Recombinant Capsella bursa-pastoris ATP synthase subunit c, chloroplastic (atpH)

What is Capsella bursa-pastoris and why is it significant for ATP synthase research?

Capsella bursa-pastoris Medik. (commonly known as shepherd's purse) is a plant species with documented antibacterial, anti-inflammatory, antioxidant, anticancer, and hepatoprotective effects . Its widespread distribution and distinctive morphological characteristics make it an accessible model organism for studying plant proteins, including chloroplastic ATP synthase components. The plant contains diverse bioactive compounds including polyphenols (634.23 mg GAE/g DW in fruits), flavonoids (23.14 mg QE/g DW in fruits), and anthocyanins (7.18 mg cyanidin/100 g DW in flowers) . These compounds may influence protein stability and function during extraction, making it essential to understand the phytochemical environment when isolating chloroplastic proteins like atpH.

How does the chloroplastic ATP synthase subunit c function in Capsella bursa-pastoris?

The chloroplastic ATP synthase subunit c (atpH) in C. bursa-pastoris functions as part of the membrane-embedded F0 portion of the ATP synthase complex. This protein forms a ring structure in the thylakoid membrane that facilitates proton translocation across the membrane. The proton gradient generated during photosynthesis drives the rotation of this c-ring, which couples with the F1 portion to synthesize ATP. In C. bursa-pastoris, as in other plants, this process is fundamental to energy production during photosynthesis, converting light energy into chemical energy stored as ATP.

What are the fundamental extraction protocols for obtaining native atpH protein from Capsella bursa-pastoris tissues?

The extraction of native atpH protein from C. bursa-pastoris typically requires careful consideration of the plant's phytochemical composition. Based on established protocols for protein extraction from plant tissues, researchers should:

• Select appropriate plant tissues (leaves are recommended due to higher chloroplast content)

• Homogenize tissue in extraction buffer containing:

  • 50 mM Tris-HCl (pH 7.5)

  • 100 mM NaCl

  • 10% glycerol

  • 1 mM EDTA

  • Protease inhibitor cocktail

• Isolate chloroplasts through differential centrifugation

• Lyse chloroplasts and separate thylakoid membranes

• Solubilize membrane proteins using mild detergents (e.g., n-dodecyl β-D-maltoside)

• Purify the atpH protein using chromatographic techniques

The extraction protocol must account for the high polyphenol content in C. bursa-pastoris tissues (344.23 mg GAE/g DW in leaves) , which can interfere with protein purification by causing oxidation and protein precipitation.

What expression systems are optimal for producing recombinant Capsella bursa-pastoris atpH protein?

For optimal expression of recombinant C. bursa-pastoris atpH, researchers should consider multiple expression systems based on research objectives:

E. coli-based expression systems:

• BL21(DE3) strain is suitable for high-yield expression when proper codon optimization is performed

• C41(DE3) or C43(DE3) strains are preferable for membrane proteins like atpH

• Expression vectors containing T7 promoter and appropriate fusion tags (His6, MBP, or SUMO) improve solubility

Plant-based expression systems:

• Transient expression in Nicotiana benthamiana using Agrobacterium-mediated transformation

• Stable transformation of Arabidopsis thaliana for functional studies in a native-like environment

Cell-free expression systems:

• Wheat germ extract systems for avoiding inclusion body formation

• E. coli S30 extract supplemented with lipid vesicles for functional membrane protein synthesis

When designing expression constructs, researchers should implement codon optimization based on the target expression system and consider including a cleavable tag for purification that minimizes interference with protein structure and function.

How can researchers optimize the purification of recombinant atpH to maintain its native conformation?

Optimizing purification of recombinant atpH while maintaining its native conformation requires a multi-step approach:

• Solubilization strategy:

  • Test multiple detergents (DDM, LDAO, CHAPS) at various concentrations

  • Evaluate solubilization efficiency using Western blotting

  • Monitor protein stability using circular dichroism during detergent screening

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Employ gradient elution to separate different binding populations

  • Include low concentrations of detergent in all buffers to prevent aggregation

• Secondary purification:

  • Size exclusion chromatography to isolate properly folded monomeric or oligomeric states

  • Ion exchange chromatography for removing contaminants with different charge profiles

• Quality assessment:

  • Analytical ultracentrifugation to confirm proper oligomerization

  • CD spectroscopy to verify secondary structure integrity

  • Thermal shift assays to evaluate protein stability

The purification protocol should be optimized with antioxidants (such as 5 mM DTT or 1 mM TCEP) to counteract potential oxidative damage from residual plant phenolic compounds that might co-purify with the protein.

What analytical techniques are most effective for structural characterization of recombinant atpH protein?

The structural characterization of recombinant atpH requires complementary techniques:

Spectroscopic methods:

• Circular dichroism (CD) spectroscopy to determine secondary structure composition

• Fluorescence spectroscopy to monitor tertiary structure and conformational changes

• Nuclear magnetic resonance (NMR) for atomic-level structural information of isotope-labeled protein

Crystallographic approaches:

• X-ray crystallography for high-resolution structural determination (requires optimization of crystallization conditions)

• Cryo-electron microscopy for visualizing the protein in its membrane environment or as part of the ATP synthase complex

Computational methods:

• Homology modeling based on known ATP synthase c-subunit structures

• Molecular dynamics simulations to study dynamic properties and lipid interactions

Biophysical techniques:

• Small-angle X-ray scattering (SAXS) for low-resolution structural information in solution

• Hydrogen-deuterium exchange mass spectrometry to probe solvent accessibility and dynamics

Each technique provides complementary information, and researchers should design a structural characterization pipeline based on specific research questions and available resources.

What assays can effectively measure the functional activity of recombinant atpH in vitro?

Functional assays for recombinant atpH should focus on both its individual properties and its role within the ATP synthase complex:

Proton translocation assays:

• Reconstitution of purified atpH into liposomes containing pH-sensitive fluorescent dyes

• Measurement of fluorescence changes upon establishing a pH gradient

• Quantification of proton flux rates under varying conditions

ATP synthesis/hydrolysis assays:

• Reconstitution of atpH with other ATP synthase subunits in proteoliposomes

• Measurement of ATP synthesis upon establishment of proton gradient

• Quantification of Pi release during ATP hydrolysis using malachite green or EnzChek assays

Binding assays:

• Isothermal titration calorimetry to measure binding affinity to other ATP synthase subunits

• Surface plasmon resonance to study interaction kinetics with partner proteins

• Microscale thermophoresis for detecting molecular interactions in near-native conditions

Structural integrity assays:

• Proteolytic susceptibility assays to assess proper folding

• Thermal stability assays using differential scanning fluorimetry

These methodologies can be adapted based on specific research questions and available equipment, with appropriate controls to validate assay specificity and sensitivity.

How can researchers effectively analyze the integration of recombinant atpH into functional ATP synthase complexes?

To analyze the integration of recombinant atpH into functional ATP synthase complexes:

• Reconstitution approaches:

  • Co-expression of atpH with other ATP synthase subunits

  • Stepwise reconstitution of purified subunits

  • Incorporation of recombinant atpH into isolated thylakoid membranes lacking endogenous atpH

• Analytical techniques for complex formation:

  • Blue native PAGE to visualize intact ATP synthase complexes

  • Sucrose density gradient ultracentrifugation to isolate assembled complexes

  • Co-immunoprecipitation to verify specific protein-protein interactions

  • Crosslinking mass spectrometry to map interaction interfaces

• Functional validation:

  • ATP synthesis assays using reconstituted complexes

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • Rotational analysis using single-molecule techniques

• Structural validation:

  • Negative stain electron microscopy to visualize complex formation

  • Cryo-electron microscopy for higher resolution structural analysis

  • Mass photometry to determine stoichiometry of component parts

Success in these analyses depends on maintaining protein stability throughout the reconstitution process, which may require optimization of buffer conditions, lipid composition, and handling procedures.

What approaches are recommended for studying the effect of plant-derived compounds on atpH function?

Given the rich phytochemical profile of C. bursa-pastoris, studying compound-protein interactions is particularly relevant:

Screening methodologies:

• Thermal shift assays to identify compounds that affect protein stability

• Activity assays in the presence of isolated plant compounds or fractionated extracts

• Isothermal titration calorimetry to measure binding affinities

• NMR or X-ray crystallography to determine binding sites

Compound selection based on C. bursa-pastoris composition:

• Flavonoids (particularly hyperoside and rutin found in high concentrations in CBP flowers, 80.0 and 110.0 mg/g respectively)

• Phenolic acids (including ferulic acid found in CBP root and leaves, 3.86 mg/g)

• Other bioactive compounds identified in the plant

Functional impact assessment:

• Proton translocation efficiency in the presence of compounds

• ATP synthesis rates with compound supplementation

• Structural changes induced by compound binding

This research direction could identify natural compounds that modulate ATP synthase activity, potentially providing insights into the plant's energy metabolism regulation and stress responses.

How should researchers interpret contradictory data between in vitro and in vivo atpH functional assays?

When facing contradictory results between in vitro and in vivo functional assays of atpH:

• Systematic comparison approach:

  • Create a detailed comparative table of experimental conditions

  • Identify key differences in protein environment (lipids, pH, ionic strength)

  • Evaluate the presence/absence of regulatory factors in different systems

• Analysis of potential artifacts:

  • Assess whether purification tags affect protein function

  • Evaluate detergent effects on protein conformation and activity

  • Consider post-translational modifications present in vivo but absent in vitro

• Reconciliation strategies:

  • Develop intermediate experimental systems (e.g., reconstituted membranes)

  • Use computational modeling to predict behavior under different conditions

  • Design experiments to specifically test hypothesized reasons for discrepancies

• Reporting recommendations:

  • Document all experimental conditions comprehensively

  • Present both sets of data with appropriate context

  • Discuss possible biological significance of the observed differences

Researchers should recognize that differences between in vitro and in vivo results may reflect biologically meaningful regulatory mechanisms rather than experimental artifacts.

What are the common pitfalls in recombinant atpH expression and purification, and how can they be overcome?

When working with C. bursa-pastoris extracts or recombinant atpH, be particularly aware of the high antioxidant content of the plant tissues. The flowers show particularly high DPPH radical scavenging activity (87.07%) and FRAP values (753.64 μmol Trolox Equivalent/100g) , suggesting significant antioxidant presence that could affect protein oxidation states during extraction.

How can researchers effectively incorporate computational approaches to supplement experimental studies of atpH?

Computational approaches can significantly enhance experimental studies of atpH:

• Structural prediction and analysis:

  • Homology modeling based on related ATP synthase c-subunits

  • Molecular dynamics simulations to study protein-membrane interactions

  • Prediction of critical residues for function through conservation analysis

  • QM/MM studies of proton translocation mechanisms

• Systems biology approaches:

  • Gene co-expression network analysis to identify functional partners

  • Metabolic flux analysis to understand the impact of atpH modifications

  • Multi-scale modeling connecting molecular function to cellular energetics

• Data integration frameworks:

  • Development of databases combining experimental data with predictions

  • Machine learning approaches to identify patterns in large-scale datasets

  • Network analysis to understand ATP synthase in the context of chloroplast biology

• Experimental design optimization:

  • In silico mutagenesis to prioritize experimental targets

  • Virtual screening of compounds for interaction studies

  • Simulation of experimental conditions to interpret results

By integrating computational and experimental approaches, researchers can develop more comprehensive models of atpH function and regulation, potentially identifying novel research directions.

How can researchers effectively study post-translational modifications of atpH in Capsella bursa-pastoris?

Studying post-translational modifications (PTMs) of atpH requires a methodical approach:

• PTM prediction and mapping:

  • In silico prediction using PTM-specific algorithms

  • Conservation analysis across species to identify likely modification sites

  • Development of site-specific antibodies for common modifications

• Mass spectrometry-based approaches:

  • Bottom-up proteomics with enrichment strategies for specific PTMs

  • Top-down proteomics to analyze intact proteoforms

  • Middle-down approaches for improved sequence coverage

  • Targeted MS methods for quantification of specific modifications

• Functional impact assessment:

  • Site-directed mutagenesis of modified residues

  • Activity assays comparing wild-type and mutant proteins

  • Structural studies to determine conformational effects

• Physiological context analysis:

  • Investigation of conditions that alter modification patterns

  • Quantitative proteomics across developmental stages or stress responses

  • Identification of enzymes responsible for adding/removing modifications

The rich phytochemical profile of C. bursa-pastoris suggests potential for interesting regulatory PTMs that may be unique to this species or modulated by its specific metabolite composition.

What methodologies are most appropriate for comparative studies between atpH from Capsella bursa-pastoris and other plant species?

For effective comparative studies of atpH across plant species:

• Sequence-based comparisons:

  • Multiple sequence alignment to identify conserved and variable regions

  • Phylogenetic analysis to understand evolutionary relationships

  • Selection pressure analysis to identify functionally important residues

• Structural comparisons:

  • Homology modeling of atpH from multiple species

  • Superimposition of structures to identify conformational differences

  • Analysis of species-specific structural features

• Functional comparisons:

  • Heterologous expression of atpH from different species

  • Standardized activity assays under identical conditions

  • Chimeric protein construction to identify functionally divergent regions

• Environmental adaptation analysis:

  • Correlation of sequence/structural differences with habitat conditions

  • Thermal stability comparisons across species from different climates

  • Investigation of species-specific regulatory mechanisms

These comparative approaches can reveal adaptations in energy metabolism across plant species and potentially identify unique features of C. bursa-pastoris atpH related to the plant's documented medicinal properties.

How can researchers leverage atpH studies to understand the relationship between energy metabolism and antioxidant capacity in Capsella bursa-pastoris?

Investigating the relationship between atpH function and antioxidant capacity:

• Integrated experimental approaches:

  • Analysis of ATP synthase activity in different plant tissues with varying antioxidant profiles

  • Correlation of ATP production capacity with antioxidant compound accumulation

  • Measurement of atpH expression and ATP synthase assembly under oxidative stress

• Oxidative damage assessment:

  • Quantification of oxidative modifications to atpH protein

  • Functional impact of oxidation on ATP synthase activity

  • Protective effects of tissue-specific antioxidants

• Metabolic network analysis:

  • Tracking carbon flux between energy production and antioxidant biosynthesis

  • Investigation of regulatory crosstalk between pathways

  • Mathematical modeling of resource allocation under varying conditions

This research direction is particularly relevant for C. bursa-pastoris given its documented high antioxidant capacity, with flowers showing strong antioxidant activity as measured by DPPH (87.07%), CUPRAC (1318.33 μmol Trolox Equivalent/mL), and FRAP (753.64 μmol Trolox Equivalent/100g) assays . Understanding how the plant balances energy metabolism and antioxidant production could provide insights into its therapeutic potential.

What are the most promising research directions for Capsella bursa-pastoris atpH studies in the context of plant adaptation to environmental stress?

The study of C. bursa-pastoris atpH offers several promising research directions:

• Stress response mechanisms:

  • Investigation of atpH regulation under various abiotic stresses

  • Analysis of how ATP synthase efficiency correlates with stress tolerance

  • Identification of stress-specific post-translational modifications

• Comparative genomics approaches:

  • Analysis of atpH sequence variations in C. bursa-pastoris populations from different environments

  • Investigation of chloroplast genome evolution in relation to energy metabolism

  • Identification of natural variants with enhanced stress tolerance

• Engineering applications:

  • Development of modified atpH variants with improved function under stress

  • Creation of sensor systems based on atpH to monitor cellular energy status

  • Integration of atpH studies with broader photosynthetic efficiency enhancement efforts

These research directions could contribute to our understanding of plant adaptation mechanisms and potentially lead to applications in agriculture and biotechnology.

How can multi-omics approaches enhance our understanding of atpH function within the broader context of Capsella bursa-pastoris biology?

Multi-omics integration provides a comprehensive framework for understanding atpH:

• Integrated omics workflow:

  • Genomics: Analysis of atpH gene structure, variants, and regulatory elements

  • Transcriptomics: Expression patterns across tissues, developmental stages, and conditions

  • Proteomics: Protein abundance, interactions, and modifications

  • Metabolomics: Correlation with energy metabolites and antioxidant compounds

  • Phenomics: Connection to plant growth, development, and stress responses

• Data integration approaches:

  • Network analysis to identify functional modules

  • Machine learning for pattern recognition across datasets

  • Systems biology modeling to predict emergent properties

• Biological insights:

  • Identification of condition-specific regulatory mechanisms

  • Discovery of novel functional interactions

  • Understanding of atpH's role in whole-plant energy homeostasis