Recombinant Illicium oligandrum ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c, chloroplastic (atpH) in Illicium oligandrum and what is its significance in photosynthetic research?

ATP synthase subunit c is a critical component of the chloroplastic ATP synthase complex in Illicium oligandrum. This protein forms part of the c-ring structure embedded in the thylakoid membrane, which rotates during ATP synthesis. The rotation is mechanically coupled to the translocation of protons across the thylakoid membrane along an electrochemical gradient. This proton movement drives the catalysis of ATP production in the F1 region of the ATP synthase complex, where three ATP molecules are synthesized for every complete rotation cycle .

The significance of studying Illicium oligandrum ATP synthase subunit c lies in understanding species-specific variations in ATP synthesis efficiency among different plant species. The number of c-subunits in the ring (stoichiometry) can vary between species, directly affecting the bioenergetic efficiency of ATP production. This makes it a valuable research target for investigations into photosynthetic energy conversion and plant adaptation mechanisms.

How is the atpH gene organized within the chloroplast genome of Illicium oligandrum?

The atpH gene encoding ATP synthase subunit c is located within the chloroplast genome of Illicium oligandrum. Based on comparative chloroplast genome analyses, the gene is situated in a conserved region typical of angiosperms. The chloroplast genome of Illicium oligandrum contains distinct regions including a large single copy (LSC) region, a small single copy (SSC) region, and two inverted repeat (IR) regions .

Analysis of the chloroplast genome structure shows that Illicium oligandrum has specific IR/SC boundaries that differ from those in other basal angiosperms. The chloroplast genome sequencing has revealed that most SSRs (Simple Sequence Repeats) in Illicium oligandrum are located in the LSC region (74.0%), followed by the SSC region (16.0%) and IR regions (5.0%) . These genomic features provide important context for understanding the evolutionary conservation and functional importance of the atpH gene.

What is the amino acid sequence and predicted structure of Illicium oligandrum ATP synthase subunit c?

The Illicium oligandrum ATP synthase subunit c consists of 81 amino acids with the sequence: MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV . This protein is highly hydrophobic, containing primarily non-polar amino acids that facilitate its integration into the thylakoid membrane.

The predicted secondary structure of the protein is predominantly α-helical, with two major transmembrane helices connected by a polar loop region. This structure is crucial for its function in the c-ring assembly and proton translocation. The high conservation of certain residues, particularly those involved in proton binding and transfer, reflects the functional importance of these amino acids in the ATP synthesis mechanism.

What are the most effective expression systems for producing recombinant Illicium oligandrum ATP synthase subunit c?

The most effective expression system for producing recombinant Illicium oligandrum ATP synthase subunit c is based on Escherichia coli platforms with codon optimization. Drawing from successful approaches with other plant ATP synthase subunits, a recommended protocol involves:

• Gene synthesis with E. coli codon optimization of the atpH gene sequence

• Cloning into an expression vector with a fusion tag (maltose-binding protein or histidine tag) to enhance solubility and facilitate purification

• Transformation into an E. coli expression strain optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3))

• Induction with IPTG at lower temperatures (16-20°C) to enhance proper folding

This approach has proven successful for the spinach ATP synthase c-subunit, where significant quantities of purified protein were obtained . The fusion tag approach is particularly valuable for membrane proteins like ATP synthase subunit c, as it increases solubility and facilitates subsequent purification steps.

What purification strategies yield the highest purity and functional integrity of the recombinant protein?

An effective purification strategy for recombinant Illicium oligandrum ATP synthase subunit c should account for its hydrophobic nature while preserving its structural integrity. Based on established protocols for similar proteins, a multi-step purification approach is recommended:

• Cell lysis using a combination of lysozyme treatment (1 mg/mL) and sonication in an appropriate buffer (e.g., 20 mM Tris-HCl pH 8.0 with protease inhibitors)

• Membrane fraction isolation through differential centrifugation

• Solubilization using mild detergents (e.g., n-dodecyl-β-D-maltoside or digitonin)

• Affinity chromatography utilizing the fusion tag (e.g., histidine tag with Ni-NTA resin)

• Size exclusion chromatography for further purification and to remove aggregates

• Optional: Tag removal using a specific protease if the tag might interfere with functional studies

Storage in a suitable buffer containing 50% glycerol at -20°C maintains protein stability for extended periods . Validation of purified protein quality should include SDS-PAGE, Western blotting with specific antibodies, and circular dichroism to confirm proper secondary structure.

