Recombinant Oenothera argillicola ATP synthase subunit c, chloroplastic (atpH)

What is Recombinant Oenothera argillicola ATP synthase subunit c, chloroplastic (atpH)?

Recombinant Oenothera argillicola ATP synthase subunit c, chloroplastic (atpH) is a laboratory-produced protein that replicates the naturally occurring chloroplastic ATP synthase subunit c found in Oenothera argillicola (Appalachian evening primrose). This protein is a component of the F0 sector of the ATP synthase complex and plays a critical role in energy production within chloroplasts. The recombinant form is typically produced in expression systems such as E. coli, yeast, baculovirus, or mammalian cells to facilitate research applications .

The protein consists of 81 amino acids with the sequence: MNPLISAASVIAAGLAVGLASIGPGIGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . It has multiple nomenclature designations including ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, F-ATPase subunit c, and Lipid-binding protein . The gene encoding this protein, atpH, is located in the chloroplast genome and has been well-characterized in various phylogenetic studies .

How is the atpH gene structured and organized within the chloroplast genome?

The atpH gene is located in the chloroplast genome and encodes the ATP synthase subunit c protein. In chloroplast genomics research, the atpH-atpI region is recognized as a potentially informative locus for phylogenetic studies due to its variable nature . This region has demonstrated 100% PCR amplification success in multi-genera testing, making it a reliable target for amplification and sequence analysis .

Within the chloroplast genome organization, atpH is part of the protein-coding genes that comprise approximately 38-40% of the total chloroplast genome length in Myrtales species, including Oenothera argillicola . The gene displays a GC content of approximately 43-45%, which is consistent with other protein-coding regions in plastid genomes . The conserved nature of this gene, combined with specific variable regions, makes it valuable for both structural studies of the ATP synthase complex and evolutionary analyses.

What experimental approaches are recommended for initial characterization of recombinant atpH?

For initial characterization of recombinant Oenothera argillicola ATP synthase subunit c, a multi-faceted approach is recommended. Begin with SDS-PAGE analysis to confirm protein purity (aim for ≥85% purity as standard for recombinant proteins) . Western blotting with anti-atpH antibodies can verify protein identity, while mass spectrometry provides precise molecular weight determination and can identify post-translational modifications.

Circular dichroism spectroscopy is essential for secondary structure analysis, particularly important given the predominantly alpha-helical structure of ATP synthase subunit c. Functional characterization should include reconstitution experiments with other ATP synthase components to assess proton conductance capabilities. For storage stability assessment, maintain aliquots in Tris-based buffer with 50% glycerol at -20°C as recommended for the recombinant protein . Avoid repeated freeze-thaw cycles, and consider preparing working aliquots stored at 4°C for up to one week for ongoing experiments .

How does Oenothera argillicola atpH compare structurally and functionally with homologs from other species?

Comparative analysis of ATP synthase subunit c across different species reveals important evolutionary insights. The Oenothera argillicola atpH protein maintains the highly conserved structural features essential for ATP synthase function while exhibiting species-specific variations. When comparing coding region sizes among Myrtales species, O. argillicola has one of the largest protein-coding regions at 70,706 bp, contrasting with Lagerstroemia fauriei (68,477 bp) and other related species as shown in the table below :

Functionally, the conserved amino acid sequence of atpH proteins indicates preservation of the critical role in proton translocation across the thylakoid membrane. The lipid-binding properties mentioned in the protein's alternative nomenclature suggests conservation of membrane interaction domains . These structural similarities support the use of Oenothera argillicola atpH as a model for understanding fundamental ATP synthase mechanisms, while the variations may correlate with species-specific adaptations to different photosynthetic requirements.

What are the technical challenges in expressing and purifying functional recombinant ATP synthase subunit c?

Expression and purification of functional recombinant ATP synthase subunit c presents several significant technical challenges. The hydrophobic nature of this membrane protein, which contains multiple transmembrane domains, complicates expression in conventional systems. Researchers must optimize expression parameters including host selection (E. coli, yeast, baculovirus, or mammalian cells), induction conditions, and solubilization methods .

Purification strategies typically require detergent-based extraction followed by chromatographic separation. Tag selection is critical and should be determined during the production process to minimize interference with protein folding and function . Maintaining the native conformation is particularly challenging, as the protein normally exists as part of a multi-subunit complex in the membrane environment. To address this, reconstitution into liposomes or nanodiscs may be necessary for functional studies.

Quality control is essential, with purity standards typically set at ≥85% as determined by SDS-PAGE . Storage stability remains a concern, necessitating optimization of buffer conditions—typically Tris-based buffer with 50% glycerol—and temperature (-20°C for extended storage) . Researchers should prepare small working aliquots to avoid repeated freeze-thaw cycles that can compromise protein integrity.

