Recombinant Phalaenopsis aphrodite subsp. formosana ATP synthase subunit b, chloroplastic (atpF)

What is the evolutionary conservation status of ATP synthase subunit b in Phalaenopsis aphrodite compared to other plant species?

ATP synthase subunit b (atpF) in Phalaenopsis aphrodite shows significant evolutionary conservation across plant species, particularly in functional domains critical for ATP synthesis. The protein contains a transmembrane domain and an extramembrane domain, with the N-terminal region bonded to the complex membrane through antiparallel α-helicals. The remaining portion extends from the membrane as an electrically charged coil structure that is crucial for binding the F1 component to the membrane-embedded F0 component . Comparative genomic studies of Phalaenopsis aphrodite subsp. formosana with other orchids reveal conservation patterns in key functional regions while allowing for species-specific adaptations in non-critical regions.

What specific domains of the atpF gene are most conserved in Phalaenopsis aphrodite subsp. formosana, and what are their functional implications?

The most conserved domains in the atpF gene of Phalaenopsis aphrodite subsp. formosana include the transmembrane α-helical regions (residues 12-26 and 33-47) and the coiled-coil domains . These regions are critical for proper insertion into the chloroplast membrane and for maintaining structural stability during ATP synthesis. The N-terminus of the b subunit, responsible for proton translocation, contains highly conserved residues that facilitate efficient H+ ion selectivity . The C-terminal domain, which binds to the oligomycin sensitivity conferral protein (OSCP) C-terminal domain, is also well-preserved across species due to its role in regulating complex rotation and binding.

What are the optimal expression systems for producing recombinant Phalaenopsis aphrodite subsp. formosana atpF protein?

For successful expression of recombinant Phalaenopsis aphrodite subsp. formosana atpF protein, heterologous expression systems should be carefully selected based on the specific research objectives. Bacterial expression systems (particularly E. coli) offer high yield and rapid production but may struggle with proper folding of plant chloroplastic proteins. For more authentic post-translational modifications, plant-based expression systems such as Nicotiana benthamiana or Arabidopsis cell cultures are recommended.

When using bacterial systems, codon optimization is essential to accommodate the different codon usage between orchids and bacteria. The addition of chloroplast transit peptide sequences from model plants can improve targeting in plant-based systems. Expression protocols should include careful temperature control (typically 18-25°C) to minimize inclusion body formation and maximize proper folding of the transmembrane regions that are critical for atpF function.

What purification challenges are specific to recombinant atpF protein, and how can they be overcome?

Purification of recombinant atpF presents several challenges due to its transmembrane domains and tendency to form stable complexes. A multi-step purification strategy is most effective, beginning with affinity chromatography using carefully positioned tags that don't interfere with protein folding or function. N-terminal His6 tags are commonly used, but C-terminal positioning may be preferable to avoid disrupting the critical N-terminal transmembrane regions .

Membrane protein extraction requires specialized detergents such as n-dodecyl-β-D-maltoside (1% w/v) or digitonin (2% w/v) as used in ATP synthase complex isolation . Size exclusion chromatography following initial affinity purification helps separate monomeric atpF from aggregates or complexes. For studying atpF in its native complex, blue native polyacrylamide gel electrophoresis (BN-PAGE) on 3-10% gradient gels has proven effective for analyzing ATP synthase complexes .

How can researchers verify the proper folding and assembly of recombinant atpF protein?

Verification of proper folding and assembly requires a combination of structural and functional analyses. Circular dichroism spectroscopy is essential for confirming the expected secondary structure, particularly the α-helical content characteristic of the b subunit. Thermal shift assays can assess protein stability under various buffer conditions.

For functional verification, reconstitution of the recombinant atpF into proteoliposomes allows assessment of its ability to participate in ATP synthesis. Single-molecule techniques can detect both ATP synthesis and ion transport simultaneously . Properly folded and functional atpF should demonstrate K+ and H+ transport capabilities that drive ATP synthesis under physiological conditions (pH 7.2, K+ = 140 mEq/L) . The reconstituted protein should show typical inhibitor sensitivity profiles, with specific F0 inhibitors blocking both ATP synthesis and ion transport.

