Recombinant Eucomis bicolor ATP synthase subunit beta, chloroplastic (atpB)

What is the function of ATP synthase subunit beta in chloroplasts?

ATP synthase subunit beta (atpB) is a crucial component of the F1Fo-ATP synthase complex in chloroplasts, which produces adenosine triphosphate (ATP) required for photosynthetic metabolism. The beta subunit forms part of the catalytic F1 region of the enzyme, specifically within the α3β3 hexamer where ATP synthesis occurs. This subunit contains nucleotide-binding domains and participates directly in the catalysis of the ADP + Pi → ATP reaction that takes place at the α-β subunit interfaces . The rotation of the c-subunit ring, driven by proton translocation across the thylakoid membrane, mechanically couples to the rotation of the γ-stalk in the F1 region, powering ATP synthesis through conformational changes in the beta subunits .

How does the atpB gene structure in Eucomis bicolor compare to other plant species?

While specific information on Eucomis bicolor atpB gene structure is limited in the provided search results, insights can be drawn from studies on other species. In Chlamydomonas reinhardtii, the chloroplast atpB gene contains three in-frame ATG codons that could potentially serve as translation initiation sites. Research has demonstrated that translation is initiated exclusively at the second ATG codon in this species . This finding suggests that the beta subunit is not synthesized with an N-terminal leader sequence prior to its assembly into a functional ATP synthase complex .

For Eucomis bicolor, a South African plant from the Asparagaceae family, similar gene structure analysis would be valuable to determine whether its atpB gene follows comparable patterns of translation initiation. Researchers studying E. bicolor atpB should consider conducting comparative genomic analyses with other monocotyledonous plants to identify conserved regulatory elements and structural features.

What are the main challenges in expressing chloroplastic proteins like atpB in heterologous systems?

Recombinant expression of chloroplastic proteins presents several unique challenges:

• Codon optimization: Chloroplast genes often have codon usage patterns that differ significantly from common expression hosts like E. coli, potentially leading to poor translation efficiency.

• Post-translational modifications: Chloroplastic proteins may require specific modifications not readily available in bacterial expression systems.

• Membrane association: As part of a membrane-associated complex, atpB may exhibit solubility issues when expressed recombinantly.

• Assembly requirements: In its native environment, atpB functions as part of a multi-subunit complex, and isolated expression may affect proper folding.

To address these challenges, researchers have developed strategies similar to those used for other ATP synthase subunits. For example, the ATP synthase c subunit from spinach chloroplasts has been successfully produced in an E. coli expression system using a synthetic gene constructed by annealing and ligating overlapping oligonucleotides, with phosphates added to the 5' end of oligonucleotides prior to annealing . This approach may be adapted for atpB expression.

What expression systems are most suitable for recombinant Eucomis bicolor atpB production?

Based on protocols developed for other chloroplastic ATP synthase subunits, bacterial expression systems, particularly E. coli, offer a practical starting point for recombinant atpB production. A methodological approach would include:

E. coli Expression System Protocol:

• Gene synthesis: Design a synthetic gene encoding Eucomis bicolor atpB optimized for E. coli codon usage.

• Vector selection: Choose a vector with an appropriate promoter (e.g., T7) and affinity tag (His6, GST, etc.) to facilitate purification.

• Expression conditions: Optimize temperature, IPTG concentration, and induction duration to maximize protein yield while minimizing inclusion body formation.

• Host strain selection: BL21(DE3) or derivatives may be appropriate, though strains with enhanced disulfide bond formation capabilities could be beneficial if the protein contains critical cysteine residues .

An expression protocol similar to that used for ATP synthase subunit c may be effective, where expression is induced with IPTG (1.0 mM) followed by a short incubation period (30 minutes) . Cell harvesting by centrifugation and storage at -80°C maintains protein integrity prior to purification.

For challenging expressions, alternative systems such as cell-free protein synthesis or expression in chloroplast-containing eukaryotes (e.g., Chlamydomonas) could be considered, though these typically yield lower protein quantities.

How can I optimize the synthetic gene design for efficient expression of Eucomis bicolor atpB?

Effective synthetic gene design significantly impacts recombinant protein expression. For Eucomis bicolor atpB, consider the following optimization strategies:

• Codon optimization: Adjust codon usage to match the preferred codons of your expression host. For E. coli expression, this typically involves avoiding rare codons while maintaining a GC content between 40-60%.

