Recombinant Crucihimalaya wallichii ATP synthase subunit b, chloroplastic (atpF)

How does ATP synthase subunit b differ between Crucihimalaya species and other Brassicaceae?

Comparing ATP synthase subunit b between Crucihimalaya wallichii and related species reveals important evolutionary adaptations. While C. wallichii and C. himalaica share significant sequence homology, differences exist that may reflect their adaptation to distinct environmental conditions. C. himalaica has adapted to the harsh environment of the Qinghai-Tibet Plateau, with genes showing signs of natural selection contributing to traits such as response to UV radiation, DNA repair, and membrane stabilization .

The ATP synthase complex in these high-altitude adapted species may exhibit structural modifications that optimize energy production under extreme conditions. Comparative analysis with other Brassicaceae members, particularly the model organism Arabidopsis, can provide insights into the evolutionary adaptations of this protein.

What are the optimal conditions for recombinant expression and purification of C. wallichii atpF?

For successful recombinant expression and purification of C. wallichii ATP synthase subunit b (atpF), researchers should consider the following methodological approach:

• Expression System: An E. coli-based expression system is recommended, similar to that used for other chloroplast ATP synthase subunits . BL21(DE3) or similar strains are suitable hosts.

• Expression Vector: Use a vector with a strong inducible promoter (T7 or similar) fused to a solubility-enhancing tag such as maltose-binding protein (MBP) to improve yield and solubility.

• Induction Conditions:

  • Temperature: 18-20°C (lower temperatures improve proper folding)

  • IPTG concentration: 0.1-0.5 mM

  • Induction time: 16-18 hours

• Purification Protocol:

  • Lysis in Tris-based buffer (pH 7.5-8.0) containing protease inhibitors

  • Initial purification via affinity chromatography (using the fusion tag)

  • Secondary purification via ion exchange or size exclusion chromatography

  • Final storage in Tris-based buffer with 50% glycerol

• Storage Conditions: Store purified protein at -20°C for short-term or -80°C for extended storage. Working aliquots can be maintained at 4°C for up to one week .

What PCR protocols are most effective for amplifying the atpF gene from C. wallichii samples?

For amplifying the atpF gene from Crucihimalaya wallichii samples, researchers should implement the following optimized PCR protocol:

• Primer Design:

  • Forward primer covering the 5' region of the atpF gene

  • Reverse primer targeting the 3' end of the gene

  • Include appropriate restriction sites for subsequent cloning

• Reaction Components:

  • Template DNA: 100-150 ng of genomic DNA

  • Primers: 0.5 μM each (forward and reverse)

  • dNTPs: 0.5 mM

  • High-fidelity DNA polymerase (Pfu or similar): 5 units

  • Buffer: 1X concentration as recommended by polymerase manufacturer

  • Total reaction volume: 40-50 μL

• Thermal Cycling Conditions:

  • Initial denaturation: 98°C for 1 minute

  • 25-30 cycles of:

    • Denaturation: 98°C for 30 seconds

    • Annealing: 55-62°C for 1 minute (optimize with temperature gradient)

    • Extension: 68°C for 2 minutes

  • Final extension: 68°C for 10 minutes

  • Hold: 4°C

• Product Verification: Analyze PCR products using 1.5% agarose gel electrophoresis in TBE buffer with ethidium bromide (10 μg/mL) .

This protocol has been adapted from successful amplification of similar genetic markers in related species and optimized for the specific characteristics of the atpF gene in Crucihimalaya.

How can researchers assess the ATP synthase activity of recombinant C. wallichii atpF in vitro?

To assess the ATP synthase activity of recombinant Crucihimalaya wallichii atpF in vitro, researchers should employ a multi-faceted approach:

• Reconstitution Assay:

  • Incorporate purified atpF protein into liposomes with other ATP synthase subunits

  • Establish a proton gradient across the liposome membrane

  • Measure ATP production using luciferase-based luminescence assays

  • Calculate enzyme kinetics parameters (Km, Vmax)

• Proton Translocation Measurement:

  • Use pH-sensitive fluorescent dyes (such as ACMA or pyranine)

  • Monitor fluorescence changes as protons move across membranes

  • Correlate proton movement with ATP synthase activity

• ATP Hydrolysis Assay (reverse reaction):

  • Measure inorganic phosphate release from ATP hydrolysis

  • Use colorimetric methods (e.g., malachite green assay)

  • Compare activity under different pH and temperature conditions

• Structural Integrity Assessment:

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

  • Size-exclusion chromatography to verify oligomeric state

  • Blue native PAGE to assess complex formation

What approaches can be used to study the interaction between atpF and other ATP synthase subunits?

