Recombinant Phaeodactylum tricornutum ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c in Phaeodactylum tricornutum and why is it important for research?

ATP synthase subunit c (atpH) in P. tricornutum is a critical component of the chloroplastic ATP synthase complex, responsible for ATP production during photosynthesis. This small, hydrophobic protein forms the c-ring in the F0 portion of ATP synthase, which is embedded in the thylakoid membrane and facilitates proton translocation. Studying recombinant atpH is important for several reasons:

• It provides insights into energy metabolism in diatoms, which contribute significantly to global primary production

• Understanding the structure and function of diatom ATP synthase can reveal evolutionary adaptations specific to marine photosynthetic organisms

• Recombinant expression systems for chloroplastic proteins can serve as models for the expression of other challenging membrane proteins

• As a relatively small protein with essential function, it serves as an excellent model for studying chloroplast genetic engineering in diatoms

Research indicates that chloroplastic proteins like atpH can be efficiently expressed using endogenous promoters such as HASP1, which shows superior activity compared to traditional fcpA promoters, especially during stationary phase growth .

How does P. tricornutum atpH differ from other model organisms' ATP synthase subunit c?

• Sequence variations: The primary sequence shows differences reflecting adaptation to the diatom chloroplast environment, which originated from secondary endosymbiosis

• Codon usage: Unlike model organisms like Chlamydomonas reinhardtii, P. tricornutum has distinct codon preferences that must be considered when designing recombinant expression systems

• Post-translational modifications: Diatom chloroplastic proteins may undergo unique post-translational modifications

• Thylakoid membrane composition: The lipid environment in diatom thylakoids differs from that of plants and green algae, potentially affecting atpH structure and function

When designing experimental systems for recombinant P. tricornutum atpH, researchers must account for these differences. For example, the chloroplast genome cloning approach described for P. tricornutum (117 kb) differs from methods used for green algae like C. reinhardtii (34.57% G+C) .

Which promoter systems are most efficient for expressing recombinant chloroplastic proteins like atpH in P. tricornutum?

Based on comparative studies, the HASP1 promoter system demonstrates superior efficiency for expressing recombinant proteins in P. tricornutum compared to the traditional fcpA promoter. Experimental data shows:

The HASP1 promoter maintains high expression levels throughout all growth phases, with particularly strong activity during the stationary phase. Transcript analysis reveals that HASP1 promoter-driven expression resulted in 3-fold higher transcript levels than fcpA during log phase and 44-fold higher levels during stationary phase .

For chloroplastic proteins like atpH, this sustained high expression is advantageous as it allows continual protein accumulation throughout the culture period. Additionally, the HASP1 promoter includes a signal peptide sequence that can be utilized for efficient protein secretion if desired, offering flexibility in experimental design .

What are the critical factors to consider when designing expression constructs for recombinant atpH?

When designing expression constructs for recombinant P. tricornutum atpH, researchers should address these critical factors:

• Promoter selection:

  • For constitutive high expression, the HASP1 promoter shows optimal activity across all growth phases

  • For growth phase-specific expression, choose between HASP1 (stationary phase) or fcpA (log phase) accordingly

• Codon optimization:

  • Align codons with P. tricornutum preferences to enhance translation efficiency

  • Avoid rare codons that might limit expression

• Signal peptide considerations:

  • For chloroplast targeting, maintain the native chloroplast transit peptide

  • For potential secretion studies, the HASP1 signal peptide has been shown to facilitate efficient protein secretion

• Reporter system integration:

  • Include a reporter (e.g., GFP) to monitor expression levels

  • Consider fusion proteins versus co-expression systems based on experimental goals

• Selectable markers:

  • Include appropriate antibiotic resistance genes for selection in P. tricornutum

• Vector backbone considerations:

  • Vector stability in P. tricornutum

  • Replication origin compatibility

Research shows that expression constructs combining the HASP1 promoter with its native signal peptide can achieve protein secretion levels up to 19-fold higher than those using the fcpA promoter during late stationary phase . For intracellular chloroplastic proteins like atpH, removing the signal peptide while maintaining the HASP1 promoter would be advisable to retain high expression while ensuring proper subcellular localization.