• mRNA secondary structure modification: Eliminate stable secondary structures in the mRNA, particularly near the ribosome binding site and start codon, which can impede translation initiation.

• Restriction site engineering: Incorporate useful restriction sites for cloning while eliminating problematic internal sites.

• Reduction of repetitive sequences: Minimize sequence repeats that could lead to recombination or polymerase slippage during PCR amplification.

A synthetic gene construction approach similar to that used for ATP synthase subunit c could be employed, where overlapping oligonucleotides (ranging from 24 to 46 bp) are annealed and ligated after phosphorylation with T4 Polynucleotide Kinase . This method allows precise control over the gene sequence while accommodating all necessary optimizations.

What factors affect solubility of recombinant atpB, and how can they be addressed?

Recombinant chloroplastic proteins often face solubility challenges due to their native membrane association or complex formation requirements. For atpB, consider these factors and solutions:

• Expression temperature: Lower temperatures (16-20°C) often promote proper folding over rapid expression, increasing solubility.

• Induction conditions: Lower IPTG concentrations (0.1-0.5 mM) and slower induction can improve solubility.

• Fusion partners: Solubility-enhancing fusion tags such as MBP (maltose-binding protein), SUMO, or Trx can significantly increase soluble yields.

• Lysis buffer optimization: Include stabilizing agents such as glycerol (5-10%), mild detergents, or specific salt concentrations based on protein characteristics.

• Co-expression strategies: Consider co-expressing atpB with chaperones or other ATP synthase subunits that may facilitate proper folding.

If inclusion bodies form despite these precautions, develop a refolding protocol using gradual dialysis from denaturing conditions (8M urea or 6M guanidine-HCl) to native buffer conditions. For refolding ATP synthase components, a stepwise reduction in denaturant concentration while maintaining an appropriate redox environment is critical, particularly given the redox-sensitivity observed in some ATP synthase subunits .

What purification strategies are most effective for recombinant atpB isolation?

Effective purification of recombinant atpB requires a multi-step approach tailored to the protein's properties. Based on protocols for other ATP synthase subunits, the following strategy is recommended:

Step 1: Initial extraction

• Resuspend cell pellets in lysis buffer containing protease inhibitors (e.g., 20 mM Tris-HCl pH 8.0 with 2% v/v Protease Inhibitor Cocktail)

• Add lysozyme (1 mg/mL) and incubate at 4°C for 1.5 hours before sonication

• Centrifuge to separate soluble fraction from inclusion bodies

Step 2: Affinity chromatography

• For His-tagged constructs, use immobilized metal affinity chromatography (IMAC) with Ni-NTA resin

• Apply sample in buffer containing 20-40 mM imidazole to reduce non-specific binding

• Elute with imidazole gradient (50-300 mM)

Step 3: Secondary purification

• Ion exchange chromatography based on the predicted isoelectric point of atpB

• Size exclusion chromatography for final polishing and buffer exchange

Step 4: Quality assessment

• SDS-PAGE and Western blotting using antibodies against atpB or the affinity tag

• Mass spectrometry to confirm protein identity and integrity

For membrane-associated proteins like ATP synthase components, including mild detergents (e.g., 0.1% DDM or CHAPS) in purification buffers may improve yields by preventing aggregation.

How can I confirm the proper folding and functionality of purified recombinant atpB?

Assessing the structural integrity and functional activity of purified recombinant atpB is critical for ensuring that the protein maintains its native properties. Several complementary approaches are recommended:

• Circular dichroism (CD) spectroscopy: Evaluate secondary structure content and compare with predicted values based on homology models or known structures of ATP synthase beta subunits.

• Thermal shift assays: Measure protein stability and the effects of different buffer conditions on melting temperature.

• Limited proteolysis: Properly folded proteins typically show discrete, reproducible digestion patterns compared to misfolded variants.

• ATP binding assays: As atpB contains nucleotide-binding domains, assess ATP binding using fluorescent ATP analogs or isothermal titration calorimetry (ITC).

• Reconstitution experiments: For definitive functional analysis, attempt to reconstitute the recombinant atpB with other ATP synthase components to restore enzymatic activity.

The ATP hydrolysis activity can be measured using a coupled enzyme assay system or by directly monitoring phosphate release. When designing functional assays, consider that chloroplast ATP synthase activity in plants is known to be regulated by redox state, with specific cysteine residues acting as redox sensors . Therefore, maintaining appropriate redox conditions during activity measurements is essential.

