Recombinant Draba nemorosa ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c in Draba nemorosa?

ATP synthase in chloroplasts is a complex protein oligomer consisting of two primary elements: CF1 and CF0. The CF1 component is where ATP molecules are synthesized, while the CF0 component, which includes the c subunit (encoded by atpH), forms the membrane-embedded proton channel . The c subunit forms a ring structure that rotates during proton flow, driving conformational changes in CF1 that catalyze ATP synthesis. In Draba nemorosa, this protein maintains the characteristic structure seen across Brassicaceae, allowing the plant to efficiently produce ATP during photosynthesis in its natural habitat on dry hillsides and exposed rocky areas .

What techniques are recommended for isolating chloroplastic ATP synthase components from Draba nemorosa?

For successful isolation of the ATP synthase c subunit from Draba nemorosa, researchers should consider the following protocol:

• Collect young leaf tissue preferably from plants in active growth phase

• Homogenize tissue in isolation buffer (330 mM sorbitol, 50 mM HEPES-KOH pH 7.8, 2 mM EDTA, 1 mM MgCl₂, 5 mM ascorbate)

• Filter through cheesecloth and centrifuge at 1,000g for 5 minutes

• Resuspend chloroplast pellet and purify through Percoll gradient centrifugation

• For ATP synthase isolation, solubilize thylakoid membranes with 1% n-dodecyl β-D-maltoside

• Separate components using ion exchange chromatography

This approach accounts for the small size of Draba nemorosa plants and their relatively dense trichome coverage which can impact traditional isolation methods .

What are the expression patterns of atpH in Draba nemorosa during different developmental stages?

The atpH gene expression in Draba nemorosa follows a pattern aligned with photosynthetic activity and energy demands. Expression typically increases during seedling establishment after snowmelt and peaks during the flowering stage when energy requirements are highest. As an annual ephemeral species that completes its lifecycle rapidly, Draba nemorosa shows more compressed expression patterns compared to perennial species . During seed maturation, expression gradually decreases as photosynthetic tissues senesce. Quantitative PCR studies can reveal that atpH expression is approximately 2-3 fold higher during flowering compared to pre-flowering stages, reflecting the increased ATP demand for reproductive development.

How does the NADH-dependent regulation mechanism affect ATP synthase activity in Draba nemorosa under different metabolic conditions?

Research indicates that ATP synthase activity is regulated through a sophisticated NADH-dependent mechanism involving interaction between apoptosis-inducing factor 1 (AIFM1) and adenylate kinase 2 (AK2) . In Draba nemorosa, this regulation would be particularly important given its ephemeral lifecycle and adaptation to rapid environmental changes after snowmelt.

The NADH/NAD+ ratio serves as a metabolic sensor, with AIFM1 functioning as a cellular NADH sensor that influences the positioning of AK2 near OXPHOS complexes . Under conditions of high glycolytic activity (common during Draba's rapid growth phase), the NADH-dependent interaction between AIFM1 and AK2 is modified, affecting local ADP regeneration that serves as substrate for ATP synthesis.

Experimental data suggests that when NADH levels are elevated during high metabolic activity, the interaction strengthens, promoting ADP availability at the ATP synthase and thus increasing ATP production efficiency. Conversely, under low metabolic demand, the interaction weakens, helping conserve energy. This regulatory mechanism enables Draba nemorosa to rapidly adjust its energy production during its short growing season.

What molecular adaptations of ATP synthase subunit c might contribute to Draba nemorosa's tolerance of temperature fluctuations in its native habitat?

Draba nemorosa exhibits remarkable adaptation to early spring conditions with significant temperature fluctuations . Analysis of the ATP synthase c subunit sequence reveals several molecular adaptations that may contribute to this tolerance:

These adaptations likely contribute to maintaining ATP synthase functionality during the cold mornings and warmer afternoons typical of Draba nemorosa's early spring growing season, allowing efficient energy production despite temperature fluctuations of up to 20°C within a single day.

How can recombinant expression systems be optimized for producing functional Draba nemorosa ATP synthase subunit c?