What are the most effective methods for genetically modifying the P. tricornutum chloroplast genome to express recombinant atpH?

Several approaches have been developed for modifying the P. tricornutum chloroplast genome, with efficiency varying based on the specific experimental goals:

• Yeast assembly-based chloroplast genome cloning:

  • PCR-based approach: The entire 117-kb chloroplast genome can be PCR-amplified as 8 overlapping fragments and assembled in yeast with 90-100% efficiency when screening just 10 yeast colonies

  • Precloned approach: Individual fragments are first cloned into plasmids before assembly, allowing greater flexibility for genome modification

• SapI restriction site integration:

  • Introducing a single SapI site into the cloned genome (e.g., within the URA3 marker) provides a landing pad for targeted integration of transgenic cassettes

  • This approach allows precise insertion of recombinant atpH constructs at a specific location

• Homologous recombination-based strategies:

  • Targeting recombinant genes to intergenic regions minimizes disruption of essential functions

  • The noncoding region between hypothetical chloroplast open reading frame 88 (f88) and ribosomal protein CL22 (rpl22) has been identified as an effective integration site

• Biolistic transformation:

  • Gold or tungsten particles coated with the engineered DNA can be used for chloroplast transformation

  • Selection using appropriate markers enables isolation of transformants

The yeast assembly approach has proven particularly effective, with assembly efficiency reaching 90-100% when screening as few as 10 yeast colonies following whole-genome assembly . This method provides a versatile platform for engineering the chloroplast genome to express recombinant proteins like atpH.

How can researchers verify successful chloroplast genome integration and expression of recombinant atpH?

Verification of successful chloroplast genome integration and expression requires a multi-step approach:

• PCR verification:

  • Design primers flanking the integration site to confirm correct insertion

  • Additional PCR reactions spanning the junctions between the insertion and flanking sequences

  • Long-range PCR to verify larger-scale genome integrity

• Transcript analysis:

  • Real-time RT-PCR to quantify atpH transcript levels

  • Compare expression levels between different constructs (e.g., HASP1 vs. fcpA promoter-driven)

  • Northern blotting for qualitative assessment of transcript size and integrity

• Protein detection:

  • Western blotting using antibodies against atpH or epitope tags

  • Fluorescence measurements if using fluorescent protein fusions

  • Mass spectrometry for detailed protein characterization

• Functional assays:

  • ATP synthesis activity measurements

  • Proton pumping assays

  • Growth rate analysis under different light conditions

Experimental data from similar recombinant protein studies show that real-time RT-PCR can reliably detect differences in transcript levels, with the HASP1 promoter generating 35-fold and 764-fold higher transcript levels compared to promoter-less controls on days 4 and 8, respectively . For protein-level verification, fluorescence measurements of GFP-tagged proteins have demonstrated that HASP1 promoter-driven expression can reach levels 300-fold higher than background in late stationary phase .

What are the optimal methods for purifying recombinant P. tricornutum atpH for structural and functional studies?

Purifying recombinant atpH presents challenges due to its hydrophobic nature and membrane localization. The following optimized protocol integrates approaches from membrane protein biochemistry with diatom-specific considerations:

• Cell disruption and membrane isolation:

  • French press or sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 1 mM DTT, and protease inhibitors

  • Differential centrifugation: low-speed centrifugation (5,000 × g, 10 min) to remove unbroken cells, followed by high-speed centrifugation (100,000 × g, 1 hour) to isolate membranes

• Detergent solubilization:

  • Screen multiple detergents for optimal extraction (n-dodecyl-β-D-maltoside, digitonin, or n-octyl-β-D-glucoside)

  • Typical conditions: 1% detergent, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 4°C, 1 hour with gentle agitation

  • Remove insoluble material by centrifugation (100,000 × g, 30 min)

• Affinity purification (if tagged):

