operates within the context of diatom-specific photosynthetic machinery .

What are the optimal conditions for recombinant expression of T. pseudonana atpH in E. coli?

For optimal recombinant expression of T. pseudonana atpH in E. coli, a systematic approach based on established protocols for similar proteins is recommended. The following conditions have proven effective for other T. pseudonana proteins and can be adapted for atpH:

Expression vector selection:

• pPROEX-HTb or pET28a vectors are suitable for expression in E. coli

• Addition of N-terminal and/or C-terminal hexahistidine tags enhances purification efficiency

• Inclusion of rTEV protease cleavage sites allows for tag removal if needed

PCR amplification and cloning strategy:

• Design primers with appropriate restriction sites (e.g., StuI/HindIII or BamHI/XhoI)

• Include hexahistidine tag sequences in the primers if not present in the vector

• Amplify the target gene from T. pseudonana genomic DNA

• Digest and ligate into the expression vector

• Transform into E. coli DH5α for plasmid propagation

Expression conditions:

• Transform expression plasmid into E. coli BL21(DE3) or similar expression strains

• Grow cultures at 37°C until OD600 reaches 0.6

• Induce with 1 mM IPTG

• Continue expression for 3-4 hours at 37°C

• Harvest cells by centrifugation and wash with 1% (w/v) NaCl

For membrane proteins like atpH, expression at lower temperatures (16-25°C) for longer durations (16-24 hours) may improve proper folding and solubility.

What purification techniques are most effective for isolating recombinant T. pseudonana atpH while maintaining protein activity?

Purifying recombinant T. pseudonana atpH while preserving its activity requires careful consideration of its membrane-associated nature. The following purification strategy is recommended:

Cell lysis protocol:

• Resuspend cell pellet in lysis buffer A (50 mM Tris-HCl, pH 8.0, 25% (w/v) sucrose, 1 mM EDTA, 10 mM DTT)

• Add lysozyme (0.5 mg/ml), DNase I (19 μg/ml), and MgCl2 (2.0 mM)

• Add equal volume of lysis buffer B (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 10 mM DTT)

• Incubate at room temperature for 1 hour

• Add EDTA to final concentration of 6.7 μM

• Flash freeze in liquid nitrogen to enhance membrane disruption

Purification scheme:

For membrane proteins like atpH, inclusion of appropriate detergents is crucial:

• Initial extraction: 1% (w/v) n-dodecyl-β-D-maltoside (DDM)

• Purification buffers: 0.02-0.05% DDM to maintain solubility

Storage conditions:

• Store purified protein in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Addition of 5-50% glycerol (typically 50%) for long-term storage

• Aliquot and store at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

How can researchers effectively validate the functional integrity of recombinant T. pseudonana atpH?

Validating the functional integrity of recombinant T. pseudonana atpH requires multiple complementary approaches:

Structural integrity assessment:

• SDS-PAGE analysis to confirm protein purity and expected molecular weight

• Western blotting using anti-His antibodies or custom antibodies against atpH

• Circular dichroism (CD) spectroscopy to assess secondary structure elements

• Limited proteolysis to evaluate proper folding

• Dynamic light scattering to check for aggregation

Functional characterization:

• Proton transport assays: Using pH-sensitive fluorescent dyes in reconstituted liposomes

• ATP hydrolysis activity: Measuring ATPase activity through phosphate release assays

• Binding affinity studies: Isothermal titration calorimetry to assess interactions with other ATP synthase subunits

• Reconstitution experiments: Integration into artificial membranes or proteoliposomes to assess function

In silico validation:

• Comparison of experimental results with predictions from the genome-scale metabolic model (iThaps987)

• Structural modeling and molecular dynamics simulations to assess stability and potential functional properties

Genetic complementation:

• Expression in ATP synthase subunit-deficient bacterial strains to test functional rescue

• If transformation protocols for T. pseudonana are available, testing complementation in atpH mutant strains

Researchers should combine multiple validation approaches to build a comprehensive assessment of recombinant atpH integrity and functionality.

How can recombinant T. pseudonana atpH be used to study diatom-specific adaptations in photosynthetic energy conversion?

Recombinant T. pseudonana atpH serves as a valuable tool for investigating diatom-specific adaptations in photosynthetic energy conversion. Diatoms have unique photosynthetic pathways that differ from green algae and plants, making their ATP synthase components particularly interesting for comparative studies.

Research applications include:

• Comparative structural biology: Crystal structure determination of recombinant atpH can reveal diatom-specific structural adaptations that may relate to function in marine environments. These structures can be compared with ATP synthase components from other photosynthetic organisms to identify unique features.

• Functional characterization under varying conditions: Recombinant atpH can be used in reconstitution studies to assess how diatom ATP synthase functions under conditions that mimic marine environments, including:

  • Variable salt concentrations

  • pH fluctuations

  • Temperature changes

  • Light intensity variations

• Interaction studies with diatom-specific components: Using techniques such as pull-down assays, surface plasmon resonance, or crosslinking studies with recombinant atpH to identify unique interaction partners in diatoms.

• Integration with genomic metabolic models: Experimental data from recombinant atpH studies can be incorporated into the genome-scale metabolic model (iThaps987) to refine predictions about energy metabolism in diatoms .

