Recombinant Atropa belladonna Photosystem II reaction center protein Z (psbZ)

What is Photosystem II reaction center protein Z (psbZ) and what is its role in Atropa belladonna?

Photosystem II reaction center protein Z (psbZ) is a low molecular weight protein component of the photosynthetic machinery in Atropa belladonna (deadly nightshade). This 62-amino acid protein (UniProt ID: P59706) is encoded by the chloroplast genome and plays a structural role in the organization of the photosystem II (PSII) complex. In A. belladonna, psbZ contributes to the stability of the PSII-light harvesting complex II (LHCII) supercomplexes and influences the phosphorylation status of PSII core proteins, thereby affecting photosynthetic efficiency under varying light conditions .

How does the amino acid sequence of A. belladonna psbZ compare with other plant species?

The A. belladonna psbZ protein consists of 62 amino acids with the sequence: MTLAFQLAVFALIATSLILLISVPVVFASPDGWSSNKNVVFSGTSLWIGLVFLVGILNSLIS . When compared to Magnolia tripetala psbZ (sequence: MTIAFQLAVFALIATSSILLISVPVVFASSDGWSSNKNVVFSGTSLWIGLVFLVAILNSLIS) , we observe high conservation with minimal differences. These variations typically occur in the transmembrane regions and may reflect evolutionary adaptations to different environmental conditions. The high degree of sequence conservation suggests the critical functional importance of psbZ across diverse plant lineages.

What expression systems are commonly used for recombinant production of A. belladonna psbZ?

Escherichia coli is the predominant expression system for recombinant A. belladonna psbZ production. This bacterial expression system offers several advantages for membrane protein production, including rapid growth, high protein yields, and established protocols for protein purification. For psbZ specifically, E. coli-based expression systems have been optimized to produce the full-length protein (amino acids 1-62) with an N-terminal His-tag to facilitate purification . Alternative expression systems such as yeast or insect cells may be considered when proper folding or post-translational modifications are critical research considerations.

What is the recommended protocol for reconstituting lyophilized recombinant A. belladonna psbZ?

For optimal reconstitution of lyophilized recombinant A. belladonna psbZ:

• Centrifuge the vial briefly (30 seconds at 10,000 × g) to collect the powder at the bottom before opening.

• Reconstitute the protein in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL.

• For long-term storage, add glycerol to a final concentration of 5-50% (typically 50% is recommended) and aliquot into smaller volumes to minimize freeze-thaw cycles.

• Store working aliquots at 4°C for up to one week and long-term storage at -20°C/-80°C .

This methodology preserves protein stability and biological activity by preventing protein aggregation and degradation that commonly occurs with membrane proteins during freeze-thaw cycles.

What purification strategies are most effective for isolating recombinant His-tagged A. belladonna psbZ?

The most effective purification strategy for His-tagged A. belladonna psbZ involves:

• Cell Lysis: Use sonication or pressure-based disruption in a buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and detergent (typically 1% n-dodecyl β-D-maltoside) to solubilize membrane proteins.

• Immobilized Metal Affinity Chromatography (IMAC): Apply the clarified lysate to a Ni-NTA column equilibrated with buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% detergent.

• Washing: Remove non-specifically bound proteins with increasing imidazole concentrations (10-40 mM).

• Elution: Collect the purified protein using buffer containing 250-300 mM imidazole.

• Size Exclusion Chromatography: Further purify the protein based on size to remove aggregates and contaminants.

This multi-step approach typically yields protein purity greater than 90% as determined by SDS-PAGE . During the entire purification process, maintaining the protein in a detergent-containing buffer is critical to prevent aggregation of this hydrophobic membrane protein.

What are the optimal conditions for functional assays involving recombinant A. belladonna psbZ?

Functional assays for recombinant A. belladonna psbZ should be conducted under conditions that mimic its native environment:

• Buffer Composition: Use 20 mM MES-NaOH (pH 6.5), 10 mM NaCl, 5 mM MgCl₂, and 0.03% n-dodecyl β-D-maltoside to maintain protein stability while preserving native-like membrane conditions.

• Temperature: Conduct assays at 25°C, which represents the optimal growth temperature for A. belladonna.

• Light Conditions: When measuring photosynthetic activity, use red light (650-680 nm) at moderate intensities (100-500 μmol photons m⁻² s⁻¹) to specifically excite PSII without causing photoinhibition.

• Electron Transport Measurements: Include appropriate electron donors (water/artificial donors) and acceptors (typically quinones) in the reaction mixture.

• Protein Reconstitution: For comprehensive functional studies, reconstitute the purified protein into liposomes with a lipid composition mimicking the thylakoid membrane (MGDG, DGDG, SQDG, and PG in ratios of 8:3:2:1).

These optimized conditions ensure that the recombinant protein maintains structural integrity and functional activity comparable to its native state within the thylakoid membrane.