  • For His-tagged constructs: Ni-NTA affinity chromatography

  • For FLAG-tagged constructs: Anti-FLAG affinity chromatography

  • Include 0.05% detergent in all buffers to maintain protein solubility

• Size exclusion chromatography:

  • Further purification and assessment of oligomeric state

  • Superdex 200 column with buffer containing 0.05% detergent, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0)

• Protein concentration:

  • Use 30 kDa MWCO concentrators with gentle centrifugation

  • Monitor for precipitation and adjust detergent concentration if necessary

It's important to note that when using the HASP1 promoter system, protein expression is highest during the stationary phase, with levels approximately 3-fold higher than those achieved with the fcpA promoter . Therefore, harvesting cells during late stationary phase is recommended for maximum yield.

What analytical techniques are most informative for characterizing the structure and function of recombinant atpH?

Comprehensive characterization of recombinant atpH requires multiple analytical approaches to understand its structure, function, and interactions:

• Structural analysis:

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

  • Nuclear magnetic resonance (NMR) for atomic-level structural insights

  • Cryo-electron microscopy for visualization of the assembled c-ring

  • X-ray crystallography for high-resolution structural determination

• Functional characterization:

  • ATP synthesis assays using reconstituted proteoliposomes

  • Proton translocation measurements using pH-sensitive fluorescent dyes

  • ATPase activity assays to assess reverse function

• Interaction studies:

  • Blue native PAGE to analyze complex assembly

  • Co-immunoprecipitation to identify interacting partners

  • Crosslinking mass spectrometry to map interaction interfaces

• Biophysical characterization:

  • Thermal stability assays using differential scanning fluorimetry

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

When designing these experiments, researchers should consider that the superior expression levels achieved with the HASP1 promoter (up to 44-fold higher transcript levels compared to fcpA promoter) can significantly impact protein yield and purity . This enhanced expression facilitates more detailed structural and functional studies that might be challenging with lower-yielding systems.

How can researchers assess the functional integrity of recombinant atpH in comparative studies with native ATP synthase?

Assessing functional integrity requires comparison of recombinant atpH with native ATP synthase across multiple parameters:

• ATP synthesis capacity:

  • Reconstitute purified recombinant atpH into liposomes with other ATP synthase subunits

  • Establish a proton gradient and measure ATP production rates

  • Compare kinetic parameters (Km, Vmax) with those of native enzyme

• Proton translocation efficiency:

  • Monitor proton movement using pH-sensitive fluorescent dyes

  • Quantify the number of protons translocated per ATP synthesized

  • Compare proton conductance with native c-rings

• Structural integrity assessment:

  • Analyze c-ring assembly using native PAGE

  • Assess subunit stoichiometry using quantitative mass spectrometry

  • Compare thermal stability profiles between recombinant and native complexes

• In vivo complementation studies:

  • Express recombinant atpH in ATP synthase-deficient mutants

  • Measure restoration of photosynthetic efficiency

  • Analyze growth rates under photoautotrophic conditions

When designing these experiments, researchers should consider optimizing expression using the HASP1 promoter, which maintains high activity throughout all growth phases, particularly during stationary phase when protein levels can be 3-fold higher than with the fcpA promoter . This ensures sufficient protein yield for comprehensive functional analyses.

What approaches are most effective for studying atpH assembly into the complete ATP synthase complex?

Investigating atpH assembly into the ATP synthase complex requires specialized techniques that span from molecular to cellular levels:

• In vitro assembly studies:

  • Co-expression of atpH with other ATP synthase subunits

  • Step-wise reconstitution experiments to determine assembly intermediates

  • Time-course analysis of complex formation using labeled subunits

• Interaction mapping:

  • Crosslinking mass spectrometry to identify interaction interfaces

  • FRET analysis to monitor subunit proximity during assembly

  • Co-immunoprecipitation with stage-specific antibodies to capture assembly intermediates

• Visualization techniques:

  • Single-particle cryo-electron microscopy of assembly intermediates

  • Super-resolution microscopy to track assembly in vivo

  • Electron tomography of chloroplast membranes

• Genetic approaches:

  • Site-directed mutagenesis of key residues to identify assembly determinants

  • Creation of conditional expression systems to control assembly kinetics

  • Isolation and characterization of assembly-defective mutants

The efficiency of assembly studies depends significantly on expression levels. The HASP1 promoter offers advantages over the fcpA promoter, with transcript levels up to 44-fold higher during stationary phase . This higher expression facilitates detection of assembly intermediates that might be present at low abundance when using less efficient promoter systems.