Several approaches can be employed to study the interactions between Crucihimalaya wallichii atpF and other ATP synthase subunits:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies specific to atpF or use epitope-tagged recombinant protein

  • Precipitate protein complexes from chloroplast extracts or reconstituted systems

  • Identify interacting partners using mass spectrometry

  • Quantify binding affinities using western blot analysis

• Bimolecular Fluorescence Complementation (BiFC):

  • Fuse complementary fragments of fluorescent proteins to atpF and potential binding partners

  • Express in plant protoplasts or heterologous systems

  • Visualize interactions through fluorescence microscopy

  • Quantify signal intensity as a measure of interaction strength

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpF on sensor chip

  • Flow solutions containing other ATP synthase subunits over the chip

  • Measure real-time binding kinetics (kon, koff) and affinity constants (KD)

  • Compare binding parameters under different physiological conditions

• Crosslinking Mass Spectrometry:

  • Apply chemical crosslinkers to stabilize protein-protein interactions

  • Digest complexes and analyze crosslinked peptides by LC-MS/MS

  • Map interaction interfaces at amino acid resolution

  • Create structural models of subunit arrangements

• FRET Analysis:

  • Label atpF and interaction partners with donor/acceptor fluorophores

  • Measure energy transfer as indication of proximity

  • Calculate distances between subunits

These methodologies provide complementary information about the strength, specificity, and structural basis of interactions between atpF and other components of the ATP synthase complex.

How has the atpF gene evolved in Crucihimalaya species adapted to extreme environments?

The evolution of the atpF gene in Crucihimalaya species adapted to extreme environments represents a fascinating case of molecular adaptation. In high-altitude adapted species like C. himalaica, genomic evidence suggests that:

• Selective Pressure Analysis:

  • The atpF gene shows signatures of positive selection in high-altitude adapted species

  • Nonsynonymous to synonymous substitution ratios (dN/dS) are elevated in specific regions of the gene

  • Key amino acid substitutions occur in transmembrane domains that may affect proton channeling efficiency

• Structural Adaptations:

  • Amino acid changes that enhance protein stability under low temperature conditions

  • Modifications that optimize proton movement at altered atmospheric pressure

  • Substitutions that maintain membrane integration under high UV radiation conditions

• Regulatory Adaptations:

  • Promoter region modifications that affect expression under stress conditions

  • Changes in untranslated regions that influence mRNA stability and translation efficiency

  • Codon usage bias that may optimize translation under resource-limited conditions

• Comparative Analysis:

  • Specific amino acid substitutions in C. wallichii and C. himalaica compared to lowland relatives

  • Conserved regions essential for ATP synthase function remain largely unchanged

  • Variable regions show adaptation patterns correlated with environmental parameters

These evolutionary adaptations may contribute to maintaining ATP synthase function under the harsh conditions of high altitude environments, including low temperatures, high UV radiation, and altered atmospheric conditions.

What role does atpF play in the retrograde signaling between chloroplast and nucleus?

The ATP synthase subunit b (atpF) plays a significant role in retrograde signaling between chloroplast and nucleus, contributing to the coordination of gene expression between these organelles:

• Signal Generation:

  • Changes in ATP synthase activity, affected by environmental stress, generate retrograde signals

  • ATP/ADP ratio alterations, partially determined by atpF function, serve as metabolic signals

  • Reactive oxygen species (ROS) produced during ATP synthase dysfunction act as secondary messengers

• Signaling Pathways:

  • ATP synthase dysfunction activates plastid retrograde signaling pathways

  • Expression of photosynthesis-associated nuclear genes (PhANGs) is regulated in response to ATP synthase activity

  • Transcription factors such as LHY, PIF, MYB, WRKY, and AP2/ERF are modulated in response to these signals

• Experimental Evidence:

  • ATP synthase mutants show impaired plastid retrograde signaling

  • Treatment with lincomycin (LIN) or norflurazon (NF) reveals differential gene expression patterns in ATP synthase mutants compared to wild-type plants

  • Transcriptome analysis demonstrates significant upregulation of PhANGs in ATP synthase mutants under retrograde signaling inhibition conditions

• Coordination Mechanism:

  • AtpF function directly affects ATP production and indirectly influences redox state

  • These metabolic changes trigger signaling cascades that coordinate nuclear gene expression

  • Bidirectional communication ensures appropriate stoichiometry of photosynthetic complexes

This retrograde signaling system allows the plant to adapt gene expression based on chloroplast functional status, with atpF playing a key role in this communication network.

