Recombinant Oenothera elata subsp. hookeri ATP synthase subunit c, chloroplastic (atpH)

What is the genetic organization of atpH in Oenothera elata subsp. hookeri?

The atpH gene in Oenothera elata subsp. hookeri is located on the plastid chromosome. The complete nucleotide sequences of five genetically distinct plastid chromosomes (I-V) of subsection Oenothera have been reported in scientific literature . In nature, these plastid genomes associate with six distinct nuclear genotypes derived from three basic nuclear genomes (A, B, or C), creating various combinations that can be either compatible or incompatible . The atpH gene is part of the genetic complement that may contribute to plastome-genome interactions affecting plant development and speciation processes.

How does atpH protein function differ among Oenothera subspecies?

The function of atpH protein may vary among Oenothera subspecies due to sequence polymorphisms that have accumulated during evolution. Studies comparing the five basic plastid genomes (I-V) have identified genes with high Ka/Ks ratios, indicating adaptive evolution . While the search results don't specifically mention atpH variations, the analytical approach used to identify functionally significant amino acid changes can be applied to atpH:

These comparative analyses help researchers understand how functional differences in atpH might contribute to physiological adaptations or incompatibilities between nuclear and plastid genomes in different Oenothera subspecies .

What are the most effective methods for isolating chloroplasts from Oenothera elata tissues?

Isolation of intact chloroplasts is critical for studying chloroplastic proteins like atpH. Based on protocols adapted for Oenothera species, researchers should implement a multi-step procedure:

• Harvest young leaves (preferably early morning) to ensure optimal chloroplast content

• Homogenize tissue in isolation buffer containing sorbitol (0.33 M), HEPES-KOH (50 mM, pH 7.8), EDTA (2 mM), and protease inhibitors

• Filter through multiple layers of cheesecloth and miracloth

• Centrifuge at low speed (200×g) to remove debris

• Collect supernatant and centrifuge at higher speed (2500×g) to pellet chloroplasts

• Resuspend in resuspension buffer and purify through Percoll gradient centrifugation

This method preserves the integrity of the chloroplast outer membrane, which is critical for maintaining protein complexes like ATP synthase in their native state. The same approach has been used successfully to isolate chloroplast outer membrane vesicles for electrophysiological studies of chloroplastic channels .

What purification strategies are most suitable for recombinant atpH protein?

Purification of recombinant atpH requires specialized approaches due to its hydrophobic nature as a membrane protein. A recommended strategy involves:

• Express with an affinity tag (His6 or GST) in an E. coli expression system optimized for membrane proteins

• Extract using mild detergents (n-dodecyl-β-D-maltoside or digitonin) to solubilize while maintaining protein structure

• Purify via affinity chromatography under native conditions

• Perform size exclusion chromatography to remove aggregates and obtain homogeneous protein

• Verify purity by SDS-PAGE and Western blotting with antibodies against the recombinant protein

When reconstituting the purified protein into liposomes for functional studies, researchers should consider that recombinant chloroplast membrane proteins typically incorporate into membranes with a preferred orientation, which may affect subsequent functional assays .

How do experimental conditions affect the functional reconstitution of atpH in liposomes?

The functional reconstitution of membrane proteins like atpH requires careful optimization of experimental parameters. Based on studies with other chloroplastic membrane proteins, researchers should consider a design of experiments (DoE) approach to systematically evaluate critical factors:

Proper reconstitution is critical for assessing the functional properties of the protein. Electrophysiological studies of reconstituted chloroplastic membrane proteins have shown that they typically incorporate with a preferred orientation, which must be considered when interpreting functional assays . Monitoring parameters like proteoliposome size, protein incorporation efficiency, and functional activity across different conditions will help establish optimal reconstitution protocols.

What are the molecular determinants of ATP binding and regulation in atpH?

