Recombinant Oenothera elata subsp. hookeri Cytochrome b559 subunit alpha (psbE)

What is the genomic organization of psbE in Oenothera elata subsp. hookeri?

The psbE gene in Oenothera elata subsp. hookeri is located in the chloroplast genome, not in the mitochondrial genome which has been extensively studied in recent research . The gene encodes the alpha subunit of Cytochrome b559, a critical component of photosystem II. While specific information about psbE organization in O. elata is limited in the available literature, genomic studies would typically involve chloroplast DNA isolation followed by a combination of short-read (Illumina) and long-read (PacBio) sequencing technologies similar to those used in the mitochondrial genome studies of O. elata .

The psbE gene is generally conserved across plant species and is typically found in a gene cluster with psbF, psbL, and psbJ. To determine the precise genomic organization, a standard protocol would include:

• DNA extraction from young O. elata tissue

• PCR amplification using primers designed to target conserved regions

• Sequencing of amplified products

• Comparative analysis with other known plant chloroplast genomes

What expression systems are most suitable for recombinant production of Oenothera elata psbE?

For recombinant production of membrane proteins like Cytochrome b559 subunit alpha, several expression systems can be considered, each with distinct advantages:

For psbE specifically, E. coli expression with specialized membrane protein vectors is often preferred, utilizing fusion partners (MBP, SUMO) to enhance solubility and membrane targeting. The protein can then be extracted using mild detergents like n-dodecyl-β-D-maltoside (DDM) that maintain structural integrity.

How can proper heme incorporation be verified in recombinant psbE protein?

Verifying proper heme incorporation into recombinant Cytochrome b559 is critical for ensuring functional protein production:

• UV-visible spectroscopy:

  • The correctly incorporated heme shows characteristic absorption peaks at approximately 559 nm in the reduced state

  • The ratio of Soret band (~410-420 nm) to protein absorbance (280 nm) indicates the degree of heme incorporation

  • Reduced minus oxidized difference spectra should show typical cytochrome features

• Quantitative analysis:

  • The extinction coefficient of heme-containing Cytochrome b559 can be used to calculate the percentage of heme incorporation

  • Pyridine hemochrome assay can provide absolute quantification of heme content

• Functional verification:

  • Redox potential measurements using potentiometric titrations

  • Electron paramagnetic resonance (EPR) spectroscopy to determine the heme coordination environment

What purification strategy yields the highest activity for recombinant psbE?

A systematic purification approach is essential for obtaining functionally active recombinant psbE:

• Cell lysis and membrane isolation:

  • Mechanical disruption (sonication or French press) of bacterial cells

  • Isolation of membrane fraction by ultracentrifugation (100,000 × g, 1 hour)

• Solubilization optimization:

  • Screening multiple detergents (DDM, digitonin, LMNG) at various concentrations

  • Typical conditions: 1% detergent, 4°C, gentle agitation for 1-2 hours

• Multi-step chromatography:

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

  • Ion exchange chromatography to remove contaminants

  • Size exclusion chromatography for final polishing and detergent exchange

• Quality control at each step:

  • SDS-PAGE and Western blot to track purity

  • Spectroscopic analysis to monitor heme retention

  • Activity assays to ensure functional preservation

What are the optimal storage conditions for maintaining stability of purified recombinant psbE?

Long-term stability of purified Cytochrome b559 requires careful consideration of storage conditions:

• Buffer composition:

  • 25-50 mM phosphate or Tris buffer, pH 7.0-7.5

  • 100-150 mM NaCl to maintain ionic strength

  • 0.02-0.05% detergent (below critical micelle concentration)

  • 10% glycerol as cryoprotectant

• Temperature conditions:

  • Short-term (1-2 weeks): 4°C with minimal freeze-thaw cycles

  • Medium-term (1-2 months): -20°C in small aliquots

  • Long-term (>2 months): -80°C with flash freezing in liquid nitrogen

• Additives for enhanced stability:

  • Reducing agents (2-5 mM DTT or β-mercaptoethanol) to prevent oxidative damage

  • Protease inhibitors to prevent degradation

  • Specific lipids (0.01-0.02% MGDG or DGDG) to mimic native environment

• Alternative approaches:

  • Lyophilization with appropriate excipients

  • Immobilization on solid supports

  • Storage in nanodiscs or liposomes for enhanced stability

Stability monitoring should be performed periodically using spectroscopic methods to assess heme retention and functional assays to verify activity maintenance.

How do mutations in the psbE gene affect electron transport in photosystem II?

Targeted mutations in the psbE gene can provide valuable insights into the role of Cytochrome b559 in photosystem II electron transport:

• Critical residues for functional analysis:

  • Histidine residues involved in heme coordination (typically His23 and His24)

  • Residues at the interface with other PSII subunits

  • Amino acids in putative proton channels or electron transfer pathways

• Functional consequences of mutations:

• Experimental approaches:

  • Site-directed mutagenesis followed by recombinant expression

  • Spectroelectrochemical analysis to measure altered redox potentials

  • Reconstitution with other PSII components to assess assembly

  • Oxygen evolution and chlorophyll fluorescence measurements under photoinhibitory conditions

• Mechanistic insights:

  • High-potential to low-potential conversion mechanisms

  • Relationship between structure and redox properties

  • Role in photoprotection and secondary electron transfer pathways

Recent research has shown that the redox potential of Cytochrome b559 can exist in multiple forms (high, intermediate, and low potential), and mutations can shift the equilibrium between these forms, providing insights into its regulatory function in photosystem II.