What methodologies are most effective for studying the ion transport function of recombinant atpF in vitro?

For studying ion transport function of recombinant atpF, proteoliposome reconstitution combined with voltage clamp techniques provides the most detailed insights. The protocol involves:

• Reconstitution of purified recombinant atpF into liposomes with defined lipid composition

• Generation of an electrochemical gradient across the proteoliposome membrane

• Simultaneous measurement of ion currents and ATP synthesis

This approach has successfully demonstrated that ATP synthase can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological conditions . Single-molecule measurements of K+-driven ATP synthesis can be achieved using bioluminescence photon detection concurrent with unitary K+ current measurements by voltage clamp. This methodology revealed a 2.7:1 K+:H+ stoichiometry in the transport process .

For higher throughput screening, fluorescent pH indicators or potassium-sensitive probes can monitor ion movement across membranes in reconstituted systems or isolated chloroplasts expressing the recombinant protein.

How can researchers assess the impact of specific amino acid mutations in recombinant atpF on ATP synthesis efficiency?

Assessment of mutation effects requires a systematic approach combining structural and functional analyses:

• Generate site-directed mutations in conserved or functionally relevant residues

• Express and purify the mutant proteins alongside wild-type controls

• Perform in vitro ATP synthesis assays using reconstituted proteoliposomes

• Measure both ATP synthesis rates and ion transport capabilities

Mutations in the transmembrane domains can significantly impact proton translocation. For example, polar edits (such as S20F, S24L, and S30L) in the N-terminus have been shown to affect the proton flow mechanism by altering H+ ion selectivity . Similarly, mutations in the coiled-coil regions (positions S72L, P76L, P83L, P84L, S131L, P136L, and T139I) can disrupt the stabilizing alpha-helical structures that prevent dissociation of the ATP synthase complex under stress conditions .

Comparative analysis between wild-type and mutant proteins should include:

What techniques are available for detecting protein-protein interactions involving recombinant atpF in the ATP synthase complex?

Several complementary techniques can effectively characterize protein-protein interactions involving recombinant atpF:

• Blue Native PAGE (BN-PAGE): This technique separates native protein complexes while preserving their interactions. Using a 3-10% gradient with either 1% n-dodecyl-β-D-maltoside or 2% digitonin for solubilization, followed by second-dimension SDS-PAGE, allows visualization of atpF interactions within the ATP synthase complex .

• Co-immunoprecipitation: Using antibodies against atpF or potential interaction partners can pull down intact complexes for subsequent analysis by mass spectrometry.

• Förster resonance energy transfer (FRET): By tagging atpF and potential interaction partners with appropriate fluorophores, researchers can detect close molecular associations in reconstituted systems or in vivo.

• Cross-linking coupled with mass spectrometry: This approach can identify precise interaction interfaces between atpF and other ATP synthase subunits, providing structural insights beyond traditional interaction mapping.

Analysis of these interactions under different physiological conditions (pH, ion concentrations, energy status) can reveal dynamic changes in the ATP synthase complex that regulate its function in response to environmental conditions.

How does the genomic organization of the atpF gene in Phalaenopsis aphrodite subsp. formosana compare to other orchid species?

Comparative genomic analyses should examine:

• Exon-intron structure of the atpF gene

• Presence of transposable elements in proximity to the gene

• Conservation of regulatory regions

The genome of Phalaenopsis aphrodite has been published , facilitating such comparative analyses. Special attention should be paid to transposable elements, as they significantly impact orchid genome structure and evolution. For instance, retrotransposons like Orchid-rt1 and Gypsy1 have been identified in Phalaenopsis orchids and may affect gene expression when inserted into introns .

What transcriptomic approaches are most useful for studying atpF expression patterns across different tissues and developmental stages?

For comprehensive transcriptomic analysis of atpF expression:

• RNA-Seq: Next-generation sequencing provides a global view of gene expression. In Phalaenopsis orchids, this approach has successfully identified expression patterns in various tissues . For atpF analysis, sampling should include photosynthetically active tissues (leaves), non-photosynthetic tissues (roots), and different developmental stages of floral organs.