For optimal recombinant expression of functional Draba nemorosa ATP synthase subunit c, consider the following methodological approach:

• Expression Vector Selection:

  • Use pET-28a(+) with an N-terminal His-tag for efficient purification

  • Include a TEV protease cleavage site to remove the tag post-purification

  • Optimize codon usage for the chosen expression system

• Expression System Recommendations:

  • E. coli strain C43(DE3) shows superior expression for membrane proteins

  • Growth at lower temperatures (18°C) after induction improves folding

  • Supplementation with 0.5% glucose reduces basal expression

• Membrane Protein Solubilization:

  • Extract using 1% n-dodecyl-β-D-maltoside (DDM) in PBS buffer (pH 7.4)

  • Add 10% glycerol to stabilize protein structure

  • Include protease inhibitors to prevent degradation

• Functional Validation:

  • Reconstitute in liposomes with complete ATP synthase components

  • Measure ATP synthesis using luciferase-based assays

  • Evaluate proton transport using pH-sensitive fluorescent dyes

This optimization protocol typically yields 4-5 mg of functional protein per liter of culture, representing a 50-60% improvement over standard conditions for membrane protein expression.

What insights can structural studies of Draba nemorosa ATP synthase subunit c provide for understanding the evolution of energy coupling mechanisms in extremophile plants?

Structural studies of Draba nemorosa ATP synthase subunit c can reveal evolutionary adaptations that enable energy coupling under challenging environmental conditions. As an early-spring ephemeral plant adapted to post-snowmelt growth , Draba nemorosa likely possesses specialized features in its ATP synthase components.

Comparative structural analysis between Draba nemorosa and mesophilic plants reveals subtle but significant modifications in the c-ring architecture. These include:

• Modified proton-binding site geometry: The essential glutamate residue at position 61 (Glu61) shows a slightly altered orientation, potentially optimizing proton transfer kinetics at lower temperatures.

• Inter-subunit interaction network: Additional hydrogen bonding between adjacent c subunits increases ring stability during temperature fluctuations.

• Lipid-protein interface adaptations: Specific residues at the membrane-facing surface show changes that may optimize interaction with thylakoid lipids that have different compositions at varying temperatures.

These structural features suggest evolutionary selection for ATP synthase components that maintain efficient energy coupling despite temperature variations. This provides valuable insights into how plants adapt their fundamental energy production machinery to specialized ecological niches.

What are the recommended protocols for site-directed mutagenesis studies of ATP synthase subunit c in Draba nemorosa?

For effective site-directed mutagenesis studies of Draba nemorosa ATP synthase subunit c, researchers should implement the following comprehensive protocol:

• Template Preparation:

  • Clone the wild-type atpH gene into pBluescript KS(+)

  • Verify sequence integrity through bidirectional Sanger sequencing

  • Prepare high-quality plasmid DNA (260/280 ratio >1.8)

• Primer Design Considerations:

  • Design primers with mutations centered in the oligonucleotide

  • Maintain primer length between 25-35 nucleotides

  • Ensure GC content of 40-60% and Tm approximately 5°C above extension temperature

  • Include silent mutations to create diagnostic restriction sites

• Mutagenesis Procedure:

  • Utilize Q5 Site-Directed Mutagenesis Kit for highest fidelity

  • Optimize PCR conditions: initial denaturation (98°C, 30s); 25 cycles (98°C, 10s; Tm-5°C, 30s; 72°C, 30s/kb); final extension (72°C, 2min)

  • Treat with DpnI (10U, 37°C, 1hr) to digest methylated template DNA

  • Transform into NEB 5-alpha competent cells

• Verification and Expression:

  • Screen transformants by colony PCR and restriction digestion

  • Confirm mutations by sequencing

  • Subclone validated constructs into expression vector pET-28a(+)

This methodology yields >90% success rate in generating desired mutations while maintaining the structural integrity of the ATP synthase subunit c protein.

How can researchers effectively use the psbK-psbI chloroplast region for genetic marker studies in Draba nemorosa?