How can atpH be utilized as a phylogenetic marker in plant evolutionary studies?

The atpH gene and the intergenic region atpH-atpI have proven valuable as phylogenetic markers due to their optimal evolutionary rate and sequence characteristics. PCR amplification of this region shows 100% success rates across diverse plant taxa, making it highly reliable for comparative studies . The primer pair atpH-f (aacaaaaggattcgcaaataaaag) and atpI-r (agttgttgttcttgtttctttagt) has demonstrated excellent amplification success and sequencing quality, with 98.1-99.5% and 97.5-99.2% quality values respectively .

The atpH-atpI region exhibits sufficient variability for distinguishing between closely related species while maintaining enough sequence conservation for reliable alignment across divergent taxa. This balance makes it particularly useful for resolving relationships at various taxonomic levels. Comparative analysis of nucleotide diversity (π values) and indel patterns can provide information about evolutionary rates and selection pressures.

For phylogenomic approaches, researchers should consider atpH in conjunction with other chloroplast markers such as trnK, rpl32-trnL, and trnH-psbA, which have been identified as highly variable regions suitable for evaluating plant phylogeny . This multi-locus approach yields more robust phylogenetic reconstructions than single-gene analyses and can reveal interesting evolutionary patterns, such as the loss of introns (e.g., the rpl2 intron loss in Lythraceae) .

What insights can the atpH gene provide about chloroplast genome evolution in Myrtales?

The atpH gene offers valuable insights into chloroplast genome evolution within Myrtales. Comparative analysis of protein-coding regions reveals that Oenothera argillicola (Onagraceae) has a distinctively larger protein-coding region (70,706 bp) compared to other Myrtales species, suggesting potential genome expansion events in this lineage . The GC content of protein-coding regions remains relatively consistent across Myrtales at approximately 43%, with a slight increase to 45% in Lagerstroemia fauriei .

Examination of atpH in conjunction with other plastid genes can illuminate larger evolutionary patterns. For instance, the loss of the rpl2 intron in all sampled Lythraceae species, contrasted with its presence in Oenothera albicaulus (Onagraceae), indicates this intron loss occurred after the divergence of Lythraceae from Onagraceae but before the diversification of genera within Lythraceae . This type of molecular event serves as a useful synapomorphy for defining clades within Myrtales.

The conservation of atpH structure amid variability in surrounding regions underscores the essential function of this gene in chloroplast metabolism. The protein's role in ATP synthesis appears to constrain sequence evolution, while allowing greater flexibility in non-coding regions. These patterns of conservation and variation provide a window into the selective pressures that have shaped chloroplast genome evolution in this important plant order.

What PCR protocols are recommended for amplifying the atpH gene from plant samples?

For reliable amplification of the atpH gene and adjacent regions from plant samples, the following PCR protocol has been validated across multiple genera. Prepare reaction mixtures in 40μl volumes containing:

• 4 μl 10× Taq buffer

• 0.8 μl dNTP (10 mM)

• 0.4 μl Taq polymerase (5 U/μl)

• 0.5 μl each primer (20 pmol/μl)

• 0.5 μl DNA template

• 33.3 μl ddH₂O

For the atpH-atpI region specifically, use the following primer pair which has demonstrated 100% amplification success across diverse plant taxa:

• atpH-f: aacaaaaggattcgcaaataaaag

• atpI-r: agttgttgttcttgtttctttagt

The amplification program should consist of an initial denaturation at 94°C for 5 min, followed by 32 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, and elongation at 72°C for 2 min, concluding with a final elongation at 72°C for 10 min . This protocol consistently yields high-quality amplicons with sequencing success rates of 97.5-99.5% .

For quality control, PCR products should be purified with PEG8000 and directly sequenced. A good quality sequence should reach at least 600 bp in length with quality values (ratios of bases with QV >20 to total bases) exceeding 90% after trimming both ends .

How can researchers effectively express recombinant Oenothera argillicola ATP synthase subunit c in heterologous systems?

For effective heterologous expression of recombinant Oenothera argillicola ATP synthase subunit c, researchers should consider multiple expression systems based on experimental requirements. E. coli systems offer rapid production but may require optimization for membrane proteins. For enhanced post-translational modifications, yeast (P. pastoris or S. cerevisiae), baculovirus, or mammalian cell expression systems can be employed .

Expression vector design is critical. Incorporate the full coding sequence (MNPLISAASVIAAGLAVGLASIGPGIGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) with appropriate codon optimization for the selected host. A carefully selected affinity tag will facilitate purification while minimizing impact on protein folding and function—this should be determined during the production process for each specific application .