What are common challenges in recombinant atpH expression and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant atpH, each requiring specific troubleshooting approaches:

• Low expression levels:

  • Challenge: Hydrophobic membrane proteins often express poorly

  • Solution: Use the HASP1 promoter, which shows 3-44 fold higher expression levels than the conventional fcpA promoter across growth phases

  • Solution: Harvest cells during stationary phase when using HASP1 promoter for maximum yield

• Protein misfolding and aggregation:

  • Challenge: Membrane proteins may aggregate during expression

  • Solution: Optimize growth temperature (typically lower temperatures reduce aggregation)

  • Solution: Co-express with chaperones or ATP synthase assembly factors

• Toxicity to host cells:

  • Challenge: Overexpression of membrane proteins can disrupt membrane integrity

  • Solution: Use inducible expression systems or controlled culture conditions

  • Solution: Balance expression levels by modifying the strength of the Shine-Dalgarno sequence

• Difficulties in purification:

  • Challenge: Maintaining solubility during extraction

  • Solution: Screen multiple detergents or use styrene-maleic acid lipid particles (SMALPs)

  • Solution: Add stabilizing lipids during purification

• Chloroplast genome instability:

  • Challenge: The A+T-rich chloroplast genome (32.15% G+C) may be unstable in E. coli

  • Solution: Use specialized strains designed for A+T-rich DNA maintenance

  • Solution: The cloned P. tricornutum chloroplast genome has been demonstrated to be stably maintained in E. coli for 60 generations

Experimental data demonstrates that using the HASP1 promoter can significantly improve expression, with GFP fluorescence levels reaching 300-fold higher than background levels during late stationary phase , making it a valuable tool for overcoming expression challenges.

How can researchers design experiments to study the role of atpH in diatom energy metabolism and photosynthetic efficiency?

Designing experiments to investigate atpH's role in diatom energy metabolism requires sophisticated approaches that connect molecular structures to cellular functions:

• Site-directed mutagenesis studies:

  • Target conserved residues in the proton-binding site

  • Create variants with altered c-subunit stoichiometry

  • Modify interface residues that connect with other ATP synthase subunits

• Conditional expression systems:

  • Develop tunable promoters to modulate atpH expression levels

  • Create inducible knockout systems using CRISPR/Cas9

  • Establish complementation systems with variant atpH proteins

• Metabolic flux analysis:

  • Trace carbon and energy flow using stable isotope labeling

  • Quantify ATP/ADP ratios under different light conditions

  • Measure NADPH production in atpH variants

• Photosynthetic efficiency measurements:

  • Chlorophyll fluorescence analysis (PAM fluorometry)

  • P700 absorbance changes to monitor electron transport

  • Oxygen evolution measurements under varying light intensities

• Environmental response studies:

  • Analyze atpH expression and ATP synthase activity under different:

    • Light conditions (intensity, quality)

    • Nutrient availability

    • Temperature regimes

    • CO₂ concentrations

For genetic manipulation experiments, the chloroplast genome cloning methods developed for P. tricornutum provide an efficient platform, with 90-100% assembly efficiency when screening as few as 10 yeast colonies . This high efficiency facilitates the generation of multiple atpH variants for comparative functional studies.

Additionally, when expressing recombinant proteins for these experiments, the HASP1 promoter provides superior expression levels throughout all growth phases, with particularly high activity during stationary phase where it can achieve 44-fold higher transcript levels than the fcpA promoter .