How can researchers validate the structural integrity of purified recombinant ATP synthase subunit c?

Validation of structural integrity for purified recombinant Illicium oligandrum ATP synthase subunit c requires multiple complementary analytical techniques:

• Circular Dichroism (CD) Spectroscopy: Essential for confirming the α-helical secondary structure characteristic of ATP synthase subunit c. The expected spectrum should show negative peaks at 208 and 222 nm, typical of α-helical proteins .

• Mass Spectrometry: For accurate molecular weight determination and verification of post-translational modifications or proteolytic processing.

• Size Exclusion Chromatography (SEC): To assess the oligomeric state and homogeneity of the purified protein.

• Fourier Transform Infrared Spectroscopy (FTIR): Provides additional structural information, particularly for membrane proteins.

• Nuclear Magnetic Resonance (NMR): For detailed structural analysis at the atomic level, though this may require isotopic labeling.

These analytical approaches collectively ensure that the recombinant protein maintains its native-like structure after the expression and purification process, which is crucial for subsequent functional studies.

What methods are available for reconstituting functional c-rings from recombinant Illicium oligandrum ATP synthase subunit c?

Reconstitution of functional c-rings from recombinant Illicium oligandrum ATP synthase subunit c requires careful manipulation of membrane protein assembly conditions. A systematic approach includes:

• Detergent-mediated reconstitution: Purified subunit c is mixed with appropriate detergents (e.g., n-dodecyl-β-D-maltoside or digitonin) at specific protein-to-detergent ratios to facilitate oligomerization.

• Liposome incorporation: The protein-detergent mixture is combined with lipids resembling the thylakoid membrane composition, followed by detergent removal through dialysis or adsorption with Bio-Beads.

• pH and ionic strength control: Assembly is typically performed under controlled pH (around 6.5-7.5) and salt concentration (100-150 mM) conditions that mimic the native environment.

• Temperature cycling: Controlled temperature shifts can facilitate proper folding and assembly of the c-ring structure.

• Time-dependent monitoring: The assembly process should be monitored over time using techniques such as blue native PAGE, electron microscopy, or atomic force microscopy to track oligomerization progress.

The successful reconstitution of c-rings opens possibilities for detailed structural studies using cryo-electron microscopy or X-ray crystallography, as well as functional characterization through proton translocation assays.

How can researchers investigate the proton translocation mechanism using recombinant Illicium oligandrum ATP synthase systems?

Investigation of proton translocation using recombinant Illicium oligandrum ATP synthase systems requires specialized biophysical techniques focusing on membrane dynamics and proton movement:

• Proteoliposome-based assays: Reconstituted c-rings or complete ATP synthase complexes are incorporated into liposomes with pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) to monitor proton movement across the membrane.

• Patch-clamp electrophysiology: For direct measurement of proton currents through reconstituted ATP synthase complexes in artificial membrane systems.

• Solid-supported membrane (SSM) electrophysiology: Allows measurement of transient currents associated with proton translocation events.

• Site-directed mutagenesis studies: Systematic modification of key residues involved in proton binding and translocation, particularly the conserved glutamate/aspartate residue essential for proton binding.

• Hydrogen/deuterium exchange mass spectrometry: For studying the accessibility and exchange rates of proton-binding sites within the protein structure.

These approaches provide complementary information about the kinetics and energetics of proton translocation, offering insights into the molecular mechanism of energy conversion in the ATP synthase complex.

