Recombinant Cycas taitungensis ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in Cycas taitungensis?

ATP synthase subunit c (atpH) in Cycas taitungensis functions as part of the membrane-embedded F0 sector of the chloroplastic ATP synthase complex. The subunit forms an oligomeric c-ring structure that constitutes the rotor of the F0 complex. This ring is mechanically coupled to the rotation of the γ-subunit in the F1 region, which drives ATP synthesis from ADP and inorganic phosphate .

The synthesis of ATP is coupled to the rotation of this c-subunit ring, which is driven by the translocation of protons across the thylakoid membrane along an electrochemical gradient. This proton gradient is established during the light reactions of photosynthesis .

In ATP synthases across different organisms, the c-ring stoichiometry (number of c-subunits per ring) varies, which directly affects the H+/ATP coupling ratio. This ratio determines the number of protons required to synthesize one ATP molecule .

How does the atpH gene differ among cycad species and other plant groups?

The atpH gene is part of the highly conserved chloroplast genome in cycads. Comparative analyses of chloroplast genomes reveal that atpH is one of the 78 protein-coding genes consistently found across various gymnosperm species .

Cycad plastomes, including that of Cycas taitungensis, demonstrate remarkable evolutionary stasis with significantly lower synonymous and nonsynonymous substitution rates compared to other gymnosperms . This conservation reflects the slow evolutionary rate of cycads, often referred to as "living fossils."

When examining gene-specific evolutionary patterns in Cycas, genes involved in photosynthetic processes (including ATP synthase components) show lower rates of nucleotide diversity and variations compared to other functional groups . Specifically, the ATP synthase genes demonstrated lower median values of dN/dS ratios, indicating strong purifying selection maintaining their function .

What are the recommended methods for recombinant expression of ATP synthase subunit c?

Based on successful approaches used for related ATP synthase subunits, the following methodology is recommended:

• Gene synthesis and vector design:

  • Design a synthetic atpH gene with codons optimized for the expression system (typically E. coli)

  • Add appropriate terminal restriction sites for cloning

  • Select an appropriate expression vector such as pMAL-c2x, pET-32a(+), or pFLAG-MAC

• Expression conditions:

  • Transform E. coli cells (T7 Express lysY/Iq or similar strains)

  • For difficult-to-express proteins, co-transform with plasmids expressing chaperone proteins (DnaK, DnaJ, and GrpE) to increase yield

  • Induce expression with IPTG (typically at 1.0 mM concentration)

• Purification strategy:

  • Use affinity chromatography methods based on fusion tags

  • For hydrophobic membrane proteins like subunit c, detergent solubilization may be necessary

  • Verify purified protein using SDS-PAGE and immunoblotting

• Storage conditions:

  • For the purified recombinant protein, store at -20°C or -80°C in a buffer containing 50% glycerol

  • Avoid repeated freezing and thawing

  • Working aliquots can be stored at 4°C for up to one week

How can researchers investigate the stoichiometric variation in ATP synthase c-rings using recombinant subunit c?

The stoichiometry of c-subunit rings varies across organisms, affecting the coupling ratio of protons translocated to ATP molecules synthesized. This ratio ranges from 3.3 to 5.0 among studied organisms . Using recombinant Cycas taitungensis atpH, researchers can investigate factors influencing this variation through:

• Reconstitution experiments:

  • Express and purify recombinant c subunits in mg quantities

  • Attempt in vitro reconstitution of the multimeric ring (cn)

  • Analyze the stoichiometry and stability of reconstituted rings under different conditions

• Site-directed mutagenesis:

  • Introduce specific mutations to investigate residues that might influence oligomerization

  • Compare the assembly properties with those of other species with different natural c-ring stoichiometries

• Hybrid assembly studies:

  • Mix recombinant c-subunits from different species to determine compatibility and preferred stoichiometry

  • Identify molecular determinants of c-ring assembly specificity

The recombinant expression system enables the application of molecular biology techniques that cannot be applied to native c-rings, providing insights into factors affecting stoichiometric variation.

What role does the atpH gene play in the evolutionary history of cycad chloroplast genomes?

The atpH gene provides valuable insights into the evolutionary history of cycad chloroplast genomes:

How does gene conversion affect the evolution of ATP synthase genes in cycad plastomes?