What approaches are most effective for reconstituting recombinant psbE with other photosystem II components?

Reconstitution of recombinant psbE with other photosystem II components requires careful methodological considerations:

• Stepwise vs. co-expression approaches:

  • Stepwise: Individual expression and purification of components followed by controlled assembly

  • Co-expression: Simultaneous production of multiple components to facilitate natural assembly

  • Hybrid: Co-expression of selected components followed by addition of others in vitro

• Membrane mimetic selection:

• Assembly monitoring techniques:

  • Blue native PAGE to visualize complex formation

  • Förster resonance energy transfer (FRET) between labeled components

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Single-particle cryo-EM to capture assembly intermediates

• Functional verification:

  • Oxygen evolution measurements

  • Electron transfer kinetics using flash photolysis

  • Thermoluminescence to assess charge recombination pathways

  • EPR spectroscopy to monitor formation of specific radical species

The most successful reconstitution strategies typically involve co-expression of psbE with its natural partner psbF (forming Cytochrome b559) followed by stepwise addition of other components in carefully optimized detergent-lipid mixtures.

How does the recombinogenic nature of the Oenothera elata genome influence expression and analysis of recombinant psbE?

The highly recombinogenic nature of the Oenothera elata genome, particularly evident in its mitochondrial genome , presents both challenges and opportunities for psbE research:

• Genetic diversity considerations:

  • O. elata shows significant recombinogenic repeat pairs (RRPs) in its mitochondrial genome

  • While the chloroplast genome (containing psbE) is generally more stable, similar recombination events may influence gene organization

  • Multiple variants of psbE may exist within a single population, requiring careful genetic characterization

• Expression strategy adaptations:

  • Codon optimization based on O. elata-specific usage patterns

  • Potential need to test multiple genetic variants to identify optimal expression constructs

  • Consideration of species-specific regulatory elements

• Sequence verification importance:

  • Thorough sequencing of expression constructs to confirm exact sequence

  • Population-level sampling to identify potential natural variants

  • Comparison with closely related species (O. biennis, O. villaricae) to identify conserved regions

• Evolutionary insights:

  • Comparative analysis of psbE across Oenothera species can reveal selective pressures

  • Understanding of species-specific adaptations in photosystem II

  • Correlation between environmental adaptations of O. elata (e.g., growth in open slopes, moist lowlands) and psbE structure

The stoichiometric analysis techniques developed for studying the mitochondrial genome of O. elata could potentially be adapted to analyze chloroplast genome variations, providing insights into psbE evolution and diversity within this species.

What isotope labeling strategies are most informative for structural studies of recombinant psbE?

Isotope labeling of recombinant psbE enables sophisticated structural analyses using various biophysical techniques:

• NMR spectroscopy applications:

• Mass spectrometry enhancements:

  • Hydrogen-deuterium exchange (HDX) to map solvent-accessible regions

  • Cross-linking MS with isotope-coded linkers for spatial constraints

  • Native MS with differentially labeled subunits to determine stoichiometry

• Neutron scattering applications:

  • Contrast variation with selectively deuterated components

  • Small-angle neutron scattering (SANS) to position subunits within complexes

  • Neutron crystallography to locate protons in proton transfer pathways

• X-ray crystallography strategies:

  • Selenomethionine labeling for phase determination

  • Heavy atom derivatives for isomorphous replacement

  • Multi-wavelength anomalous dispersion (MAD) phasing

For membrane proteins like psbE, combinatorial approaches are particularly powerful. For example, selective methyl labeling in a deuterated background can provide critical distance constraints through NMR, while parallel HDX-MS experiments can map conformational changes upon complex formation with other photosystem II components.

How can recombinant psbE be used to study the high-potential to low-potential transition of Cytochrome b559?

The ability of Cytochrome b559 to exist in multiple redox potential forms (high, intermediate, and low potential) is crucial for its proposed photoprotective function in photosystem II:

• Structural determinants of redox potential:

  • Axial ligand identity and coordination geometry

  • Heme microenvironment hydrophobicity

  • Protein conformational states

  • Lipid interactions and membrane positioning

• Experimental approaches to study potential transitions:

• Recombinant psbE modifications for mechanistic insights:

  • Site-directed mutagenesis of heme pocket residues

  • Incorporation of non-natural amino acids as spectroscopic probes

  • Creation of chimeric proteins with segments from high/low potential variants

  • Systematic lipid composition studies using nanodiscs with defined lipid mixtures

• Correlation with functional states:

  • Photoprotection efficiency under various conditions

  • Alternative electron transfer pathways

  • Interaction with extrinsic proteins under stress conditions

Recent hypotheses suggest that the redox potential transitions of Cytochrome b559 may serve as a "molecular fuse" to protect photosystem II from photodamage. Recombinant psbE provides a controlled system to test these hypotheses through systematic structural and functional modifications.