What methods can determine if recombinant atpB maintains its native conformation and interaction capabilities?

Confirming that recombinant atpB maintains its native structure and interaction capabilities requires specialized analytical techniques:

• Native PAGE: Compare migration patterns with native atpB extracted from Eucomis bicolor chloroplasts.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS): Determine the oligomeric state of purified atpB.

• Surface plasmon resonance (SPR) or bio-layer interferometry (BLI): Measure interaction kinetics between atpB and other ATP synthase subunits.

• Pull-down assays: Identify binding partners from chloroplast extracts using the recombinant protein as bait.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map protein dynamics and conformational changes upon ligand binding or under different conditions.

• Cryo-electron microscopy: For structural validation if sufficient quantities of protein can be produced.

When studying recombinant ATP synthase components, researchers should be aware that in the complete enzyme complex, the conformational state of subunits like atpB is influenced by interactions with other components, particularly during the catalytic cycle. Isolated subunits may adopt slightly different conformations compared to their assembled state.

How does redox regulation affect ATP synthase function in chloroplasts, and how can this be studied in recombinant atpB?

Redox regulation represents a critical control mechanism for chloroplast ATP synthase. The available literature indicates:

• Established redox regulation: Chloroplast ATP synthase activity, particularly its ATP hydrolytic function, is regulated by the formation and reduction of disulfide bonds . This mechanism has been well-established in plant systems.

• Functional impact: Redox state changes do not affect ATP binding rates to the catalytic sites or torque for rotation but instead cause pauses in the catalytic process due to ADP inhibition .

• Structural basis: In chloroplasts, redox-sensitive amino acid sequences in the γ-subunit mediate this regulation .

To study redox regulation in recombinant Eucomis bicolor atpB, consider these approaches:

• Cysteine mapping: Identify all cysteine residues in the atpB sequence and assess their conservation across species.

• Site-directed mutagenesis: Create cysteine-to-serine mutants to evaluate the role of specific residues in redox sensitivity.

• Differential oxidation assays: Compare ATP synthesis/hydrolysis activity under reducing (DTT, β-mercaptoethanol) and oxidizing (H2O2, diamide) conditions.

• Redox titrations: Determine the mid-point redox potential of regulatory thiols using defined ratios of oxidized/reduced glutathione or other redox couples.

• Mass spectrometry analysis: Use differential alkylation strategies to identify cysteine residues susceptible to oxidative modifications.

What post-translational modifications have been identified in chloroplastic atpB, and how might they impact recombinant protein studies?

While the search results don't explicitly detail post-translational modifications (PTMs) specific to Eucomis bicolor atpB, research on ATP synthase from other organisms provides valuable insights into potential modifications and their functional significance:

• Phosphorylation: Several ATP synthase subunits undergo phosphorylation, which can modulate enzyme activity and assembly.

• Oxidative modifications: Cysteine residues in ATP synthase subunits can undergo various oxidative modifications including disulfide formation, S-glutathionylation, and S-nitrosation .

• Processing: The search results indicate that in Chlamydomonas reinhardtii, the N-terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon , suggesting potential N-terminal processing.

These PTMs can significantly impact recombinant protein studies in several ways:

• Expression system limitations: Bacterial systems like E. coli lack many of the enzymes responsible for eukaryotic PTMs, potentially yielding proteins with different properties than their native counterparts.

• Functional assessment: Absence of critical PTMs may lead to misleading results in functional assays.

• Structural differences: PTMs can influence protein folding, stability, and interaction capabilities.

To address these challenges, researchers should:

• Characterize native PTMs: Analyze PTMs on atpB isolated from Eucomis bicolor chloroplasts using mass spectrometry.

• Consider eukaryotic expression systems: For critical PTMs, yeast or insect cell systems may provide more appropriate modifications.

• Implement in vitro modification: For well-characterized PTMs, consider enzymatic or chemical modification of the purified recombinant protein.

• Control redox environment: Given the established redox sensitivity of chloroplast ATP synthase, carefully control and document the redox conditions during all experimental procedures .

How can recombinant atpB be used to study the stoichiometry and assembly of ATP synthase complexes?

Recombinant atpB provides a valuable tool for investigating ATP synthase complex assembly and stoichiometry through several sophisticated approaches:

• Reconstitution studies: Purified recombinant atpB can be combined with other ATP synthase subunits to study the assembly process and requirements. This approach has been applied for other ATP synthase subunits, such as the c-subunit from spinach chloroplasts .