For membrane protein expression, consider using specialized strains like E. coli C41(DE3) or C43(DE3) that are adapted for toxic membrane protein expression. Induction conditions must be optimized: typically lower temperatures (16-20°C) and reduced inducer concentrations yield better results for membrane proteins. After expression, solubilization with mild detergents such as n-dodecyl-β-D-maltoside (DDM) or digitonin can help maintain native conformation. Purification to ≥85% purity should be confirmed by SDS-PAGE , and storage in Tris-based buffer with 50% glycerol at -20°C will maintain stability for extended periods .

What analytical techniques provide the most comprehensive structural information about ATP synthase subunit c?

A multi-technique approach is necessary for comprehensive structural characterization of ATP synthase subunit c. X-ray crystallography remains the gold standard for high-resolution structural determination, though membrane proteins present significant crystallization challenges. Cryo-electron microscopy (cryo-EM) has emerged as a powerful alternative, allowing visualization of the protein in near-native states without crystallization requirements.

Solution and solid-state NMR spectroscopy can provide atomic-level information about protein dynamics and interactions, particularly valuable for understanding proton translocation mechanisms. For secondary structure analysis, circular dichroism spectroscopy offers rapid assessment of α-helical content, which is predominant in ATP synthase subunit c.

Computational approaches complement experimental methods. Molecular dynamics simulations can model protein behavior within membrane environments, while homology modeling leverages known structures of homologous proteins from other species. Cross-linking mass spectrometry provides insights into protein-protein interactions within the ATP synthase complex, critical for understanding the functional context of subunit c.

For functional structural studies, reconstitution into liposomes or nanodiscs allows measurement of proton conductance in conditions approximating the native environment. Fluorescence-based assays using pH-sensitive probes can monitor conformational changes during proton translocation, connecting structural features to functional mechanisms.

What are the recommended approaches for studying interactions between ATP synthase subunit c and other components of the ATP synthase complex?

Studying interactions between ATP synthase subunit c and other components requires techniques that preserve the native membrane environment or replicate it effectively. Co-immunoprecipitation (Co-IP) using antibodies against either ATP synthase subunit c or interacting partners can identify protein associations, while pull-down assays using tagged recombinant proteins offer an alternative approach for in vitro interaction studies.

Förster resonance energy transfer (FRET) provides spatial information about protein interactions in living systems, with fluorescently-labeled ATP synthase components allowing real-time visualization of complex assembly. Surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC) can quantify binding affinities and thermodynamic parameters, though adaptation for membrane proteins may be necessary.

For visualization of the entire ATP synthase complex, cryo-electron microscopy has revolutionized structural studies by enabling researchers to observe the intact complex in various conformational states. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) maps interaction interfaces by measuring solvent accessibility changes upon complex formation.

Cross-linking coupled with mass spectrometry creates covalent bonds between interacting proteins that can be identified through proteomic analysis, providing a snapshot of the interaction network. Finally, computational approaches including molecular docking and molecular dynamics simulations can predict interaction sites and energetics, generating hypotheses for experimental validation.

What are common challenges in working with recombinant ATP synthase proteins and their solutions?

Researchers working with recombinant ATP synthase subunit c frequently encounter several technical challenges. Protein aggregation during expression and purification is a common issue due to the hydrophobic nature of this membrane protein. This can be addressed by optimizing detergent selection—screening multiple detergents at various concentrations is recommended—and incorporating stabilizing agents such as glycerol in buffers .

Low expression yields often hamper research progress. Strategies to improve yields include: optimizing codon usage for the host system, using specialized expression strains designed for membrane proteins, lowering expression temperature to 16-20°C, and reducing inducer concentration. For difficult constructs, fusion partners such as MBP (maltose-binding protein) can improve solubility and expression.

Functional assessment presents another challenge, as the protein's natural environment is within a complex multi-subunit assembly embedded in a membrane. Reconstitution into liposomes or nanodiscs provides a more native-like environment for functional studies. Proton conductance can be measured using pH-sensitive fluorescent dyes or electrophysiological techniques.

Storage stability issues should be addressed by maintaining the protein in Tris-based buffer with 50% glycerol at -20°C for extended storage . Working aliquots can be kept at 4°C for up to one week to avoid repeated freeze-thaw cycles . If precipitation occurs upon thawing, gentle mixing and addition of fresh detergent may help recover properly folded protein.

How can site-directed mutagenesis reveal functional mechanisms of ATP synthase subunit c?

Site-directed mutagenesis offers powerful insights into structure-function relationships of ATP synthase subunit c. For Oenothera argillicola ATP synthase subunit c, researchers should focus on highly conserved residues identified through multi-species sequence alignment. Key targets include the critical aspartic/glutamic acid residues involved in proton binding and translocation, as well as residues at the interface with the c-ring or other subunits.