ATP binding and regulation in chloroplastic proteins involves specific molecular motifs. While direct information about atpH is not provided in the search results, insights can be drawn from studies of other chloroplastic ATP-binding proteins:

• ATP binding sites in chloroplastic membrane proteins often feature conserved motifs such as the FX₄K type (F-X₄-K) that have been identified in other chloroplastic proteins

• The C-terminal region of membrane proteins frequently contains ATP binding domains that are accessible from the intermembrane space

• ATP binding can cause dramatic changes in channel properties, including reversal potential and ion selectivity

For atpH specifically, researchers should examine:

• Conserved sequence motifs that might participate in ATP binding

• Conformational changes induced by ATP binding using spectroscopic methods

• Effects of ATP concentration on protein function using dose-response curves (half-maximal changes in other chloroplastic proteins occur at approximately 70 μM ATP)

How does atpH contribute to plastome-genome incompatibility (PGI) in Oenothera species?

Plastome-genome incompatibility (PGI) represents a significant aspect of speciation in Oenothera. Several lines of evidence suggest chloroplastic proteins like atpH may play roles in this phenomenon:

• Studies of Oenothera plastomes have revealed a remarkable number of genes with high Ka/Ks ratios, indicating adaptive evolution that may contribute to species differentiation

• Sequence polymorphisms, particularly in intergenic regions, have been proposed as possible sources for PGI in Oenothera

• Specific loci, such as the bidirectional promoter region between psbB and clpP, have been linked to compartmental PGI in interspecific hybrids

To investigate atpH's potential role in PGI, researchers should:

• Compare atpH sequences across compatible and incompatible plastome-genome combinations

• Analyze expression levels of atpH in hybrid tissues showing compatibility versus incompatibility

• Evaluate protein-protein interactions between atpH and nuclear-encoded partners

• Create transgenic lines with altered atpH to test for rescue of incompatible phenotypes

These approaches could reveal whether atpH contributes to Dobzhansky-Muller incompatibilities that drive speciation processes in Oenothera .

What analytical techniques best capture the structural dynamics of atpH in different lipid environments?

Understanding the structural dynamics of membrane proteins like atpH requires sophisticated analytical approaches. Based on methodologies used for similar proteins, researchers should consider:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map solvent-accessible regions and conformational changes

• Solid-state NMR to analyze protein structure in membrane environments

• Cryo-electron microscopy for high-resolution structural determination

• Molecular dynamics simulations to predict protein behavior in different lipid compositions

• Fluorescence resonance energy transfer (FRET) to monitor dynamic structural changes upon substrate binding

Each technique provides complementary information about how the protein functions in different lipid environments, which is particularly relevant for atpH as its activity may be influenced by the lipid composition of the chloroplast membrane. The same reconstitution approaches used in electrophysiological studies of chloroplastic channels can be adapted for these structural analyses .

How does post-translational modification affect atpH function?

Post-translational modifications (PTMs) can significantly impact protein function. For chloroplastic proteins like atpH, several PTMs may be relevant:

• Phosphorylation: May regulate ATP synthase assembly or activity

• Acetylation: Could affect protein stability or interactions with other subunits

• Oxidative modifications: May occur under stress conditions, affecting protein function

To investigate PTMs in atpH:

• Use mass spectrometry-based proteomics to identify modification sites

• Generate site-directed mutants to mimic or prevent specific modifications

• Compare modification patterns between different physiological states or stress conditions

• Analyze the impact of modifications on protein-protein interactions within the ATP synthase complex

Unlike some other chloroplastic processes, RNA editing does not appear to play a significant role in the function of plastid genes in Oenothera, distinguishing it from other plant models like Solanacean cybrids .

What factors should be optimized in experimental designs for studying recombinant atpH?

Experimental design optimization is critical for studying recombinant proteins. A systematic approach using Design of Experiments (DoE) methodology can enhance experimental efficiency and reliability . For atpH research, key factors to consider include:

Multiple Linear Regression analysis can then be used to identify the most significant factors affecting experimental outcomes, similar to approaches used for optimization of extraction procedures . Surface response plots can visualize the effects of each variable on protein yield or activity, guiding researchers to optimal conditions.

How can researchers effectively analyze atpH interactions with other ATP synthase subunits?