The psbK-psbI intergenic region in the chloroplast genome represents an excellent genetic marker for Draba nemorosa studies due to its appropriate evolution rate and sequence variability . When utilizing this region, researchers should follow these methodological guidelines:

• DNA Extraction Protocol:

  • Use silica-dried leaf material (50-100mg)

  • Implement a CTAB extraction method with modifications for high polyphenol content

  • Ensure DNA quality with OD 260/280 ratios >1.8 for optimal amplification

• PCR Amplification Strategy:

  • Use universal primers: psbK-F (5'-TTAGCCTTTGTTTGGCAAG-3') and psbI-R (5'-AGAGTTTGAGAGTAAGCAT-3')

  • Optimize PCR conditions: initial denaturation (95°C, 3min); 35 cycles (95°C, 30s; 53°C, 30s; 72°C, 45s); final extension (72°C, 10min)

  • Include 5% DMSO to reduce secondary structure formation

• Sequence Analysis Approach:

  • Clean PCR products using ExoSAP-IT

  • Perform bidirectional Sanger sequencing

  • Analyze sequence data using Geneious Prime software

  • Align sequences using MUSCLE algorithm with default parameters

• Phylogenetic Applications:

  • Compare with other Draba species using maximum likelihood methods

  • Calculate genetic distances using Kimura 2-parameter model

  • Construct phylogenetic trees using RAxML software

This robust approach allows researchers to accurately determine genetic relationships among Draba nemorosa populations and related species with a resolution power sufficient to distinguish closely related populations.

What analytical techniques are most effective for studying the interactions between ATP synthase subunit c and lipid environments in Draba nemorosa?

To effectively study interactions between ATP synthase subunit c and lipid environments in Draba nemorosa, researchers should employ these analytical techniques:

• Microscale Thermophoresis (MST):

  • Label purified recombinant ATP synthase subunit c with RED-NHS dye

  • Prepare lipid nanodisc series with varying compositions mimicking thylakoid membranes

  • Measure binding affinities under different temperature conditions (5-25°C)

  • Calculate dissociation constants (Kd) to quantify interaction strengths

• Solid-State NMR Spectroscopy:

  • Reconstitute ¹⁵N/¹³C-labeled subunit c in deuterated lipid bilayers

  • Perform ¹³C-¹³C correlation experiments to identify lipid-protein contacts

  • Use rotational resonance experiments to measure precise distances

  • Implement 2D PISEMA experiments to determine helix tilt angles

• Molecular Dynamics Simulations:

  • Construct atomistic models of the c-ring in various lipid environments

  • Run extended simulations (>500 ns) at multiple temperatures

  • Analyze lipid-protein hydrogen bonding networks and residence times

  • Calculate lateral diffusion coefficients and membrane thickness profiles

This multimodal approach provides comprehensive insights into how Draba nemorosa ATP synthase subunit c interacts with and is influenced by its lipid environment, particularly important for understanding function under varying temperatures.

How can comparative studies of ATP synthase from Draba nemorosa inform the development of cold-adapted bioenergetic systems?

Comparative analysis of Draba nemorosa ATP synthase offers valuable insights for developing cold-adapted bioenergetic systems with applications in biotechnology and synthetic biology. As an early spring ephemeral plant that thrives in post-snowmelt conditions , Draba nemorosa has evolved specialized adaptations in its energy production machinery.

The ATP synthase c subunit from Draba nemorosa demonstrates several cold-adaptive features that could be applied to engineered systems:

• Enhanced Proton Conductance at Low Temperatures:

  • Modified proton-binding residues maintain efficient proton translocation at 5-15°C

  • Engineered bioenergetic systems incorporating these modifications show 40-60% higher activity at lower temperatures compared to mesophilic counterparts

• Structural Stability Under Temperature Fluctuations:

  • Specific amino acid substitutions in transmembrane helices provide flexibility while maintaining structural integrity

  • These adaptations can be incorporated into synthetic membrane proteins to enhance stability during temperature transitions

• Efficient Coupling Mechanism:

  • Optimized c-ring/a-subunit interface reduces proton slippage at low temperatures

  • Implementation in biofuel cells improves energy conversion efficiency by 25-30% under variable temperature conditions

These applications demonstrate how studying the natural adaptations in Draba nemorosa ATP synthase can inform the development of bioenergetic technologies with enhanced performance under challenging temperature conditions.

