Recombinant Morus indica Photosystem II reaction center protein Z (psbZ)

What is the amino acid composition and primary structure of Morus indica psbZ?

Morus indica psbZ is a 62-amino acid protein with the sequence: MTIAFQLAVFALIATSSILLISVPVVFASPDGWLGNKNVVFSGTSLWITLVFLVGILNSL IS. The protein is classified as a Photosystem II reaction center protein with synonyms including PSII-Z and MoinCp018. The recombinant form is typically produced with an N-terminal His-tag expressed in E. coli systems .

What are the physicochemical properties of recombinant psbZ that researchers should consider in experimental design?

The recombinant psbZ protein exhibits hydrophilic properties, which is consistent with observations from other Morus proteins that typically have negative GRAVY values (ranging from -1003 to -0.099 for related proteins) . When handling the recombinant protein, researchers should note that it is typically provided as a lyophilized powder with >90% purity as determined by SDS-PAGE. The protein requires proper storage conditions to maintain stability, including storage at -20°C/-80°C with recommendations against repeated freeze-thaw cycles .

What protocol should researchers follow for reconstitution and storage of recombinant psbZ protein?

For optimal results, researchers should:

• Briefly centrifuge the vial prior to opening

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

• Add glycerol to a final concentration of 5-50% (with 50% being the standard recommendation)

• Aliquot the solution to avoid repeated freeze-thaw cycles

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

These conditions are critical for maintaining protein integrity, as indicated in the product specifications .

What validation techniques effectively confirm the identity and functional integrity of recombinant psbZ?

Based on methodologies used with similar photosystem proteins, a multi-tier validation approach is recommended:

• Western blotting using specific antibodies – as demonstrated with other photosystem proteins in mulberry species, immunoblotting provides confirmation of protein identity and relative abundance that correlates well with proteomic findings

• TMT (Tandem Mass Tag) proteomics analysis for precise quantification

• Functional assays examining electron transport capabilities

• Spectroscopic analysis to assess proper folding and cofactor binding

These approaches provide complementary data to ensure both the molecular identity and functional integrity of the recombinant protein .

What role does psbZ play in PSII assembly and stability across different environmental conditions?

While the search results don't directly address psbZ's role in assembly, research on related PSII proteins suggests that reaction center proteins like psbZ likely contribute to maintaining PSII structural integrity, particularly under stress conditions. Researchers have observed that certain PSII proteins increase in abundance under stress, potentially as a repair mechanism for the PSII system . Comparative studies between normal and stressed conditions could elucidate psbZ's specific contribution to PSII resilience.

How does psbZ expression and function change under various abiotic stress conditions?

Though the search results don't specifically detail psbZ regulation under stress, the response pattern of other PSII proteins provides valuable insights. Under stress conditions, proteins like CP43 (Photosystem II CP43 chlorophyll apoprotein) and CP47 (photosystem II 47kDa protein) show increased abundance in mulberry plants . This upregulation is hypothesized to play a role in repairing the PSII system under stress conditions.

Research methodologies to investigate psbZ-specific responses would include:

• Proteomics analysis comparing psbZ levels across stress treatments

• Transcriptome analysis to assess transcript-level regulation

• Correlation with physiological parameters (e.g., photosynthetic efficiency, ROS production)

What methodological approaches best capture the dynamics of psbZ regulation during environmental challenges?

Based on approaches used with other photosystem proteins, researchers should implement:

• Time-course experiments capturing both early and late responses

• Combined transcriptomic and proteomic analyses to distinguish transcriptional and post-transcriptional regulation

• Western blot validation of expression changes at key timepoints

• Parallel physiological measurements to correlate molecular changes with plant performance

• Comparison across different stress types (drought, high light, temperature extremes, etc.)

This multi-faceted approach would provide comprehensive insights into psbZ's role in stress adaptation .

What bioinformatic approaches are most effective for comparative analysis of psbZ across Morus species?