• Quantitative RT-PCR: For targeted validation of expression patterns identified through RNA-Seq. This approach should use carefully designed primers specific to atpF to avoid cross-amplification with closely related genes.

• In situ hybridization: To visualize tissue-specific expression patterns within complex organs, particularly important for understanding spatial regulation in heterogeneous tissues.

Expression analysis should correlate atpF levels with energy demands across tissues and developmental stages. In Phalaenopsis orchids, meristematic tissues tend to show enrichment of genes involved in energy metabolism due to their high proliferation activity .

How does RNA editing affect the atpF transcript in chloroplasts, and what are the functional consequences?

RNA editing plays a crucial role in modifying organellar transcripts, including those encoding ATP synthase components. In chloroplasts, C-to-U editing is most common and can significantly alter protein structure and function.

RNA editing in atpF transcripts can result in amino acid changes with significant functional implications. For example, serine-to-leucine (S24L, S30L) or serine-to-phenylalanine (S20F) substitutions in the N-terminal region can alter the proton flow mechanism by affecting H+ ion selectivity rather than causing dramatic structural changes . These subtle modifications enable the ATP synthase to adapt to changes in proton motive force under different environmental conditions.

Next-generation sequencing (NGS) is an effective tool for comprehensively detecting RNA editing sites in organellar transcripts . Analysis of RNA editing in atpF should examine:

• Conservation of editing sites across orchid species

• Tissue-specificity of editing patterns

• Environmental factors that may influence editing efficiency

• Structural consequences of edited amino acids using predictive modeling

How can recombinant atpF be used to develop biosensors for monitoring energy metabolism in plant cells?

Recombinant atpF can serve as a foundation for developing sophisticated biosensors to monitor cellular energy status:

• Fluorescent protein fusions: By creating functional fusions of atpF with fluorescent proteins, researchers can monitor ATP synthase localization and assembly dynamics in living cells. Care must be taken to position the fluorescent tag where it minimally disrupts function, potentially at the C-terminus or within non-critical loops.

• FRET-based sensors: Engineered atpF variants can incorporate fluorophore pairs that undergo FRET in specific conformational states, allowing real-time monitoring of ATP synthase activity in response to changing metabolic conditions.

• Electrochemical biosensors: Immobilization of purified recombinant atpF in electrode systems can create biosensors that detect changes in proton concentration or membrane potential, providing insights into energy metabolism.

These biosensors can address fundamental questions in plant biology, including how energy metabolism adapts to environmental stresses and developmental transitions. Applications could extend to monitoring chloroplast function in engineered crop plants or detecting alterations in energy metabolism associated with plant diseases.

What structural modeling approaches provide the most accurate predictions of atpF conformational changes during ATP synthesis?

Advanced structural modeling approaches for atpF include:

• Homology modeling: Using high-resolution structures of ATP synthase b subunits from model organisms as templates. Three-dimensional models of mitochondrial ATP synthase have revealed distinct conformational states reflecting differences in the central stalk and F1-catalytic domain .

• Molecular dynamics simulations: To predict conformational changes during the catalytic cycle, particularly the movement of the b subunit as it coordinates with other components of the ATP synthase complex.

• Cryo-electron microscopy data integration: Electron cryo-microscopy has determined three distinct states of mitochondrial ATP synthase , providing templates for modeling conformational changes in the plant chloroplastic version.

Structural models should focus on the critical domains of atpF, including:

• The N-terminal transmembrane α-helices (residues 12-26 and 33-47)

• The small turn in the long α-helix at residues 48-54

• The coiled-coil regions that provide structural stability

These models can predict how specific amino acid changes affect the stability and function of the ATP synthase complex, particularly under stress conditions like drought or high salinity .

How does the interaction between recombinant atpF and translocase complexes affect protein import into chloroplasts?

The interaction between ATP synthase components and chloroplast protein import machinery represents a complex research area with significant implications for chloroplast biogenesis. Research approaches should include:

• Co-immunoprecipitation studies: To identify physical interactions between atpF and components of the TOC/TIC (Translocon at the Outer/Inner envelope membrane of Chloroplasts) complexes.

• In vitro protein import assays: Using isolated chloroplasts and recombinant precursor proteins to assess how alterations in atpF affect import efficiency.

