Recombinant Capsella bursa-pastoris ATP synthase subunit b, chloroplastic (atpF)

What is the evolutionary significance of studying ATP synthase subunit b in Capsella bursa-pastoris?

ATP synthase subunit b (atpF) in C. bursa-pastoris represents an interesting target for evolutionary studies due to the species' hybrid nature. C. bursa-pastoris is an allopolyploid species derived from two parental species: Capsella rubella and Capsella orientalis, making its chloroplast proteins valuable for understanding hybrid speciation events. Mitochondrial genome analyses have shown that C. bursa-pastoris likely inherited its organellar genomes from C. orientalis as the maternal progenitor species, whereas nuclear genomic contributions came from both parental species . When studying the chloroplastic atpF protein, researchers should consider this evolutionary context, as it influences the interpretation of structural and functional variations. The analysis approach should include comparative sequence alignment with both parental species to identify conserved and divergent regions that may reflect adaptation following hybridization.

What expression systems are most suitable for producing recombinant Capsella bursa-pastoris atpF protein?

For expressing recombinant C. bursa-pastoris ATP synthase subunit b, E. coli-based expression systems remain the most widely used due to their efficiency and relative simplicity. Typically, researchers use BL21(DE3) or Rosetta strains with expression vectors containing T7 promoters. The protein is often tagged with a polyhistidine tag for purification purposes . The expression protocol involves:

• Cloning the atpF coding sequence into an appropriate expression vector (pET series recommended)

• Transforming the construct into the bacterial host

• Inducing expression with IPTG (0.1-1.0 mM) at lower temperatures (16-25°C) to enhance proper folding

• Harvesting cells and lysing using sonication or pressure-based methods

• Purifying the recombinant protein using Ni-NTA affinity chromatography

This approach typically yields 2-5 mg of purified protein per liter of bacterial culture. Alternative expression systems such as yeast or insect cells may be considered if post-translational modifications are essential for functional studies.

How can RNA editing sites in atpF be identified and what are their functional implications?

RNA editing is a critical post-transcriptional modification in chloroplast genes, including atpF. Based on methodologies applied to the mitochondrial genome of C. bursa-pastoris, RNA editing sites can be identified through:

• Extraction of total RNA from fresh leaf tissue using RNA isolation kits

• rRNA depletion to enrich for mRNA content

• cDNA synthesis and high-throughput sequencing (RNAseq)

• Comparative analysis of genomic DNA and RNA sequences to identify C to U editing sites

Studies on C. bursa-pastoris mitochondrial genes revealed that most genes contain RNA editing sites that lead to non-synonymous changes of amino acids, potentially affecting protein function . For atpF specifically, researchers should:

• Map all RNA editing sites

• Determine if editing is tissue-specific or developmentally regulated

• Assess conservation of editing sites between C. bursa-pastoris and related species

• Evaluate the impact of editing on protein structure using predictive modeling

RNA editing may significantly influence ATP synthase assembly and function, potentially contributing to adaptive traits in this cosmopolitan weed species.

What approaches can be used to study the integration of recombinant atpF into functional ATP synthase complexes?

Studying the integration of recombinant atpF into functional ATP synthase complexes requires sophisticated biochemical and biophysical approaches:

Researchers should first isolate intact chloroplasts from C. bursa-pastoris leaves and extract thylakoid membranes. These preparations can be used as a source of native ATP synthase components for reconstitution experiments with the recombinant atpF protein. Additionally, heterologous expression systems can be employed to co-express multiple ATP synthase subunits for complex assembly studies. The functional integrity of reconstructed complexes can be assessed through ATP hydrolysis or synthesis assays using fluorescent ATP analogs or coupled enzyme systems.

How does atpF contribute to doxorubicin resistance mechanisms in Capsella bursa-pastoris?

Recent studies have shown that water extracts of C. bursa-pastoris can mitigate doxorubicin-induced cardiotoxicity . While the direct involvement of atpF has not been established, ATP synthase function is crucial for maintaining cellular energy homeostasis during oxidative stress. To investigate potential connections:

• Perform comparative proteomics on doxorubicin-treated and untreated C. bursa-pastoris cells

• Analyze atpF expression levels using qRT-PCR under doxorubicin treatment

• Assess ATP synthase activity in response to doxorubicin exposure

• Investigate whether flavonoid glycosides identified in C. bursa-pastoris extracts (such as luteolin-7-O-glucoside and isoquercitrin at concentrations of 133.41 μg/g and 131.22 μg/g respectively) interact with ATP synthase

The research approach should incorporate both in vitro enzyme activity assays and cellular models to comprehensively evaluate how atpF and the broader ATP synthase complex may contribute to stress response mechanisms. Special attention should be given to the MAPK-Nrf2 signaling pathway, which has been implicated in the cardioprotective effects of C. bursa-pastoris extracts .

What are the optimal conditions for analyzing ATP synthase activity in recombinant atpF studies?

When analyzing ATP synthase activity in studies involving recombinant atpF, researchers should consider the following methodological approach:

• Buffer composition: Use 50 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl₂

• Temperature control: Maintain assays at 30°C for optimal activity

• Substrate concentrations: 1-5 mM ATP for hydrolysis studies; 1-5 mM ADP and 5-10 mM inorganic phosphate for synthesis studies

• Coupling enzymes: For ATP production measurement, use glucose-6-phosphate dehydrogenase and hexokinase to couple ATP production to NADPH generation

• Inhibitor controls: Include oligomycin (5-10 μg/mL) as a specific inhibitor control

The experimental design should incorporate:

• Concentration gradients of recombinant atpF protein to assess dose-dependent effects

• Time-course measurements to determine initial rates

• Comparisons between wild-type and mutated versions of recombinant atpF

• Controls with heat-inactivated enzyme

Activity should be monitored spectrophotometrically or using luminescence-based ATP detection assays. Results should be normalized to protein concentration and expressed as specific activity (μmol ATP/min/mg protein).

How can researchers effectively analyze subgenome-specific variations in atpF expression in Capsella bursa-pastoris?

Analyzing subgenome-specific variations in atpF expression requires sophisticated genomic and transcriptomic approaches that account for the allopolyploid nature of C. bursa-pastoris:

• Genome-specific primer design: Design primers that target unique SNPs distinguishing the O (C. orientalis-derived) and R (C. rubella-derived) subgenomes

• Subgenome phasing: Use long-read sequencing technologies (such as PacBio SMRT) to accurately phase subgenome-specific sequences

• RNA-seq analysis: Implement bioinformatic pipelines specifically designed for polyploid transcriptomes:

  • Map reads to a combined reference containing both subgenomes

  • Filter for uniquely mapping reads to avoid bias

  • Quantify expression levels using subgenome-aware counting methods

When analyzing subgenome contributions, researchers should be aware that mapping bias can occur due to unequal efficiency of mapping reads belonging to the O and R subgenomes . To mitigate this:

• Perform simulation studies by combining sequences of parental genomes into a single reference

• Map reads from each parental species to validate mapping specificity

• Apply correction factors based on mapping rates to adjust expression values

This approach enables accurate quantification of subgenome-specific expression patterns, which is crucial for understanding the evolutionary trajectory of atpF following polyploidization.

What approaches are most effective for studying post-translational modifications of atpF in Capsella bursa-pastoris?

Post-translational modifications (PTMs) of atpF can significantly impact its function within the ATP synthase complex. To study these effectively:

• Sample preparation:

  • Extract total protein from chloroplasts using non-denaturing conditions

  • Enrich for atpF using immunoprecipitation or affinity purification

  • Digest purified protein with multiple proteases (trypsin, chymotrypsin) to increase coverage

• Analytical methods:

  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with high-resolution instruments

  • Multiple reaction monitoring (MRM) for targeted analysis of specific modifications

  • Top-down proteomics for intact protein analysis

• PTM prediction and validation:

  • Utilize computational tools to predict potential modification sites

  • Generate site-specific antibodies against predicted modifications

  • Perform site-directed mutagenesis to create non-modifiable variants

• Functional assessment:

  • Compare enzyme kinetics between modified and unmodified forms

  • Analyze structural changes using circular dichroism or fluorescence spectroscopy

  • Assess impact on protein-protein interactions within the ATP synthase complex

This comprehensive approach allows researchers to not only identify PTMs but also understand their functional significance in the context of ATP synthase assembly and activity.

How does the recombinant atpF from Capsella bursa-pastoris compare functionally to its orthologues in parental species?