Gene conversion plays a significant role in the evolution of chloroplast genes in cycads, including ATP synthase genes:

• GC-biased gene conversion (gBGC):

  • Cycad plastomes show evidence of GC-biased gene conversion, particularly in the inverted repeat (IR) regions

  • This mechanism leads to reduced substitution rates and GC-biased mutations

• Mutation patterns:

  • In IR regions, frequencies of A-to-G and T-to-C transitions are higher than G-to-A and C-to-T transitions

  • In single-copy regions, these transition types do not significantly differ in frequency

• Plastome-wide mutation rectification:

  • Evidence suggests that gBGC in cycads can rectify plastome-wide mutations

  • This contributes to the evolutionary stasis observed in cycad plastomes

• Functional implications:

  • This evolutionary conservation mechanism may help maintain the functionality of essential genes like atpH

  • The slow evolution rate may reflect adaptation to stable, ancient environmental conditions

These findings suggest that gene conversion has played a crucial role in maintaining the structural and functional integrity of ATP synthase genes throughout cycad evolution.

What are common challenges in expressing and purifying recombinant ATP synthase subunit c?

Researchers may encounter several challenges when working with recombinant ATP synthase subunit c:

• Expression challenges:

  • Low expression levels due to the hydrophobic nature of membrane proteins

  • Potential toxicity to host cells

  • Protein aggregation and inclusion body formation

• Recommended solutions:

  • Co-express with chaperone proteins (DnaK, DnaJ, GrpE) to improve folding and solubility

  • Use fusion tags to enhance solubility (MBP, thioredoxin)

  • Optimize induction conditions (lower temperature, reduced IPTG concentration)

  • Consider cell-free expression systems for highly toxic proteins

• Purification difficulties:

  • Detergent selection for membrane protein solubilization

  • Maintaining protein stability during purification

  • Removing fusion tags without affecting protein structure

• Storage considerations:

  • Store in 50% glycerol to prevent freezing damage

  • Avoid repeated freeze-thaw cycles

  • For working aliquots, store at 4°C for maximum one week

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

Verifying the structural integrity of recombinant atpH protein is critical for functional studies:

• Biophysical characterization:

  • Circular dichroism (CD) spectroscopy to confirm alpha-helical secondary structure

  • Size-exclusion chromatography to assess oligomeric state

  • Dynamic light scattering to check for aggregation

• Functional verification:

  • Reconstitution into liposomes to assess proton translocation activity

  • Assembly assays with other ATP synthase subunits to verify interaction capability

  • Comparative analysis with native protein using proteoliposome-based assays

• Structural analysis:

  • Negative-stain electron microscopy to visualize c-ring formation

  • Cross-linking studies to analyze subunit-subunit interactions

  • Mass spectrometry to confirm protein integrity and post-translational modifications

What experimental approaches can resolve contradictions in ATP synthase structure-function relationships?

Researchers investigating contradictions in ATP synthase structure-function relationships can employ several approaches:

• Comparative genomic analysis:

  • Analyze the atpH gene across diverse cycad species to identify conserved and variable regions

  • Compare with other plant groups to understand evolutionary patterns

  • Examine selection pressures using dN/dS ratio analysis

• Structure-guided mutagenesis:

  • Introduce specific mutations to test hypotheses about structure-function relationships

  • Create chimeric proteins with sequences from species having different c-ring stoichiometries

  • Measure functional effects on proton translocation and ATP synthesis

• Advanced imaging techniques:

  • Use cryo-electron microscopy to determine high-resolution structures

  • Apply atomic force microscopy to study c-ring mechanics

  • Employ single-molecule techniques to observe real-time conformational changes

• Evolutionary biochemistry approaches:

  • Reconstruct ancestral sequences to understand evolutionary transitions

  • Test hypotheses about adaptive changes in ATP synthase structure

  • Correlate structural variations with ecological or physiological adaptations

How does Cycas taitungensis ATP synthase subunit c compare to other photosynthetic organisms?

Comparative analysis of ATP synthase subunit c across photosynthetic organisms reveals important evolutionary insights:

*Note: The exact c-ring stoichiometry for Cycas taitungensis has not been determined from the available search results.

Key observations:

• Cycad ATP synthase genes show remarkably slow evolutionary rates with strong purifying selection

• The conservation of ATP synthase structure reflects its fundamental importance in energy metabolism

• Variations in c-ring stoichiometry across species represent adaptations to different energetic demands

What insights do plastome analyses provide for understanding ATP synthase evolution in cycads?