What are the key considerations when studying the c-ring stoichiometry of Illicium oligandrum ATP synthase?

Studying c-ring stoichiometry of Illicium oligandrum ATP synthase requires careful consideration of both structural analysis techniques and biological context:

• Imaging techniques: Atomic force microscopy, electron microscopy, and X-ray crystallography can provide direct visualization of the c-ring structure and determination of subunit count.

• Mass determination: Native mass spectrometry can determine the precise molecular weight of intact c-rings, allowing calculation of the number of subunits.

• Bioenergetic measurements: The H⁺/ATP ratio can be experimentally determined and used to infer c-ring stoichiometry, as this ratio equals n/3 (where n is the number of c-subunits).

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry can reveal subunit organization and interfaces within the c-ring.

• Evolutionary and ecological context: The c-ring stoichiometry should be interpreted in the context of the plant's ecological niche, as variations in c-ring size affect the bioenergetic efficiency and may represent adaptations to specific environmental conditions.

The c-ring stoichiometry is particularly significant because it determines the number of protons required for the synthesis of ATP molecules. The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits, which directly impacts the bioenergetic efficiency of photosynthesis .

How do sequence variations in Illicium oligandrum ATP synthase subunit c correlate with functional differences compared to other species?

Sequence variations in Illicium oligandrum ATP synthase subunit c can significantly impact its functional properties when compared to other plant species. These correlations can be analyzed through:

• Multiple sequence alignment: Comparison of the Illicium oligandrum sequence with other species reveals conservation patterns and unique residues. Key areas of interest include the proton-binding site, transmembrane helices, and subunit-subunit interaction regions.

• Structure-function mapping: Identification of residues that differ from conserved positions and analysis of their potential impact on protein function, stability, or assembly using molecular modeling.

• Evolutionary rate analysis: Calculation of selection pressures on different regions of the protein to identify functionally important sites under purifying selection versus adaptively evolving sites.

• Experimental validation: Site-directed mutagenesis to convert Illicium oligandrum-specific residues to those found in other species, followed by functional characterization to directly assess the impact of these variations.

Comparative analyses have shown that minor sequence variations, especially in the transmembrane regions, can influence c-ring assembly, affecting both the stoichiometry and stability of the ring structure. These variations may represent adaptations to specific environmental conditions or metabolic requirements of different plant species.

What computational methods can predict interactions between Illicium oligandrum ATP synthase subunit c and other ATP synthase components?

Computational prediction of interactions between Illicium oligandrum ATP synthase subunit c and other components of the ATP synthase complex can utilize several complementary approaches:

• Homology modeling: Generation of structural models based on known ATP synthase structures from other species, with sequence-specific adaptations for Illicium oligandrum.

• Molecular docking: Prediction of protein-protein interactions between the c-subunit and other ATP synthase components (particularly subunits a, b, and ε) through docking algorithms.

• Molecular dynamics simulations: Analysis of the stability and dynamics of predicted interactions in a simulated membrane environment over nanosecond to microsecond timescales.

• Coevolutionary analysis: Identification of coevolving residue pairs between interacting subunits, which often indicate direct physical contacts between proteins.

• Electrostatic surface mapping: Calculation of electrostatic potential surfaces to identify complementary charged regions between interacting partners.

These computational predictions should guide experimental approaches such as cross-linking studies, mutagenesis, and interaction assays to validate the predicted interfaces and understand their functional significance in the context of ATP synthesis.

How can researchers resolve contradictory findings in ATP synthase subunit c research across different species?

Resolving contradictory findings in ATP synthase subunit c research requires systematic analysis and standardized approaches:

• Methodological standardization: Develop consistent protocols for expression, purification, and functional characterization to minimize technique-dependent variations.

• Multi-species comparative studies: Perform parallel analyses of ATP synthase subunit c from different species under identical conditions to directly assess species-specific differences.

• Integration of structural and functional data: Combine structural studies (e.g., cryo-EM, X-ray crystallography) with functional assays to correlate structural variations with functional differences.