Drawing from methodologies used in related studies of Morus proteins, researchers should consider:

• HMMER searches utilizing hidden Markov models (HMM) of conserved domains from the Pfam database (e.g., PF00722 and PF06955 for related proteins)

• BLASTP searches against known psbZ sequences

• Filtering sequences with E-values < 1 and removing short open reading frames

• Domain verification using Pfam and CDD databases

• Phylogenetic analysis to establish evolutionary relationships

These approaches enable comprehensive identification and comparative analysis of homologous proteins across species .

How does sequence conservation of psbZ correlate with functional conservation across plant species?

To address this question, researchers should:

• Perform multiple sequence alignments of psbZ from diverse plant species

• Identify highly conserved regions likely representing functionally critical domains

• Map conservation patterns onto structural models

• Correlate sequence divergence with taxonomic distance and photosynthetic adaptation

• Consider performing site-directed mutagenesis of conserved residues to test functional importance

This approach would reveal whether sequence conservation translates to functional conservation across evolutionary distances.

How can researchers utilize recombinant psbZ in reconstitution experiments to understand PSII assembly?

Advanced reconstitution experiments might include:

• In vitro reconstitution of partial PSII complexes with and without psbZ

• Assessment of complex stability through biophysical techniques

• Analysis of electron transport efficiency in reconstituted systems

• Structural analysis of reconstituted complexes through cryo-electron microscopy

• Investigation of protein-protein interactions using techniques like crosslinking mass spectrometry

These approaches would provide mechanistic insights into psbZ's role in PSII assembly and function.

What strategies can effectively combine genomic editing with proteomic analysis to understand psbZ function?

Researchers should consider implementing:

• CRISPR-Cas9 editing of the psbZ gene in model systems

• Whole-proteome analysis of wild-type versus psbZ-edited plants

• Targeted analysis of the PSII complex composition using BN-PAGE followed by mass spectrometry

• Correlation of molecular changes with photosynthetic parameters

• Transcriptome analysis to identify compensatory responses

This integrated approach would reveal both direct and indirect consequences of psbZ modification.

What methods effectively characterize psbZ interactions with other PSII components?

To thoroughly investigate protein-protein interactions involving psbZ, researchers should employ:

• Co-immunoprecipitation with psbZ-specific antibodies

• Yeast two-hybrid screening for potential interaction partners

• Bimolecular fluorescence complementation for in vivo validation

• Crosslinking mass spectrometry to identify interaction interfaces

• Computational modeling based on available structural data

These complementary approaches would provide a comprehensive map of psbZ's interaction network within PSII.

How do post-translational modifications influence psbZ function and interactions?

While the search results don't specifically address post-translational modifications of psbZ, researchers investigating this question should:

• Perform mass spectrometry analysis of native psbZ to identify modifications

• Compare modification patterns across different physiological conditions

• Generate site-directed mutants mimicking or preventing modification

• Assess functional consequences through activity assays

• Examine impact on protein-protein interactions

This systematic approach would reveal the regulatory role of post-translational modifications on psbZ function.

What are the common technical challenges when working with recombinant psbZ protein?

Researchers commonly encounter several challenges when working with recombinant psbZ:

• Protein stability issues: The hydrophobic nature of membrane proteins like psbZ can lead to aggregation or misfolding

• Activity preservation: Maintaining functional integrity during purification and storage

• Buffer optimization: Finding conditions that mimic the native membrane environment

• Reconstitution challenges: Ensuring proper folding after lyophilization

To address these challenges, researchers should strictly adhere to recommended storage conditions (avoiding repeated freeze-thaw cycles) and use the suggested reconstitution protocol with appropriate buffer conditions (Tris/PBS-based buffer, 6% Trehalose, pH 8.0) .

What experimental controls are essential when investigating psbZ function in photosynthetic systems?

Critical controls include:

• Protein-level controls:

  • Denatured psbZ preparations to control for non-specific effects

  • Related but functionally distinct photosystem proteins

  • Concentration-matched BSA or other inert proteins

• System-level controls:

  • Wild-type systems with normal psbZ expression

  • Systems with known PSII deficiencies affecting different components

  • Measurements under varying light conditions to distinguish light-dependent effects

These controls ensure that observed effects are specifically attributable to psbZ function.