• Fluorescent protein localization: Tracking the import of reporter proteins in plants with modified atpF expression.

Evidence from research on chloroplast envelope ATPases indicates their importance for protein accumulation in chloroplasts. For example, AtFtsH12 is required for the accumulation of cytosol-translated chloroplast proteins . In protoplasts with defective AtFtsH12, GFP fusion proteins showed reduced chloroplast localization and aberrant accumulation in the cytosol .

This research direction could provide valuable insights into how energy metabolism and protein import are coordinated during chloroplast biogenesis and adaptation to environmental changes.

What are the most common technical challenges in expressing recombinant chloroplastic proteins from orchids, and how can they be overcome?

Expression of recombinant chloroplastic proteins from orchids presents several technical challenges:

• Codon usage optimization: Orchid genes often contain codon biases that reduce expression efficiency in common host systems. Solution: Synthesize codon-optimized gene sequences based on the preferred codon usage of the expression host.

• Protein toxicity: Membrane proteins like atpF can be toxic when overexpressed. Solution: Use tightly regulated inducible expression systems and reduce expression temperature to 16-20°C to slow protein synthesis and improve folding.

• Transit peptide processing: Chloroplastic proteins contain transit peptides that may not be properly processed in heterologous systems. Solution: Express mature proteins without transit peptides or use homologous transit peptides from the expression host.

• Inclusion body formation: Hydrophobic membrane proteins often form inclusion bodies. Solution: Optimize solubilization conditions using different detergents like n-dodecyl-β-D-maltoside (1% w/v) or digitonin (2% w/v) and consider fusion partners that enhance solubility.

• Low protein yield: Orchid-derived proteins often express at lower levels than those from model organisms. Solution: Screen multiple expression hosts, optimize media compositions, and consider using orchid-specific expression elements.

How can researchers design experiments to distinguish between ATP synthesis driven by H+ versus K+ transport through recombinant atpF?

Designing experiments to distinguish between H+ and K+-driven ATP synthesis requires careful manipulation of ion gradients and specific inhibitors:

• Ion-selective conditions: Create proteoliposomes with defined internal and external ion compositions. By selectively creating either H+ or K+ gradients while eliminating the other, researchers can isolate the contribution of each ion to ATP synthesis.

• Simultaneous measurements: Use systems capable of measuring both ATP synthesis (via bioluminescence) and ion currents (via voltage clamp) from the same preparation . This approach has successfully demonstrated K+-driven ATP synthesis concurrent with unitary K+ currents.

• Inhibitor profiles: Apply specific inhibitors of F0 that block both ATP synthesis and ion transport to confirm the role of atpF in the observed activities .

• Mutagenesis approach: Introduce mutations that specifically alter binding sites for H+ or K+ to create variants with selective ion preferences.

Experimental data from mammalian F1Fo-reconstituted proteoliposomes have shown that ATP synthase can utilize both ΔΨm-driven H+- and K+-transport for ATP synthesis under physiological conditions (pH 7.2, K+ = 140 mEq/L) . Similar approaches can be applied to orchid recombinant atpF to determine its ion specificity and stoichiometry.

What strategies can address the challenge of studying transient protein-protein interactions involving atpF during the catalytic cycle?

Capturing transient protein-protein interactions in the dynamic ATP synthase complex requires specialized approaches:

• Time-resolved crosslinking: Apply rapid crosslinking reagents at different time points during the catalytic cycle to "freeze" transient interactions for subsequent analysis by mass spectrometry.

• Single-molecule FRET: By labeling interaction partners with appropriate fluorophores, researchers can detect conformational changes and protein associations in real-time during ATP synthesis or hydrolysis.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique identifies regions of proteins that become protected from exchange during interactions, revealing binding interfaces with temporal resolution.

• Structure-guided mutations: Introduce mutations at predicted interaction interfaces and assess their impact on complex formation and function to validate structural models.

• Computational molecular dynamics: Simulate the catalytic cycle to predict transient interactions that can then be validated experimentally.

These approaches can reveal how the b subunit coordinates with other components during the catalytic cycle, particularly how its long α-helical structure provides both stability and flexibility during the rotational movements essential for ATP synthesis.