Plastome analyses provide several key insights into ATP synthase evolution in cycads:

• Evolutionary stasis:

  • Cycad plastomes, including ATP synthase genes, show significantly lower substitution rates compared to other gymnosperms

  • This evolutionary stasis has persisted since the common ancestor of cycads, approximately 270 million years ago

• Selection patterns:

  • ATP synthase genes demonstrate lower rates of nucleotide diversity compared to other functional groups

  • Low dN/dS ratios indicate strong purifying selection

• Genomic organization:

  • ATP synthase genes are part of conserved gene clusters in cycad plastomes

  • Comparative analysis reveals structural stability across evolutionary time

• Mutation patterns:

  • GC-biased gene conversion influences the evolution of cycad plastome genes

  • This mechanism contributes to the evolutionary stasis observed in ATP synthase genes

These findings suggest that the structural and functional constraints on ATP synthase have remained remarkably stable throughout cycad evolution, reflecting the ancient and conserved nature of the photosynthetic machinery in these "living fossils."

How can recombinant ATP synthase subunit c be used to study c-ring assembly and function?

Recombinant ATP synthase subunit c provides a valuable tool for investigating c-ring assembly and function:

• In vitro reconstitution studies:

  • Express and purify recombinant c subunits in sufficient quantities

  • Attempt reconstitution of the multimeric ring under controlled conditions

  • Analyze factors affecting assembly efficiency and stability

• Stoichiometry determination:

  • Investigate the natural stoichiometry of Cycas taitungensis c-rings

  • Compare with other species to understand evolutionary patterns

  • Identify molecular determinants of c-ring stoichiometry

• Structure-function relationships:

  • Introduce specific mutations to test hypotheses about proton translocation

  • Create chimeric c-subunits to identify regions important for function

  • Analyze effects on proton conductance and ATP synthesis

• Evolutionary studies:

  • Reconstruct ancestral c-subunit sequences

  • Test hypotheses about adaptive changes in c-ring structure

  • Correlate structural variations with ecological adaptations

The ability to produce recombinant c-subunits enables numerous experimental approaches that cannot be applied to native c-rings, providing new insights into the structure, function, and evolution of this essential component of the ATP synthase complex.

What are promising research avenues for understanding the regulatory roles of ATP synthase in cycad photosynthesis?

Several promising research directions could enhance our understanding of ATP synthase regulation in cycad photosynthesis:

• Transcriptional regulation:

  • Investigate transcriptional control of atpH expression under different environmental conditions

  • Analyze promoter regions and transcription factors regulating ATP synthase genes

  • Compare regulatory mechanisms between cycads and other plant groups

• Post-translational modifications:

  • Identify potential phosphorylation, acetylation, or other modifications of ATP synthase subunits

  • Determine how these modifications affect enzyme activity under different conditions

  • Develop methods to isolate native ATP synthase complexes from cycad chloroplasts

• Evolutionary adaptations:

  • Correlate ATP synthase structure with cycad habitat and photosynthetic requirements

  • Investigate potential adaptations to stress conditions (drought, high temperature)

  • Compare with other ancient plant lineages to identify conserved regulatory mechanisms

• Structural biology approaches:

  • Determine high-resolution structures of cycad ATP synthase complexes

  • Compare with structures from other organisms to identify unique features

  • Use structural information to guide functional studies

How might techniques from synthetic biology advance our understanding of ATP synthase subunit c?

Synthetic biology approaches offer innovative ways to study ATP synthase subunit c:

• Designer c-rings:

  • Create synthetic c-rings with defined stoichiometry

  • Investigate the relationship between c-ring size and ATP synthesis efficiency

  • Design hybrid systems combining features from different species

• Biosensor development:

  • Engineer ATP synthase-based biosensors for proton gradient detection

  • Develop real-time monitoring systems for ATP synthesis activity

  • Create reporter systems for studying ATP synthase assembly

• Minimal systems approaches:

  • Reconstitute simplified ATP synthase systems with minimal components

  • Identify essential features required for function

  • Test hypotheses about the evolution of ATP synthase complexity

• Orthogonal translation:

  • Incorporate unnatural amino acids into ATP synthase subunits

  • Investigate specific residues involved in proton translocation

  • Develop new methods for studying membrane protein dynamics

What implications does the evolutionary conservation of ATP synthase have for understanding cycad biology and photosynthetic adaptation?

The remarkable evolutionary conservation of ATP synthase in cycads has several important implications: