Recombinant Aethionema grandiflora ATP synthase subunit b, chloroplastic (atpF)

What is ATP synthase subunit b (atpF) and its role in chloroplast function?

ATP synthase subunit b is a critical component of the chloroplastic ATP synthase complex, which produces adenosine triphosphate (ATP) required for photosynthetic metabolism. The ATP synthase complex consists of two primary regions: a membrane-extrinsic region designated as 'F1' and a membrane-intrinsic region designated as 'F0' . Subunit b is part of the F0 region and forms part of the stationary b/b'-stalk that connects the F0 and F1 regions, mechanically coupling them for efficient ATP production .

The b-subunit serves as a structural element that anchors the catalytic F1 portion to the membrane-embedded F0 portion. This structural role is essential for maintaining the spatial configuration necessary for the rotational catalysis mechanism that drives ATP synthesis. The rotation of the c-subunit ring (driven by proton translocation) is mechanically coupled to ATP synthesis via this b-subunit connection, allowing the conversion of the proton gradient energy into chemical energy in the form of ATP .

What experimental approaches are most suitable for analyzing atpF gene expression in Aethionema grandiflora?

Analysis of atpF gene expression in Aethionema grandiflora requires specialized approaches that account for chloroplast-specific gene regulation. The following methodological approaches are recommended:

• RNA Extraction and RT-qPCR Analysis:

  • Use specialized RNA extraction protocols optimized for plant tissues rich in polysaccharides and phenolic compounds

  • Design primers specific to the Aethionema grandiflora atpF sequence, avoiding regions with high similarity to other ATP synthase subunits

  • Normalize expression using stable chloroplast reference genes

• Chloroplast Isolation and Protein Analysis:

  • Implement differential centrifugation techniques to isolate intact chloroplasts

  • Perform western blotting with antibodies specific to conserved regions of atpF

  • Assess protein levels under different environmental conditions (light/dark, stress conditions)

• Transcriptome Analysis:

  • Employ RNA sequencing approaches with specific attention to chloroplast transcripts

  • Compare atpF expression patterns with other photosynthetic genes

  • Analyze splicing patterns, as chloroplast genes may undergo post-transcriptional modifications

The molecular phylogenetic approaches used in Aethionema studies can be adapted for expression analysis, building on techniques used for other Brassicaceae species .

What expression systems are most suitable for producing recombinant Aethionema grandiflora atpF protein?

Multiple expression systems have been employed for the recombinant production of Aethionema grandiflora ATP synthase subunit b, each with distinct advantages for different research applications:

For most academic research applications, E. coli systems offer a practical starting point, especially when coupled with optimization strategies like chaperone co-expression . The CSB-EP392380AUK1 preparation utilizes E. coli expression systems, while other variants use alternative hosts .

For membrane proteins like ATP synthase subunit b, co-expression with chaperone proteins (such as DnaK, DnaJ, and GrpE) can substantially improve folding and yield, as demonstrated with other ATP synthase subunits .

What strategies can improve solubility and folding of recombinant atpF protein?

Membrane proteins like ATP synthase subunit b present significant challenges for recombinant expression due to their hydrophobic nature. The following methodological approaches can enhance solubility and proper folding:

• Fusion Tag Selection:

  • MBP (maltose-binding protein) fusion significantly improves solubility while maintaining functionality

  • The pMAL-c2x expression vector system has been successfully used for other ATP synthase subunits and can be adapted for atpF

  • Biotinylation tags (like AviTag) can enhance stability and enable specific applications as demonstrated in the CSB-EP392380AUK1-B preparation

• Chaperone Co-expression:

  • Co-transformation with plasmids expressing chaperone proteins (DnaK, DnaJ, and GrpE) substantially increases quantities of difficult-to-produce membrane proteins

  • The pOFXT7KJE3 plasmid system has proven effective for ATP synthase subunits

• Expression Condition Optimization:

  • Lower induction temperature (16-20°C) promotes proper folding by slowing synthesis

  • Reduced IPTG concentration (0.1-0.3 mM) minimizes aggregation

  • Addition of membrane-mimetic compounds (detergents) during expression

• Membrane Fraction Recovery:

  • Specialized extraction protocols using mild detergents like n-dodecyl-β-D-maltoside (DDM)

  • Stepwise solubilization protocols to maintain native-like structure

These strategies have been effectively applied to other chloroplast ATP synthase subunits and can be adapted specifically for Aethionema grandiflora atpF .

What purification approaches yield the highest purity recombinant atpF protein for structural studies?

Purification of recombinant atpF protein at >85% purity (as achieved in commercial preparations ) requires a multi-step approach optimized for membrane proteins:

• Initial Capture:

  • Affinity chromatography using the fusion tag (His-tag, MBP, or Avi-tag biotinylation)

  • For MBP fusions, amylose resin provides high specificity and mild elution conditions

  • For biotinylated proteins, streptavidin matrices offer exceptional binding specificity

• Intermediate Purification:

  • Ion exchange chromatography to separate based on charge differences

  • Hydrophobic interaction chromatography to leverage the membrane protein's hydrophobic nature

• Polishing Steps:

  • Size exclusion chromatography to separate monomeric from aggregated forms

  • Removal of fusion tags using specific proteases (TEV, Factor Xa) followed by reverse affinity chromatography

• Quality Assessment:

  • SDS-PAGE with Coomassie staining to verify >85% purity

  • Western blotting with anti-atpF antibodies to confirm identity

  • Mass spectrometry to verify intact mass and sequence coverage

For structural studies, additional detergent exchange steps may be necessary to transition the protein into detergent systems compatible with crystallization or cryo-EM analysis.

How can site-directed mutagenesis be used to study functional domains in atpF?

Site-directed mutagenesis represents a powerful approach for dissecting the structure-function relationships of ATP synthase subunit b. The following methodological framework is recommended:

• Target Selection Based on Structural Prediction:

  • Focus on conserved residues identified through multiple sequence alignment of atpF across plant species

  • Target the interface between subunit b and other ATP synthase components (particularly important for the b/b'-stalk function)

  • Analyze predicted transmembrane domains versus soluble regions

• Mutagenesis Strategy:

  • Employ QuikChange or Q5 site-directed mutagenesis protocols

  • Design mutations that probe specific hypotheses:

    • Alanine scanning of conserved regions to identify essential residues

    • Charge-reversal mutations (Asp→Arg) to disrupt electrostatic interactions

    • Conservative substitutions to assess structural tolerance

• Functional Assessment:

  • Complement approaches must be developed to assess function:

    • In vitro reconstitution with other ATP synthase subunits

    • Proton translocation assays in liposome systems

    • ATP synthesis measurements using reconstituted systems

• Structural Impact Analysis:

  • Circular dichroism to assess secondary structure changes

  • Limited proteolysis to identify altered structural dynamics

  • Thermal stability assays to quantify folding energy changes

This approach parallels successful strategies applied to other ATP synthase components, where mutation of specific residues revealed critical insights into proton translocation and subunit interactions .

What methods are effective for studying protein-protein interactions between atpF and other ATP synthase subunits?

Understanding interactions between atpF and other ATP synthase components is crucial for comprehending the complex's assembly and function. Multiple complementary approaches are recommended:

• Co-Immunoprecipitation Studies:

  • Express recombinant atpF with different tags (His, FLAG, Avi-biotin)

  • Use tag-specific antibodies to pull down atpF and associated proteins

  • Identify interaction partners through mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpF on sensor chips via biotin-streptavidin coupling (available with Avi-tag biotinylated atpF )

  • Measure binding kinetics with potential interaction partners

  • Quantify affinity constants for different subunit interactions

• Crosslinking Mass Spectrometry:

  • Apply chemical crosslinkers with different spacer lengths

  • Digest crosslinked complexes and analyze by tandem mass spectrometry

  • Map interaction interfaces at amino acid resolution

• Förster Resonance Energy Transfer (FRET):

  • Generate fluorescently labeled atpF and partner proteins

  • Measure energy transfer as an indicator of proximity

  • Perform in-membrane FRET to assess interactions in a native-like environment

• Yeast Two-Hybrid Adaptations:

  • Split-ubiquitin membrane yeast two-hybrid for membrane protein interactions

  • Bait constructs using soluble domains of atpF

  • Screening against libraries of other ATP synthase components

These approaches provide complementary data that together build a comprehensive interaction map of atpF within the ATP synthase complex.

How can researchers reconstitute functional ATP synthase complexes using recombinant atpF?

Reconstitution of functional ATP synthase complexes represents an advanced research goal that enables mechanistic studies. The following methodology is recommended:

• Component Preparation:

  • Express and purify all necessary ATP synthase subunits individually

  • Ensure appropriate detergent selection for each component

  • Verify purity and folding of each component before assembly

• Stepwise Assembly Protocol:

  • Begin with formation of subcomplexes (e.g., c-ring assembly)

  • Add membrane components (including atpF) in detergent micelles

  • Incorporate F1 components under controlled conditions

• Membrane Reconstitution:

  • Select appropriate lipid composition mimicking chloroplast membranes

  • Use controlled detergent removal methods:

    • Bio-Beads for gentle detergent adsorption

    • Dialysis for gradual detergent reduction

    • Cyclodextrin-mediated detergent extraction

• Functional Verification:

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • ATP synthesis measurement under proton gradient conditions

  • Rotational analysis using single-molecule techniques

This approach builds on successful reconstitution methodologies developed for ATP synthase subunits from other organisms, such as the ATP synthase c1 subunit from spinach .

What spectroscopic techniques provide the most valuable structural information about recombinant atpF?

Multiple spectroscopic approaches can reveal critical structural information about recombinant Aethionema grandiflora atpF:

For comprehensive structural characterization, combining multiple techniques provides complementary information that overcomes the limitations of any single approach.

How can researchers analyze the proton translocation function of recombinant atpF in experimental systems?

• Liposome Reconstitution Assays:

  • Incorporate purified recombinant atpF into liposomes along with other essential F0 components

  • Create an artificial proton gradient using different pH buffers inside/outside

  • Monitor proton movement using pH-sensitive fluorescent dyes (e.g., ACMA, pyranine)

• Patch-Clamp Electrophysiology:

  • Reconstitute atpF-containing complexes into planar lipid bilayers

  • Measure ion conductance across the membrane

  • Assess the impact of mutations or inhibitors on proton channel function

• ATP Synthesis Coupling Measurements:

  • Assemble complete ATP synthase complexes including recombinant atpF

  • Establish proton gradients using light-driven systems (when coupled with bacteriorhodopsin)

  • Quantify ATP production using luciferase-based luminescence assays

• Site-Specific Probes:

  • Introduce environment-sensitive fluorescent labels at specific positions in atpF

  • Monitor conformational changes during proton translocation

  • Correlate structural dynamics with functional states

These approaches build upon established methodologies for studying the closely related ATP synthase subunit c, adapting them for the structural role of subunit b in the stationary stalk .

What bioinformatic approaches are most useful for analyzing atpF sequence-structure-function relationships?

Computational analysis provides valuable insights into atpF structure and function without requiring extensive experimental resources:

• Evolutionary Analysis:

  • Multiple sequence alignment of atpF across diverse plant species

  • Identification of conserved residues indicating functional importance

  • Calculation of selection pressures (dN/dS) to identify regions under evolutionary constraints

  • Phylogenetic analysis building on established approaches for Aethionema species

• Structural Prediction:

  • Ab initio modeling of transmembrane regions

  • Homology modeling based on related ATP synthase structures

  • Molecular dynamics simulations in membrane environments

  • Prediction of protein-protein interaction interfaces

• Functional Domain Annotation:

  • Identification of transmembrane domains using hydropathy analysis

  • Prediction of post-translational modification sites

  • Mapping of conserved functional motifs

  • Coevolution analysis to identify residues that evolve in a coordinated manner

• Integration with Experimental Data:

  • Mapping of mutational data onto structural models

  • Correlation of conservation patterns with experimental phenotypes

  • Development of testable hypotheses for experimental validation

These computational approaches can leverage the growing thermochemical and structural data available through resources like the Active Thermochemical Tables (ATcT) , which provide accurate thermodynamic parameters that may inform structural stability predictions.

How can recombinant Aethionema grandiflora atpF contribute to evolutionary studies of photosynthetic organisms?

Recombinant atpF provides valuable tools for investigating evolutionary relationships and adaptations in photosynthetic organisms:

• Molecular Phylogenetics:

  • Use atpF sequences to refine phylogenetic relationships within Brassicaceae

  • Incorporate structural information to understand selective pressures

  • Compare with other plastid markers (like trnL-trnF) to resolve evolutionary relationships

• Functional Evolution Studies:

  • Reconstruct ancestral atpF sequences through computational methods

  • Express and characterize ancestral proteins to study functional evolution

  • Analyze how changes in atpF correlate with speciation events or environmental adaptations

• Comparative Biochemistry:

  • Compare biochemical properties of atpF across species with different photosynthetic efficiencies

  • Identify structural adaptations that correlate with environmental niches

  • Study co-evolution of interacting subunits within the ATP synthase complex

• Endosymbiotic Evolution:

  • Investigate the evolution of chloroplast ATP synthase components compared to bacterial counterparts

  • Study gene transfer events between chloroplast and nuclear genomes affecting atpF

The established phylogenetic frameworks for Aethionema using both nuclear (ITS) and plastid markers (trnL-trnF) provide an excellent foundation for placing atpF evolution in context .

What emerging technologies will advance research on ATP synthase subunit b in the next decade?

Several cutting-edge technologies are poised to transform research on ATP synthase subunit b in the coming years:

• Cryo-Electron Microscopy Advances:

  • Higher resolution structures of complete ATP synthase complexes

  • Time-resolved cryo-EM to capture conformational states during catalysis

  • Visualizing atpF interactions within the native complex environment

• Single-Molecule Approaches:

  • FRET-based rotation measurements of reconstructed ATP synthase

  • Force microscopy to measure mechanical coupling through the b-subunit stalk

  • Single-particle tracking in synthetic membrane systems

• Artificial Intelligence Applications:

  • Improved protein structure prediction with AlphaFold-like systems

  • Machine learning analysis of sequence-structure-function relationships

  • Automated design of optimized mutations for specific functional properties

• In Cell Structural Biology:

  • Cryo-electron tomography of ATP synthase in intact chloroplasts

  • In-cell NMR to study structural dynamics in native environments

  • Correlative light and electron microscopy for functional-structural connections

• Synthetic Biology Approaches:

  • Designer ATP synthase complexes with modified subunit b

  • Biosensor development using engineered atpF proteins

  • Minimal synthetic systems to study fundamental mechanisms

These technologies will build on established methodological frameworks while offering unprecedented resolution and insight into dynamic processes.

How can researchers design effective control experiments when working with recombinant atpF?

• Expression and Purification Controls:

  • Empty vector controls processed identically to atpF constructs

  • Wild-type protein as a positive control for functional assays

  • Step-by-step quality control during purification process

• Structural Integrity Controls:

  • Circular dichroism comparison with predicted spectra

  • Limited proteolysis patterns compared to native protein

  • Thermal stability assays to verify proper folding

• Functional Assay Controls:

  • Known ATP synthase inhibitors as negative controls

  • Reconstitution with established functional components as positive controls

  • Mutant variants with predicted functional defects

• System-Specific Controls:

  • Liposome-only controls for reconstitution experiments

  • Non-specific protein controls for binding experiments

  • Host-cell background controls for interaction studies

• Statistical Design Considerations:

  • Appropriate biological and technical replicates

  • Randomization of sample processing order

  • Power analysis to determine required sample sizes

This control framework ensures that experimental results can be interpreted with confidence and distinguishes specific effects from artifacts or background noise.

What are common challenges in purifying recombinant atpF and how can they be addressed?

Researchers face several challenges when purifying recombinant ATP synthase subunit b, with specific solutions for each:

These solutions draw from successful approaches used with other membrane proteins, including related ATP synthase subunits .

How can researchers troubleshoot protein misfolding issues with recombinant atpF?

Protein misfolding represents a significant challenge for membrane proteins like ATP synthase subunit b. A systematic troubleshooting approach includes:

• Diagnostic Assessment:

  • Use size-exclusion chromatography to quantify aggregation

  • Perform thermal shift assays to assess stability

  • Apply CD spectroscopy to compare with predicted secondary structure

  • Evaluate functionality through binding or activity assays

• Expression Optimization:

  • Screen lower induction temperatures (16°C, 20°C, 25°C)

  • Test reduced inducer concentrations

  • Evaluate different media formulations

  • Co-express with chaperone systems like the combination of DnaK, DnaJ, and GrpE

• Construct Redesign:

  • Analyze sequence for aggregation-prone regions

  • Consider truncated constructs focusing on specific domains

  • Test different fusion partners (MBP has shown success with ATP synthase subunits )

  • Engineer disulfide bonds to stabilize tertiary structure

• Solubilization Strategies:

  • Systematic detergent screening (12+ detergent types)

  • Test mixed detergent systems

  • Evaluate solubilization directly from membrane fraction

  • Consider alternative membrane-mimetic systems (nanodiscs, amphipols)

• Refolding Protocols:

  • Develop dilution refolding methods using chaperones

  • Employ on-column refolding techniques

  • Test cyclodextrin-assisted refolding for membrane proteins

These approaches align with successful strategies used for other challenging membrane proteins and ATP synthase components .

What strategies can improve the success of functional reconstitution experiments with atpF?

Functional reconstitution of ATP synthase subunit b into membrane systems presents unique challenges requiring specialized approaches:

• Lipid Composition Optimization:

  • Test lipid mixtures mimicking chloroplast membranes

  • Evaluate various phospholipid:protein ratios (typically 50:1 to 500:1)

  • Consider adding specific lipids known to interact with ATP synthase

  • Screen lipid headgroup compositions for optimal reconstitution

• Protein Incorporation Methods:

  • Compare detergent-mediated reconstitution vs. direct incorporation

  • Evaluate different detergent removal rates (fast vs. slow)

  • Test oriented vs. random incorporation strategies

  • Optimize protein:lipid ratios for functional complexes

• Complex Assembly Approaches:

  • Pre-form subcomplexes before membrane incorporation

  • Test stepwise vs. single-step reconstitution

  • Evaluate different order of component addition

  • Optimize buffer conditions for complex stability

• Functional Validation:

  • Implement multiple complementary assays

  • Compare activity to native controls when available

  • Quantify reconstitution efficiency through protein recovery

  • Assess complex integrity via analytical techniques

• Troubleshooting Tools:

  • Freeze-fracture electron microscopy to visualize protein incorporation

  • Fluorescence recovery after photobleaching (FRAP) to assess mobility

  • Sucrose gradient centrifugation to separate proteoliposomes

  • Dye leakage assays to verify membrane integrity

These methodological approaches build on established protocols for ATP synthase reconstitution while addressing the specific challenges of working with subunit b as part of the stationary stalk.