• Stoichiometry analysis: The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits in the ATP synthase ring . Similar stoichiometric relationships could be investigated for atpB within the F1 complex.

• Labeled subunit incorporation: Fluorescently labeled recombinant atpB can be used to track the integration of the subunit into complexes using techniques like single-molecule fluorescence microscopy.

• Structure-function studies: Site-directed mutagenesis of recombinant atpB can help identify critical residues for complex assembly and function, particularly when combined with reconstitution assays.

• Crosslinking approaches: Engineered cysteine residues or photo-activatable crosslinkers can be introduced into recombinant atpB to capture transient interactions during assembly.

The development of a recombinant expression system for atpB enables the application of molecular biology techniques that cannot otherwise be applied to native ATP synthase complexes. This capability is particularly valuable for investigating factors that influence the stoichiometric variation of intact rings and other aspects of complex assembly .

What are the current challenges in understanding the regulatory mechanisms of chloroplastic ATP synthase, and how can recombinant atpB contribute to this field?

Understanding chloroplastic ATP synthase regulation presents several significant challenges where recombinant atpB studies could provide valuable insights:

Current Challenges:

• Integration of multiple regulatory mechanisms: Chloroplast ATP synthase is regulated through various mechanisms including proton gradient sensing, redox regulation, and potentially metabolite binding.

• Structural basis of regulation: The precise conformational changes underlying regulatory responses remain incompletely characterized.

• Species-specific differences: Regulatory mechanisms may vary between plant species, particularly those adapted to different environmental conditions.

• Dynamic responses: How ATP synthase activity rapidly adapts to changing light conditions and metabolic demands is not fully understood.

Contributions of Recombinant atpB Research:

• Mutational analysis: Systematic mutation of potential regulatory sites in recombinant atpB can help identify key residues involved in allosteric regulation.

• Interaction studies: Recombinant atpB can be used to identify potential regulatory proteins or metabolites that interact directly with the beta subunit.

• Structural studies: High-yield recombinant expression facilitates structural biology approaches (X-ray crystallography, cryo-EM) to visualize different conformational states of atpB.

• Comparative analysis: Expression of atpB from different plant species, including Eucomis bicolor, allows comparative studies of regulatory mechanisms across evolutionary diverse plants.

• Redox sensitivity mapping: Given the established redox regulation of chloroplast ATP synthase , recombinant atpB provides a platform to systematically investigate the impact of redox changes on beta subunit function.

By addressing these challenges through recombinant protein studies, researchers can develop more complete models of ATP synthase regulation that account for environmental responsiveness and metabolic integration within the chloroplast.

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) be applied to study the conformational dynamics of recombinant atpB?

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) offers powerful insights into protein dynamics and conformational changes relevant to atpB function:

Methodological Approach:

• Sample preparation: Purified recombinant atpB is diluted into deuterated buffer (typically D2O) for various time intervals (seconds to hours).

• Exchange quenching: The exchange is quenched by lowering pH (to ~2.5) and temperature (to 0°C).

• Proteolytic digestion: The protein is digested with an acid-stable protease (typically pepsin).

• LC-MS analysis: Resulting peptides are separated by liquid chromatography and analyzed by mass spectrometry to determine deuterium incorporation levels.

• Data analysis: Specialized software calculates deuterium uptake for each peptide and maps these onto the protein structure.

Applications for atpB Research:

• Nucleotide binding dynamics: HDX-MS can reveal conformational changes in atpB upon ATP, ADP, or Pi binding, providing insight into the catalytic mechanism.

• Subunit interactions: Compare deuterium exchange patterns of isolated atpB versus atpB in complex with other F1 subunits to identify interaction interfaces.

• Redox-induced changes: Given the redox sensitivity of chloroplast ATP synthase , HDX-MS can map conformational changes induced by different redox conditions.

• Allosteric networks: Identify regions of atpB that show altered exchange dynamics upon binding events at distant sites, revealing allosteric communication networks.

• Membrane association effects: Compare exchange patterns of atpB in solution versus membrane-associated environments to understand lipid effects on protein dynamics.

This technique is particularly valuable for studying the dynamic properties of ATP synthase components that undergo significant conformational rearrangements during the catalytic cycle.

What are common challenges in initial expression screening for atpB, and how can they be systematically addressed?