The unique photosynthetic electron flow patterns observed in T. pseudonana, particularly the preference for linear electron flow over cyclic electron flow under normal conditions, suggest that ATP synthase may have adapted to operate efficiently within this specific electron transport context .

What insights can mutagenesis studies of recombinant T. pseudonana atpH provide about ATP synthase evolution in diatoms?

Mutagenesis studies of recombinant T. pseudonana atpH can reveal crucial insights into ATP synthase evolution in diatoms, which have a unique evolutionary history due to secondary endosymbiosis.

Key research approaches:

• Site-directed mutagenesis targeting conserved residues: By mutating highly conserved amino acids in atpH and measuring the effects on function, researchers can identify residues that are universally critical for ATP synthase function across diverse organisms.

• Mutation of diatom-specific residues: Identifying and mutating amino acids that are unique to diatom atpH can reveal adaptations specific to the diatom lineage and their marine environment.

• Domain swapping experiments: Replacing segments of T. pseudonana atpH with corresponding regions from other organisms (e.g., cyanobacteria, green algae, or other stramenopiles) can help determine which domains confer diatom-specific properties.

Evolutionary insights that can be gained:

• The genome-scale metabolic model comparison between T. pseudonana and P. tricornutum revealed 183 unique enzymes in T. pseudonana, suggesting significant metabolic divergence even among diatoms . Similar divergence might exist in ATP synthase components.

• Understanding structural adaptations that enable ATP synthase to function efficiently with the C3-type photosynthetic carbon fixation pathway prevalent in diatoms .

• Identification of adaptations related to the marine environment, including potential mechanisms for coping with fluctuating ion concentrations, light availability, and temperature.

• Insights into how ATP synthase components co-evolved with the unique violaxanthin-diadinoxanthin pathway that appears to be preferred in T. pseudonana .

How can information from the T. pseudonana genome-scale metabolic model inform research on ATP synthase function in diatom energy metabolism?

Integration of ATP synthase in metabolic networks:

Research strategies enabled by the model:

The model also suggests that T. pseudonana has metabolic potential for producing various compounds through engineering approaches , indicating that manipulation of energy metabolism through ATP synthase could be a valuable strategy for enhancing production of target compounds.

What are the most promising approaches for studying the regulation of ATP synthase assembly in T. pseudonana?

Studying the regulation of ATP synthase assembly in T. pseudonana presents unique challenges due to the complex architecture of diatom chloroplasts derived from secondary endosymbiosis. Several promising approaches can advance this area:

• Proteomic time-course studies: Using pulse-chase labeling combined with mass spectrometry to track the assembly process of the ATP synthase complex, with particular focus on the incorporation of atpH.

• Identification of assembly factors: The genome-scale metabolic model (iThaps987) can help identify potential candidate genes involved in ATP synthase assembly through guilt-by-association network analyses .

• Fluorescence microscopy techniques: Developing fluorescently tagged versions of atpH and other ATP synthase subunits to visualize the assembly process in vivo.

• Conditional expression systems: Creating T. pseudonana strains with inducible expression of atpH to study the temporal aspects of ATP synthase assembly.

• Comparative genomics: Leveraging the unique enzymes identified in T. pseudonana compared to P. tricornutum to identify potential diatom-specific assembly factors or regulatory proteins.

The successful expression and purification strategies developed for recombinant T. pseudonana proteins provide a foundation for producing and studying multiple ATP synthase components and their interactions during the assembly process.

How might environmental factors specific to marine habitats influence the structure and function of T. pseudonana ATP synthase?

The marine environment presents unique challenges that likely influence the structure and function of T. pseudonana ATP synthase. These environmental factors and their potential impacts include:

Environmental influences on ATP synthase:

The genome-scale metabolic model of T. pseudonana reveals that this diatom possesses unique metabolic adaptations compared to other diatoms like P. tricornutum , suggesting that its energy generation systems, including ATP synthase, may have evolved specific features to thrive in its ecological niche.

Future research should focus on characterizing how recombinant atpH and the assembled ATP synthase complex respond to these environmental variables, potentially revealing adaptations that could inform biotechnological applications or provide insights into diatom evolution.

What potential exists for engineering T. pseudonana ATP synthase components for biotechnological applications?

The biotechnological potential of engineered T. pseudonana ATP synthase components extends beyond basic research into various applied fields. The genome-scale metabolic model (iThaps987) has already suggested the potential of T. pseudonana for producing industrially useful compounds , and engineering ATP synthase could enhance these capabilities:

Potential biotechnological applications:

Engineering approaches:

• Structure-guided mutagenesis: Using insights from recombinant atpH studies to make targeted modifications that enhance specific properties.

• Directed evolution: Developing screening systems to select for variants of atpH with improved properties.

• Heterologous expression: Testing hybrid ATP synthase complexes with components from different organisms to achieve desired properties.

• Model-guided engineering: Using the genome-scale metabolic model (iThaps987) to predict the system-wide effects of ATP synthase modifications .

The successful expression and purification protocols established for T. pseudonana recombinant proteins provide the foundation for producing and testing engineered variants of ATP synthase components, facilitating these biotechnological applications.