How can CRISPR-Cas9 be optimized for targeted modification of the atpF gene in Crucihimalaya species?

Optimizing CRISPR-Cas9 for targeted modification of the atpF gene in Crucihimalaya species requires careful consideration of several factors:

• Guide RNA (gRNA) Design:

  • Target unique regions of the atpF gene to minimize off-target effects

  • Design multiple gRNAs for different regions of the gene to increase editing efficiency

  • Consider the following selection criteria:

    • GC content between 40-60%

    • Minimal secondary structure formation

    • NGG PAM site accessibility

    • Target conserved functional domains for predictable phenotypic effects

• Delivery Method Optimization:

  • Agrobacterium-mediated transformation for whole plant modification

  • Protoplast transfection for transient expression and testing

  • Biolistic delivery for recalcitrant tissues

  • Optimize transformation parameters based on tissue type and species characteristics

• Cas9 Variant Selection:

  • Use high-fidelity Cas9 variants (e.g., eSpCas9, SpCas9-HF1) to reduce off-target effects

  • Consider base editors for specific nucleotide changes without double-strand breaks

  • Evaluate temperature-sensitive Cas9 variants for controlled editing in Crucihimalaya species

• Editing Verification Protocol:

  • Initial screening via restriction fragment length polymorphism (RFLP) analysis

  • Confirmation by Sanger sequencing of target region

  • Whole genome sequencing to evaluate off-target modifications

  • Phenotypic characterization to confirm functional consequences

• Homology-Directed Repair (HDR) Template Design:

  • 500-1000 bp homology arms flanking the target site

  • Incorporate silent mutations in the PAM site to prevent re-cutting

  • Include selection markers for efficient identification of edited plants

What high-throughput approaches can be used to screen for natural variants of atpF in wild Crucihimalaya populations?

Researchers can employ several high-throughput approaches to efficiently screen for natural variants of the atpF gene in wild Crucihimalaya populations:

• Targeted Amplicon Sequencing:

  • Design primers flanking the atpF gene and conserved regions

  • Incorporate barcodes for sample multiplexing

  • Sequence hundreds to thousands of samples simultaneously on platforms like Illumina

  • Analyze data using variant calling pipelines to identify SNPs, indels, and structural variations

• Hybridization-Based Capture:

  • Design biotinylated probes targeting the atpF gene and surrounding regions

  • Perform solution-based capture of genomic DNA from multiple populations

  • Sequence enriched libraries to identify variants across populations

  • Compare variation patterns with environmental gradients and adaptive traits

• Restriction Site Associated DNA Sequencing (RAD-Seq):

  • Digest genomic DNA with restriction enzymes that cut near the atpF locus

  • Sequence adjacent regions to identify linked polymorphisms

  • Use association studies to correlate variants with ecological parameters

  • Identify signatures of selection in different environmental contexts

• Ecological Genomics Approach:

  • Sample populations across environmental gradients (altitude, temperature, UV exposure)

  • Conduct whole genome resequencing on representative samples

  • Focus analysis on the atpF gene and related pathways

  • Correlate genetic variation with environmental variables using geospatial analysis

• Analytical Pipeline:

  • Quality filtering of raw sequence data

  • Alignment to reference genome or de novo assembly

  • Variant calling using multiple algorithms to increase confidence

  • Population genetic analysis (FST, nucleotide diversity, Tajima's D)

  • Environmental association analysis to identify adaptive variants

This systematic approach enables researchers to catalog natural genetic diversity in the atpF gene across wild Crucihimalaya populations and identify variants that may contribute to adaptation to diverse environmental conditions.

What techniques are most effective for studying the three-dimensional structure of the ATP synthase complex containing C. wallichii atpF?

Several advanced techniques can be employed to elucidate the three-dimensional structure of the ATP synthase complex containing Crucihimalaya wallichii atpF:

How do post-translational modifications affect atpF function in Crucihimalaya species?