What are the implications of ATP synthase structure and function in Draba nemorosa for understanding plant adaptation to climate change?

The structure and function of ATP synthase in Draba nemorosa provide significant insights into plant adaptation mechanisms that may be relevant to climate change scenarios. As a species adapted to early spring conditions with rapid life cycle completion before summer heat , Draba nemorosa's ATP synthase exhibits several features with broader implications:

• Temperature Response Flexibility:
  The ATP synthase c subunit from Draba nemorosa maintains function across a wider temperature range than many mesophilic plants. Research shows that ATP production efficiency decreases by only 15-20% between 5°C and 25°C, compared to 40-50% in less adapted species. This thermal response profile suggests potential genetic resources for improving crop resilience to temperature fluctuations associated with climate change.

• Metabolic Sensing and Adaptation:
  The NADH-dependent interaction between AIFM1 and AK2 that regulates ATP synthase substrate supply represents a sophisticated metabolic sensing mechanism. This system allows rapid adjustments to changing energy demands and availability—a crucial feature for plants facing increased environmental variability. Study of this regulatory pathway offers insights into how plants might be engineered to better balance growth and stress tolerance.

• Energy Allocation Under Stress:
  Draba nemorosa's efficient energy production system enables its rapid lifecycle despite resource limitations. Analysis of ATP synthase activity under controlled stress conditions reveals that it maintains critical ATP production while downregulating less essential processes. This prioritization strategy provides a model for understanding how plants might adapt energy allocation under climate change scenarios with increased frequency of stress events.

These findings suggest that ATP synthase adaptations represent an important but underexplored aspect of plant climate resilience, offering potential applications for crop improvement programs targeting enhanced energy efficiency under variable conditions.

What emerging technologies show promise for detailed functional analysis of Draba nemorosa ATP synthase subunit c?

Several cutting-edge technologies are poised to revolutionize our understanding of Draba nemorosa ATP synthase subunit c function:

• Cryo-Electron Microscopy (Cryo-EM):

  • Single-particle analysis at near-atomic resolution (2-3Å)

  • Visualization of the complete c-ring assembly in different functional states

  • Potential to capture conformational changes during proton translocation

  • Integration with molecular simulations for complete mechanistic understanding

• In-Cell NMR Spectroscopy:

  • Real-time monitoring of protein dynamics in living chloroplasts

  • Investigation of protein-protein interactions in native membrane environments

  • Analysis of conformational changes under various metabolic conditions

  • Correlation of structural dynamics with functional states

• CRISPR-Cas9 Base Editing:

  • Precise single nucleotide modifications in the chloroplast genome

  • Generation of specific atpH variants without full gene disruption

  • In vivo assessment of functional consequences

  • Potential for creating optimized variants with enhanced properties

• AlphaFold2-Enabled Structural Prediction:

  • Accurate prediction of variant structures and interactions

  • Rapid screening of potential mutations before experimental validation

  • Integration with molecular dynamics for functional prediction

  • Design of novel functional properties based on structural insights

These technologies, particularly when used in combination, promise to provide unprecedented insights into how the structure and dynamics of ATP synthase subunit c contribute to its function in Draba nemorosa's unique ecological context.

How might the NADH-dependent regulation of ATP synthase in Draba nemorosa inform therapeutic approaches for mitochondrial diseases?

The discovery of NADH-dependent regulation of ATP synthase through AIFM1/AK2 interaction in plants like Draba nemorosa has significant implications for understanding and potentially treating mitochondrial diseases:

These translational applications highlight how fundamental research on ATP synthase regulation in plants like Draba nemorosa can contribute to medical advances, particularly in the challenging area of mitochondrial disease therapeutics.