How can recombinant A. belladonna psbZ be used to study interactions with other PSII subunits?

To study interactions between recombinant A. belladonna psbZ and other PSII subunits, researchers should implement a multi-faceted approach:

• Co-immunoprecipitation: Utilize the His-tag on recombinant psbZ to pull down interacting partners from thylakoid membrane preparations or reconstituted systems. The precipitated complexes can be analyzed by mass spectrometry to identify specific interaction partners.

• Microscale Thermophoresis (MST): This technique measures protein-protein interactions by detecting changes in the movement of fluorescently labeled molecules in a temperature gradient. For psbZ studies, label the recombinant protein with a fluorescent dye and titrate with potential binding partners to determine binding affinities.

• Chemical Cross-linking Coupled with Mass Spectrometry: Apply chemical cross-linkers to stabilize transient interactions between psbZ and other PSII components, followed by digestion and mass spectrometric analysis to map specific interaction sites.

• Surface Plasmon Resonance (SPR): Immobilize His-tagged psbZ on a sensor chip and measure real-time binding kinetics with other PSII subunits to determine association and dissociation constants.

• Reconstitution Experiments: Co-reconstitute psbZ with other recombinant PSII subunits in liposomes and assess the functional properties of the reconstituted complex compared to systems lacking psbZ.

These methods provide complementary information about the structural and functional relationships between psbZ and other components of the photosynthetic machinery, illuminating its role in PSII assembly and function.

What strategies can be employed to investigate the role of psbZ in photosynthetic efficiency under stress conditions?

Investigating psbZ's role in photosynthetic efficiency under stress conditions requires a comprehensive experimental design:

• Comparative Studies with Mutant Systems:

  • Generate A. belladonna lines with modified psbZ using CRISPR/Cas9 technology similar to the methodology employed for H6H gene editing

  • Compare photosynthetic parameters between wild-type and mutant plants under various stress conditions

• Reconstitution Experiments with Stress Factors:

  • Reconstitute proteoliposomes containing recombinant psbZ under different stress conditions (high light, temperature extremes, salt stress)

  • Measure electron transport rates, oxygen evolution, and fluorescence parameters

• Site-Directed Mutagenesis:

  • Create specific mutations in the recombinant psbZ protein based on sequence analysis and structural predictions

  • Assess how these mutations affect protein stability and function under stress conditions

• Protein-Lipid Interaction Analysis:

  • Examine how psbZ interacts with thylakoid membrane lipids under stress conditions

  • Determine if altered lipid compositions affect psbZ stability or function

• Time-Resolved Spectroscopy:

  • Monitor energy transfer and electron transport kinetics in systems with wild-type versus modified psbZ

  • Identify rate-limiting steps in photosynthetic electron transport under stress conditions

This multi-level approach provides mechanistic insights into how psbZ contributes to stress tolerance and photosynthetic resilience in A. belladonna.

How does recombinant psbZ from A. belladonna compare to psbZ from other medicinal plants in terms of structure-function relationships?

Structure-function comparisons between A. belladonna psbZ and psbZ from other medicinal plants reveal important evolutionary adaptations:

To investigate these structure-function relationships:

• Homology Modeling and Molecular Dynamics: Generate structural models of psbZ from different species and simulate their behavior in membrane environments.

• Functional Complementation: Express psbZ from different medicinal plants in A. belladonna psbZ knockout lines to assess functional equivalence.

• Spectroscopic Analysis: Compare the spectroscopic properties of PSII complexes containing psbZ variants to identify differences in energy transfer efficiency.

• Cross-Species Reconstitution: Reconstitute psbZ from different species with core PSII components to measure differences in complex assembly and stability.

These comparative studies provide insights into how structural variations in psbZ contribute to species-specific adaptations in photosynthetic efficiency across medicinal plants.

What are common challenges in purifying recombinant A. belladonna psbZ and how can they be addressed?

Researchers frequently encounter several challenges when purifying recombinant A. belladonna psbZ:

• Poor Solubility: Being a membrane protein, psbZ has limited solubility in aqueous solutions.

  • Solution: Use appropriate detergents (n-dodecyl β-D-maltoside or digitonin) at concentrations above their critical micelle concentration. Alternatively, employ solubilization tags like SUMO or MBP.

• Low Expression Levels: Membrane proteins often express poorly in heterologous systems.

  • Solution: Optimize expression conditions (temperature, induction parameters), use specialized E. coli strains (C41/C43), or codon-optimize the gene sequence for the expression host.

• Protein Aggregation: psbZ tends to aggregate during purification.

  • Solution: Maintain detergent above CMC throughout all purification steps, avoid freeze-thaw cycles, and consider adding glycerol (5-10%) to stabilize the protein.

• Contaminating Proteins: E. coli proteins may co-purify with His-tagged psbZ.