• Environmental context consideration: Evaluate the impact of experimental conditions (pH, temperature, ionic strength) on protein behavior, as these may differentially affect proteins from different species.

• Collaborative cross-validation: Establish multi-laboratory collaborations to independently verify key findings using complementary techniques.

By applying these strategies, researchers can distinguish genuine biological differences in ATP synthase subunit c properties across species from methodological artifacts or context-dependent variations, leading to a more coherent understanding of this essential component of bioenergetic systems.

What are the major technical challenges in producing functional recombinant Illicium oligandrum ATP synthase subunit c?

The production of functional recombinant Illicium oligandrum ATP synthase subunit c faces several significant technical challenges:

• Membrane protein expression barriers: As a hydrophobic membrane protein, the c-subunit often forms inclusion bodies or aggregates during heterologous expression, reducing yields of functional protein.

• Detergent selection complexity: Identifying the optimal detergent or detergent mixture for solubilization and purification that maintains native-like structure without disrupting functional properties.

• Oxidative damage susceptibility: The protein contains methionine residues susceptible to oxidation during purification, potentially affecting structural integrity and function.

• Oligomerization control: Ensuring the formation of properly sized c-rings rather than non-physiological aggregates during reconstitution experiments.

• Functional validation limitations: Challenges in developing reliable assays to confirm that the recombinant protein retains native-like proton translocation capability and can integrate properly with other ATP synthase components.

These challenges necessitate careful optimization of expression conditions, including the use of specialized E. coli strains designed for membrane protein expression, codon optimization, and the strategic use of fusion partners to enhance solubility and facilitate purification .

What emerging technologies might advance research on ATP synthase subunit c structure and function?

Several emerging technologies hold promise for advancing research on Illicium oligandrum ATP synthase subunit c:

• Cryo-electron microscopy advancements: Improved detectors and processing algorithms are enabling atomic-resolution structures of membrane protein complexes, potentially revealing detailed c-ring architecture.

• Advanced lipid nanodiscs: New nanodisc systems with controlled lipid compositions provide more native-like environments for membrane protein reconstitution and functional studies.

• Single-molecule biophysics: Techniques such as single-molecule FRET and high-speed AFM allow direct observation of conformational changes and rotation dynamics in ATP synthase components.

• Microfluidic platforms: Novel microfluidic devices for membrane protein crystallization and functional characterization can improve throughput and reduce sample requirements.

• Computational approaches: Enhanced molecular dynamics simulations with specialized force fields for membrane proteins enable longer timescale simulations of proton translocation and conformational changes.

These technologies, used in combination, will likely provide unprecedented insights into the structural dynamics and functional mechanisms of ATP synthase subunit c, potentially revealing species-specific adaptations in Illicium oligandrum.

How might understanding Illicium oligandrum ATP synthase subunit c contribute to broader research in bioenergetics and plant adaptation?

Research on Illicium oligandrum ATP synthase subunit c has significant implications for several broader research areas:

• Evolutionary adaptation of energy conversion systems: Understanding species-specific variations in c-ring stoichiometry can reveal how plants adapt their bioenergetic efficiency to different environmental conditions.

• Chloroplast genome evolution: The organization and conservation of the atpH gene provide insights into the evolutionary history and selective pressures on chloroplast genomes in different plant lineages .

• Biomimetic energy conversion applications: Detailed understanding of the proton translocation mechanism could inspire development of artificial molecular motors and energy conversion devices.

• Plant stress responses: ATP synthase efficiency directly impacts a plant's ability to maintain energy homeostasis under stress conditions, making it relevant to agricultural research.

• Comparative bioenergetics: Species-specific variations in ATP synthase components contribute to our understanding of how different organisms optimize energy conversion processes for their ecological niches.

By studying this specific protein from Illicium oligandrum, researchers gain insights applicable to understanding fundamental bioenergetic principles that transcend individual species, while also appreciating the unique adaptations that have evolved in different photosynthetic organisms.