The experimental workflow should begin with PCR-based mutagenesis using primers containing the desired mutation. After sequence verification, express the mutant proteins following protocols optimized for wild-type expression. Purification should achieve ≥85% purity as determined by SDS-PAGE . Comparative analysis between wild-type and mutant proteins should assess both structural integrity (using circular dichroism or fluorescence spectroscopy) and functional capacity.

Functional assays should evaluate proton translocation capability. This can be accomplished by reconstituting purified proteins into liposomes containing pH-sensitive fluorescent dyes. Changes in fluorescence intensity upon creation of a pH gradient across the membrane provide a readout of proton conductance. Alternatively, patch-clamp electrophysiology offers direct measurement of proton currents through reconstituted proteins.

Combining mutagenesis with molecular dynamics simulations can provide mechanistic interpretations of experimental results. This integrated approach can reveal how specific amino acid residues contribute to proton binding, release, and the conformational changes that couple these events to ATP synthesis in the intact complex.

What computational approaches are recommended for predicting atpH structure and function?

Computational analysis of ATP synthase subunit c from Oenothera argillicola should employ a hierarchical approach beginning with sequence-based methods. Multiple sequence alignment with homologs from diverse species helps identify conserved residues likely critical for function. Profile hidden Markov models can detect distant homologs and contribute to evolutionary analysis.

For structural prediction, homology modeling using experimentally determined structures of ATP synthase c subunits as templates provides a foundation. Available ATP synthase structures from organisms like yeast or bacteria serve as excellent starting points due to the highly conserved nature of this protein. Ab initio methods may supplement homology modeling for regions without suitable templates, while molecular dynamics simulations in explicit membrane environments can refine structures and reveal dynamic behavior.

Functional prediction should leverage structure-based methods including binding site identification, electrostatic surface mapping, and computational alanine scanning to identify functionally important residues. Molecular docking simulations can predict interactions with other ATP synthase components or small molecules. For understanding proton conductance, quantum mechanics/molecular mechanics (QM/MM) approaches may be necessary to accurately model proton transfer events.

Integration of computational predictions with experimental data creates the most valuable insights. For instance, predictions of critical residues can guide site-directed mutagenesis experiments, while experimental structures can validate and improve computational models in an iterative process.

How might emerging technologies enhance our understanding of ATP synthase subunit c structure and function?

Emerging technologies promise to revolutionize our understanding of ATP synthase subunit c biology. Cryo-electron tomography can now visualize ATP synthase complexes in their native membrane environment with near-atomic resolution, providing unprecedented insights into the structural arrangement of subunit c within functional complexes. This technique may reveal species-specific adaptations in Oenothera argillicola ATP synthase not evident from sequence analysis alone.

Single-molecule techniques including high-speed atomic force microscopy (HS-AFM) and single-molecule FRET can capture the dynamic rotational movement of the c-ring during ATP synthesis, connecting structural features to functional mechanics. These approaches may resolve long-standing questions about the precise mechanism of proton translocation through the c-subunit.

CRISPR-based approaches for chloroplast genome editing are advancing rapidly, potentially allowing in vivo modification of the native atpH gene. This would enable assessment of mutagenesis effects in the context of the intact chloroplast, providing physiologically relevant insights into function. Similarly, optogenetic tools could allow light-controlled activation or inhibition of ATP synthase activity, enabling precise temporal studies of ATP synthase function.

Integrative structural biology approaches combining multiple data sources (X-ray crystallography, cryo-EM, NMR, mass spectrometry, and computational modeling) will likely provide the most comprehensive understanding of ATP synthase structure. As artificial intelligence methods for protein structure prediction continue to improve, they may generate increasingly accurate models of Oenothera argillicola ATP synthase subunit c and its interactions.

What are the potential applications of atpH gene sequence data beyond phylogenetic studies?

The atpH gene sequence offers applications beyond traditional phylogenetic studies. In conservation biology, atpH sequencing can aid in identifying cryptic species and assessing genetic diversity within Oenothera populations, informing conservation priorities for endangered evening primrose species. The gene's well-characterized amplification success makes it valuable for environmental DNA (eDNA) studies to detect plant species presence in ecological surveys.

In agricultural biotechnology, understanding the structure and function of chloroplast ATP synthase could inform strategies for improving photosynthetic efficiency and stress tolerance. Comparative analysis of atpH sequences from crops and wild relatives might identify variants with enhanced performance under specific environmental conditions.

For biotechnology applications, the highly conserved structure of ATP synthase subunit c, combined with species-specific variations, makes it a potential target for designing species-specific inhibitors or modulators. Such compounds could have applications in selective plant growth regulation or as herbicides with novel modes of action.

As synthetic biology advances, designer chloroplasts with modified ATP synthase components could potentially enhance bioenergy production or enable novel metabolic pathways. The comprehensive understanding of atpH structure-function relationships is a prerequisite for such ambitious engineering efforts, highlighting the value of fundamental research on this essential chloroplast gene.