  • Solution: Implement a two-step purification strategy combining IMAC with size exclusion chromatography or ion exchange chromatography.

• Proteolytic Degradation: Small membrane proteins are susceptible to proteolysis.

  • Solution: Include protease inhibitors in all buffers, work at 4°C, and minimize purification duration.

Addressing these challenges requires systematic optimization of expression and purification protocols tailored to the specific properties of A. belladonna psbZ.

How can researchers assess the quality and functionality of purified recombinant A. belladonna psbZ?

Comprehensive quality assessment of purified recombinant A. belladonna psbZ should include:

• Purity Assessment:

  • SDS-PAGE analysis should demonstrate >90% purity

  • Western blotting with anti-His antibodies confirms identity

  • Mass spectrometry verifies the correct sequence and potential modifications

• Structural Integrity:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Thermal shift assays to assess protein stability

  • Size exclusion chromatography to evaluate monodispersity

• Functional Verification:

  • Reconstitution into liposomes followed by electron transport measurements

  • Binding assays with known interaction partners

  • Fluorescence quenching experiments to assess pigment-protein interactions

• Activity Monitoring:

  • Measure the ability to enhance PSII assembly when added to membrane preparations

  • Assess impact on oxygen evolution rates in reconstituted systems

  • Monitor phosphorylation patterns of PSII core proteins in the presence of psbZ

A comprehensive quality assessment using these methods ensures that the purified protein maintains native-like properties and is suitable for downstream structural and functional studies.

What statistical approaches are most appropriate for analyzing data from experiments involving recombinant A. belladonna psbZ?

When analyzing experimental data involving recombinant A. belladonna psbZ, the following statistical approaches are recommended:

• For Binding and Interaction Studies:

  • Non-linear regression analysis to fit binding data to appropriate models (one-site binding, cooperative binding)

  • Scatchard or Hill plots to determine binding cooperativity

  • Statistical comparison of dissociation constants (Kd) using t-tests or ANOVA when comparing multiple conditions

• For Functional Assays:

  • Repeated measures ANOVA to assess differences in activity under varying conditions

  • Multivariate analysis to evaluate the relationships between multiple parameters (e.g., light intensity, temperature, pH) and functional output

  • Non-parametric tests (Mann-Whitney U) when data do not meet assumptions of normality

• For Structural Studies:

  • Cluster analysis to identify structural similarities or differences

  • Principal component analysis to reduce dimensionality of complex structural data

  • Bootstrapping to estimate confidence intervals for structural parameters

• For Time-Course Experiments:

  • Time series analysis to identify trends and cyclical patterns

  • Mixed-effects models to account for both fixed and random effects in longitudinal data

  • Survival analysis techniques for protein stability studies

• Data Visualization:

  • Heat maps for displaying interaction matrices

  • Hierarchical clustering for grouping similar experimental conditions

  • Box plots and violin plots for comparing distributions across experimental groups

Proper statistical analysis ensures reliable interpretation of experimental results and facilitates comparison with published literature on photosystem proteins.

How might CRISPR/Cas9 technology be applied to study psbZ function in A. belladonna?

CRISPR/Cas9 technology offers powerful approaches to elucidate psbZ function in A. belladonna:

The methodological approach would follow protocols similar to those used for AbH6H gene editing in A. belladonna , with appropriate modifications to target the psbZ gene in the chloroplast genome.

What emerging technologies might enhance our understanding of A. belladonna psbZ structure and function?

Several emerging technologies promise to advance our understanding of A. belladonna psbZ:

• Cryo-Electron Microscopy (cryo-EM): This technology now permits structural determination of membrane protein complexes at near-atomic resolution. Application to PSII complexes containing psbZ would reveal precise structural arrangements and interaction interfaces.

• Single-Molecule Fluorescence Resonance Energy Transfer (smFRET): This technique can monitor conformational changes in psbZ under different conditions in real-time, providing insights into dynamic aspects of its function.

• Native Mass Spectrometry: Advances in native MS now enable analysis of intact membrane protein complexes, potentially revealing the stoichiometry and stability of psbZ-containing assemblies.

• Artificial Intelligence for Protein Structure Prediction: Tools like AlphaFold2 can generate highly accurate structural models of psbZ and its interactions with other PSII components, guiding experimental design.

• Nanodiscs Technology: This system provides a more native-like membrane environment for reconstitution studies, potentially revealing functional aspects masked in detergent-solubilized systems.

• Time-Resolved X-ray Free Electron Laser (XFEL) Crystallography: This emerging technique allows visualization of ultrafast structural changes during photosynthetic electron transport, potentially revealing the dynamic role of psbZ during photosynthesis.

Integration of these technologies into A. belladonna psbZ research would provide unprecedented insights into its structural dynamics and functional significance within the photosynthetic apparatus.