Initial expression screening for chloroplastic proteins like atpB frequently encounters several challenges. Here's a systematic troubleshooting approach:

Challenge 1: Low or no expression

• Potential causes: Poor codon optimization, toxic protein effects, unstable mRNA

• Solutions:

  • Verify plasmid sequence integrity

  • Test multiple expression strains (BL21, Rosetta, C41/C43 for membrane proteins)

  • Reduce expression temperature to 15-18°C

  • Test different media formulations (TB, auto-induction)

  • Confirm protein expression using Western blot with anti-His or anti-atpB antibodies

Challenge 2: Insoluble protein/inclusion bodies

• Potential causes: Rapid expression rate, improper folding, hydrophobic regions

• Solutions:

  • Lower IPTG concentration (0.1-0.5 mM) and induction temperature

  • Add solubility-enhancing additives to growth medium (glycerol, sorbitol)

  • Co-express with molecular chaperones (GroEL/ES, DnaK)

  • Test fusion partners known to enhance solubility (MBP, SUMO)

  • Develop inclusion body isolation and refolding protocols

Challenge 3: Protein degradation

• Potential causes: Protease susceptibility, improper folding

• Solutions:

  • Use protease-deficient strains

  • Include protease inhibitors (as used in the ATP synthase subunit c purification protocol)

  • Optimize harvest timing to capture maximum expression before degradation

  • Maintain low temperatures during all processing steps

Challenge 4: Low yield

• Potential causes: Inefficient transcription/translation, cellular toxicity

• Solutions:

  • Optimize induction parameters (OD600, induction duration)

  • Test different promoter systems

  • Perform fed-batch cultivation to achieve higher cell densities

  • Consider cell-free protein synthesis for toxic proteins

For systematic optimization, employ a design of experiments (DOE) approach to efficiently identify optimal conditions across multiple variables. Document all attempts with standardized protocols and analysis methods to enable meaningful comparisons between conditions.

How can I develop an effective refolding protocol for atpB recovered from inclusion bodies?

When soluble expression strategies fail to yield sufficient quantities of properly folded atpB, developing an inclusion body refolding protocol becomes necessary. Here's a methodical approach:

Step 1: Isolation and purification of inclusion bodies

• Harvest cells and resuspend in lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA)

• Disrupt cells by sonication or mechanical methods

• Collect inclusion bodies by centrifugation (10,000-15,000 × g, 10 minutes)

• Wash inclusion bodies with detergent buffer (lysis buffer + 0.5% Triton X-100) to remove membrane fragments

• Perform additional washes with detergent-free buffer to remove the detergent

Step 2: Solubilization of inclusion bodies

• Dissolve inclusion bodies in denaturing buffer (8 M urea or 6 M guanidine-HCl, 50 mM Tris-HCl pH 8.0, 10 mM DTT)

• Clarify the solution by centrifugation (20,000 × g, 20 minutes)

• If using affinity tags, perform initial purification under denaturing conditions

Step 3: Refolding optimization
Test multiple refolding methods with small-scale screens:

• Dilution method: Rapidly dilute denatured protein into refolding buffer (typically 50-100 fold)

• Dialysis method: Gradually remove denaturant using step-wise or continuous dialysis

• On-column refolding: For His-tagged proteins, immobilize on Ni-NTA resin and gradually decrease denaturant concentration

• Pulse renaturation: Add denatured protein in pulses to refolding buffer

Critical parameters to optimize:

• Protein concentration (typically 0.01-0.1 mg/mL for refolding)

• Temperature (4°C, 10°C, room temperature)

• pH (7.0-8.5 range)

• Additives that promote folding:

  • Glycerol (10-20%)

  • L-arginine (0.5-1 M)

  • Non-detergent sulfobetaines (NDSB-201, 0.5-1 M)

  • Redox couples (GSH/GSSG) at various ratios to facilitate disulfide bond formation/rearrangement, important given the redox sensitivity of ATP synthase components

Step 4: Refolding assessment
Monitor refolding efficiency using:

• Turbidity measurements to detect aggregation

• Circular dichroism to assess secondary structure

• Intrinsic fluorescence to monitor tertiary structure

• Functional assays (ATP binding/hydrolysis)

For ATP synthase components, proper refolding may require the presence of other subunits or specific lipids to recapitulate the native environment. Consider co-refolding with other F1 components or refolding in the presence of liposomes.