Understanding protein-protein interactions within the ATP synthase complex requires multiple complementary approaches:

• Co-immunoprecipitation with antibodies against atpH or other subunits

• Crosslinking studies to capture transient interactions

• Fluorescence techniques (FRET, FLIM) to monitor interactions in native membranes

• Surface plasmon resonance (SPR) to measure binding kinetics

• Native PAGE to analyze intact complexes

When analyzing these interactions, researchers should consider that chloroplastic proteins may have species-specific interaction patterns that reflect coevolution between nuclear and plastid genomes . Comparing interaction profiles between compatible and incompatible plastome-genome combinations could reveal mechanisms underlying plastome-genome incompatibility.

What are the best approaches for studying the evolutionary history of atpH across Oenothera species?

Evolutionary analysis of atpH across Oenothera species requires comprehensive bioinformatic approaches:

• Sequence collection: Gather atpH sequences from all available Oenothera plastomes

• Multiple sequence alignment: Align sequences with high accuracy using algorithms optimized for conserved genes

• Phylogenetic analysis: Construct trees using maximum likelihood or Bayesian methods

• Selection analysis: Calculate Ka/Ks ratios to identify signatures of selection

• Structural mapping: Map variable sites onto protein structural models to assess functional significance

How can researchers address solubility issues with recombinant atpH protein?

Membrane proteins like atpH frequently present solubility challenges during recombinant expression and purification. Advanced troubleshooting strategies include:

• Fusion tags: Test multiple solubility-enhancing tags (MBP, SUMO, Trx)

• Expression conditions: Lower induction temperature (16-20°C) and inducer concentration

• Detergent screening: Systematically test different detergent classes for optimal solubilization

• Lipid addition: Include specific lipids during extraction that stabilize the native structure

• Protein engineering: Consider designing truncated versions that maintain core functional domains

When assessing solubility, researchers should monitor not just the presence of protein in the soluble fraction, but also its functional state. Electrophysiological methods, similar to those used for other chloroplastic membrane proteins, can verify that the solubilized protein maintains native-like functionality .

What strategies can resolve contradictory experimental results in atpH functional studies?

Contradictory results in functional studies can arise from multiple sources. A systematic troubleshooting approach includes:

• Protein orientation: Verify protein orientation in reconstituted systems, as unidirectional incorporation can lead to asymmetric functional properties

• Lipid environment: Test whether functional properties depend on specific lipid compositions

• Post-translational modifications: Assess whether the recombinant protein lacks critical modifications present in native protein

• Interacting partners: Determine if contradictory results stem from the presence/absence of other subunits or regulatory factors

• Experimental conditions: Standardize buffer compositions, pH, and ion concentrations across experiments

The search results highlight how chloroplastic membrane proteins can exhibit different properties depending on their orientation and the presence of regulatory molecules like ATP . Researchers should carefully document these parameters to facilitate comparison between studies.

How might synthetic biology approaches enhance atpH research?

Synthetic biology offers powerful tools for advancing atpH research:

• Directed evolution: Create libraries of atpH variants to select for desired properties

• Minimal ATP synthase: Engineer simplified versions of the complex to isolate subunit functions

• Orthogonal expression systems: Develop systems that allow simultaneous expression of multiple variants

• Biosensors: Create atpH-based sensors that report on ATP levels or membrane potential

• Chimeric proteins: Design fusion proteins that combine domains from different species to test functional hypotheses

These approaches could help resolve fundamental questions about the role of atpH in plastome-genome incompatibility by allowing researchers to test specific hypotheses about sequence-function relationships that have emerged from comparative genomic analyses of Oenothera plastomes .

What emerging technologies will advance structural studies of atpH?

Several cutting-edge technologies show promise for advancing structural studies of membrane proteins like atpH:

• Cryo-electron tomography: Provides structural information in cellular contexts

• Integrative modeling: Combines data from multiple experimental methods

• AlphaFold and related AI systems: Predicts structures with increasing accuracy

• Single-molecule methods: Captures rare conformational states

• Time-resolved structural methods: Reveals dynamic structural changes during function

These technologies will be particularly valuable for understanding how atpH functions within the larger ATP synthase complex and how its structure might contribute to the species-specific phenomena observed in Oenothera plastome-genome interactions .