The functional comparison between recombinant atpF from C. bursa-pastoris and its orthologues from parental species (C. rubella and C. orientalis) provides crucial insights into evolutionary consequences of hybridization. To perform this comparison:

• Express and purify recombinant atpF proteins from all three species using identical protocols

• Compare biochemical parameters:

  • Enzyme kinetics (Km, Vmax, catalytic efficiency)

  • pH and temperature optima

  • Stability under various conditions

  • Binding affinity to other ATP synthase subunits

• Integrate structural analyses:

  • Circular dichroism to assess secondary structure differences

  • Intrinsic fluorescence to probe tertiary structure variations

  • Hydrogen-deuterium exchange mass spectrometry to identify regions of differential flexibility

What bioinformatic pipelines are recommended for analyzing atpF sequence variation across Capsella populations?

To analyze atpF sequence variation across different Capsella populations, researchers should implement a multi-step bioinformatic pipeline:

• Data acquisition:

  • Obtain sequence data from diverse geographic populations

  • Include multiple accessions per population for statistical power

  • Generate high-quality consensus sequences using PacBio SMRT sequencing for accurate long-read data

• Sequence alignment and analysis:

  • Perform multiple sequence alignment using MAFFT or MUSCLE

  • Identify SNPs, insertions, deletions, and structural variants

  • Calculate nucleotide diversity (π) and population differentiation (FST)

• Evolutionary analysis:

  • Apply coalescent-based Bayesian analysis to infer demographic parameters

  • Identify signatures of selection using dN/dS ratio analysis

  • Reconstruct phylogenetic relationships using maximum likelihood or Bayesian approaches

• Visualization and interpretation:

  • Generate haplotype networks to visualize relationships between variants

  • Map variations to protein structure to assess functional implications

  • Correlate sequence variations with ecological or geographic factors

This pipeline allows for comprehensive characterization of atpF diversity patterns, which can be interpreted in the context of C. bursa-pastoris' rapid global expansion and adaptation to diverse environments .

How might CRISPR-Cas9 genome editing be applied to study atpF function in Capsella bursa-pastoris?

CRISPR-Cas9 genome editing offers powerful opportunities for functional studies of atpF in C. bursa-pastoris. A comprehensive research approach would include:

• Design considerations:

  • Target specific domains within atpF to create partial loss-of-function alleles

  • Design multiple guide RNAs to account for potential off-target effects

  • Consider subgenome-specific editing strategies for the allopolyploid genome

• Transformation protocol:

  • Use Agrobacterium-mediated transformation with floral dip method

  • Select transformants using antibiotic or herbicide resistance markers

  • Confirm editing events using targeted sequencing

• Functional characterization of mutants:

  • Analyze growth phenotypes under various conditions (light intensities, temperature stress)

  • Measure photosynthetic parameters (electron transport rate, ATP synthesis capacity)

  • Assess chloroplast ultrastructure using transmission electron microscopy

• Molecular analyses:

  • Quantify ATP synthase complex assembly using Blue Native PAGE

  • Measure ATP synthesis rates in isolated chloroplasts

  • Perform RNA-seq to identify compensatory transcriptional responses

What are the potential applications of understanding atpF function in developing stress-resistant crop varieties?

Understanding atpF function in C. bursa-pastoris could inform strategies for enhancing stress resistance in crops, particularly within the Brassicaceae family. Research applications include:

• Oxidative stress tolerance:

  • The water extract of C. bursa-pastoris has demonstrated protective effects against doxorubicin-induced oxidative damage through the Nrf2 signaling pathway

  • ATP synthase function is critical for maintaining energy homeostasis during stress

  • Variants of atpF with enhanced stability under oxidative conditions could be identified and introduced into crop species

• Methodology for translational research:

  • Identify natural variants of atpF in C. bursa-pastoris populations adapted to different environments

  • Characterize their functional properties under controlled stress conditions

  • Test promising variants in crop species using transgenic approaches or precision breeding

• Integration with other stress response mechanisms:

  • Investigate the relationship between ATP synthase function and antioxidant systems

  • Explore how flavonoid glycosides like luteolin-7-O-glucoside (133.41 μg/g) and isoquercitrin (131.22 μg/g) in C. bursa-pastoris might interact with ATP synthase to confer protection

  • Develop metabolic engineering strategies that combine enhanced ATP synthase function with increased production of protective secondary metabolites