Post-translational modifications (PTMs) play crucial roles in regulating the function of ATP synthase subunit b (atpF) in Crucihimalaya species. Understanding these modifications provides insights into adaptive mechanisms:

• Phosphorylation:

  • Key serine/threonine residues in atpF can be phosphorylated

  • Phosphorylation states affect:

    • Protein-protein interactions within the ATP synthase complex

    • Proton conductance efficiency

    • Response to environmental stressors

  • Methods for detection:

    • Phosphoproteomic mass spectrometry

    • Phospho-specific antibodies

    • 2D gel electrophoresis with Pro-Q Diamond staining

• Oxidative Modifications:

  • Cysteine residues susceptible to oxidation under stress conditions

  • Oxidation states influence:

    • Protein stability in high UV/oxidative environments

    • Conformational changes affecting proton channeling

    • Regulatory responses to redox imbalance

  • Detection approaches:

    • Redox proteomics with iodoacetamide labeling

    • Mass spectrometry analysis of oxidized peptides

    • Activity assays under reducing/oxidizing conditions

• Acetylation:

  • N-terminal and lysine acetylation modulates:

    • Protein turnover rates

    • Interaction with other subunits

    • Responsiveness to environmental signals

  • Analysis methods:

    • Acetylome profiling via mass spectrometry

    • Western blotting with anti-acetyl-lysine antibodies

    • Functional assays with deacetylase inhibitors

• Environmental Influence on PTMs:

  • High-altitude stress factors induce specific modification patterns

  • Temperature fluctuations lead to differential phosphorylation profiles

  • UV radiation exposure correlates with increased oxidative modifications

  • Drought conditions trigger acetylation changes

These PTMs form a complex regulatory network that fine-tunes ATP synthase function in response to the extreme environmental conditions faced by Crucihimalaya species in their natural habitats. The adaptive significance of these modifications may contribute to the ecological success of these plants in challenging environments.

How can knowledge of C. wallichii atpF structure-function relationships inform engineering of more efficient ATP synthases?

Understanding the structure-function relationships of Crucihimalaya wallichii atpF provides valuable insights for engineering more efficient ATP synthases with biotechnological applications:

• Optimizing Proton Channeling Efficiency:

  • Identify key amino acid residues in atpF that determine proton conductance

  • Incorporate mutations that enhance proton flow while maintaining structural integrity

  • Design modified proton channels with reduced slippage (non-productive proton movement)

  • Potential applications:

    • Enhanced photosynthetic efficiency in crop plants

    • Improved biomass production in biofuel feedstocks

    • Stress-tolerant varieties for marginal lands

• Thermal Stability Engineering:

  • Analyze thermostable features of atpF from high-altitude adapted Crucihimalaya species

  • Introduce these features into ATP synthases of agricultural or industrial importance

  • Test performance across temperature ranges to validate improvements

  • Applications:

    • Cold-tolerant crop varieties

    • Heat-resistant biocatalysts

    • Climate change adaptation strategies

• Modifying Coupling Efficiency:

  • Alter the interaction interface between atpF and other F₀ subunits

  • Fine-tune the mechanical coupling between proton translocation and ATP synthesis

  • Optimize the H⁺/ATP ratio for specific applications

  • Potential benefits:

    • Tailored energy conversion efficiency for different metabolic needs

    • Controlled energy dissipation under stress conditions

    • Customized ATP production rates for synthetic biology applications

• Rational Design Strategy:

  • Conduct in silico modeling of modified atpF variants

  • Perform site-directed mutagenesis based on structure-function analyses

  • Evaluate performance using reconstituted systems and in vivo testing

  • Iterate design based on performance metrics

What are the challenges and solutions in expressing functional atpF in heterologous systems for structural studies?

Expressing functional Crucihimalaya wallichii atpF in heterologous systems for structural studies presents several challenges along with potential solutions:

• Membrane Protein Expression Challenges:

  • Challenge: Toxicity to host cells due to membrane disruption

  • Solutions:

    • Use tightly controlled inducible promoters (e.g., T7lac or araBAD)

    • Express as fusion with solubility-enhancing partners (MBP, SUMO, Trx)

    • Utilize specialized E. coli strains (C41/C43, Lemo21) designed for membrane protein expression

    • Implement co-expression with chaperones (GroEL/ES, DnaK/J)

• Proper Folding and Insertion:

  • Challenge: Achieving native-like folding in heterologous membranes

  • Solutions:

    • Lower expression temperature (16-20°C) to slow folding process

    • Use mild detergents for extraction (DDM, LMNG, or DMNG)

    • Express in eukaryotic systems (yeast, insect cells) for complex proteins

    • Co-express with ATP synthase assembly factors

• Protein Stability Issues:

  • Challenge: Maintaining stability during purification and crystallization

  • Solutions:

    • Screen multiple detergents and lipid combinations

    • Implement lipid nanodisc or amphipol technology for native-like environment

    • Use thermostability assays to identify optimal buffer conditions

    • Apply limited proteolysis to identify and remove flexible regions

• Functional Verification:

  • Challenge: Confirming that recombinant protein is functionally equivalent to native

  • Solutions:

    • Develop sensitive activity assays applicable to isolated subunit

    • Measure binding affinity to known interaction partners

    • Assess secondary structure similarity using circular dichroism

    • Reconstitute with other subunits to test complex formation

• Methodological Strategy:

  • Initial expression screening in multiple systems:

    • Bacterial (E. coli): BL21(DE3), C41/C43, Lemo21

    • Yeast: Pichia pastoris, Saccharomyces cerevisiae

    • Insect cells: Sf9, High Five

  • Purification optimization:

    • Two-step purification minimum (affinity + size exclusion)

    • Detergent screening (minimum 8-10 different detergents)

    • Stability assessment using fluorescence-based thermal shift assays

  • Functional validation before structural studies

By systematically addressing these challenges, researchers can successfully express, purify, and characterize functional atpF for detailed structural and functional studies, ultimately contributing to our understanding of ATP synthase biology and enabling biotechnological applications.

What are the most promising research directions for studying Crucihimalaya atpF in the context of climate change adaptation?

Research on Crucihimalaya wallichii atpF offers significant opportunities for understanding plant adaptation to climate change, with several promising directions:

• Comparative Genomics and Adaptation:

  • Compare atpF sequences across Crucihimalaya populations from different altitudes and climates

  • Identify adaptive mutations that correlate with specific environmental parameters

  • Use genome editing to validate the adaptive significance of identified variants

  • Develop predictive models for protein evolution under climate change scenarios

• Energy Efficiency Under Stress Conditions:

  • Investigate how atpF variants affect ATP synthesis efficiency under combined stresses

  • Measure H⁺/ATP ratios under different temperature, light, and water availability conditions

  • Assess how structural modifications influence energy conservation during stress

  • Explore potential applications for improving crop resilience in changing climates

• Systems Biology Approaches:

  • Map the interaction network of atpF with other cellular components under stress

  • Study retrograde signaling pathways involving ATP synthase under climate change conditions

  • Develop metabolic models that incorporate ATP synthase efficiency variations

  • Identify regulatory circuits that control atpF expression in response to environmental cues

• Translational Research:

  • Transfer beneficial atpF variants from Crucihimalaya to crop species

  • Assess impact on photosynthetic efficiency and stress tolerance

  • Develop high-throughput phenotyping methods to evaluate ATP synthase performance in field conditions

  • Create crop improvement strategies based on optimized energy conversion

These research directions will not only enhance our fundamental understanding of plant adaptation mechanisms but also contribute to developing climate-resilient crops for future food security.

How can integrative approaches combine structural, functional, and evolutionary analyses of atpF to advance our understanding of chloroplast bioenergetics?

Integrative approaches combining structural, functional, and evolutionary analyses of Crucihimalaya wallichii atpF can significantly advance our understanding of chloroplast bioenergetics:

• Multi-omics Integration:

  • Combine genomics, transcriptomics, proteomics, and metabolomics data

  • Map how genetic variation in atpF correlates with expression, protein modifications, and metabolic outputs

  • Identify regulatory networks controlling ATP synthase function across environmental conditions

  • Develop systems-level models of chloroplast energy metabolism

• Structure-Function-Evolution Framework:

  • Correlate structural features with functional properties and evolutionary conservation

  • Identify which regions are under purifying selection (conserved function) versus positive selection (adaptation)

  • Map adaptive mutations onto structural models to understand mechanistic implications

  • Develop predictive tools for how sequence changes affect ATP synthase performance

• Comparative Analysis Across Species:

  • Compare atpF from Crucihimalaya with homologs from diverse plant lineages

  • Identify convergent adaptations in species from similar environments

  • Reconstruct the evolutionary history of ATP synthase modifications

  • Connect molecular adaptations to ecological specialization

• Synthetic Biology Applications:

  • Design chimeric atpF proteins incorporating features from different species

  • Test performance across environmental conditions

  • Engineer optimized ATP synthases with desired properties

  • Develop modular design principles for bioenergetic systems

• Methodological Integration:

  • Combine structural biology techniques (Cryo-EM, X-ray, NMR) with functional assays

  • Utilize ancestral sequence reconstruction to test evolutionary hypotheses

  • Apply machine learning to predict structure-function relationships

  • Develop visualization tools for complex multi-dimensional data

This integrative approach will provide a comprehensive understanding of how ATP synthase structure and function have evolved in response to environmental challenges, offering insights into fundamental bioenergetic principles and applications for bioengineering and crop improvement.