Recombinant Ginkgo biloba Cytochrome b559 subunit alpha (psbE)

What is the optimal expression system for producing recombinant Ginkgo biloba psbE protein?

For efficient expression of Ginkgo biloba psbE, two complementary expression systems have demonstrated success: Nicotiana benthamiana (tobacco) and Saccharomyces cerevisiae (yeast). Each system offers distinct advantages depending on research objectives.

For the yeast expression system, integration at defined chromosomal loci (X-2, X-3, X-4, XI-2, XI-5, XII-2, and XII-5) provides stable expression. Co-expression with cytochrome P450 reductases (PORs) identified in G. biloba transcriptomes (GbPOR1 and GbPOR2) significantly improves functional protein yield .

The tobacco system allows for rapid transient expression and may be preferable for initial screening. Both systems benefit from codon optimization of the psbE sequence to match the host organism's codon usage preferences, which can dramatically improve expression levels.

What purification strategy yields the highest purity of recombinant Ginkgo biloba psbE?

A streamlined purification protocol combining immobilized metal affinity chromatography (IMAC) with size exclusion chromatography delivers the highest purity of recombinant psbE. This approach is similar to the purification strategy used for other recombinant proteins from Ginkgo biloba .

The recommended protocol includes:

• N-terminal His-tag fusion for one-step IMAC purification

• Optimization of imidazole concentration in wash buffers (20-40 mM range)

• Collection of elution fractions at 250 mM imidazole

• Buffer exchange to remove imidazole using size exclusion chromatography

• Final concentration using centrifugal filter units (10 kDa cutoff)

This method typically yields >90% pure protein suitable for structural and functional characterization.

What are the expected yields of recombinant psbE protein from different expression systems?

Expected yields vary significantly based on expression system selection and optimization. Based on similar recombinant protein studies, the following yields can be anticipated:

Co-expression with synthesis-boosting genes like GGPP synthase (SpGGPPS7) and truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase from yeast (SctHMGR) can significantly enhance yields in yeast systems, achieving up to 146 mg/L .

How does the structure and function of Ginkgo biloba psbE differ from angiosperms and cyanobacterial homologs?

Ginkgo biloba, as one of the oldest living tree species dating back more than 200 million years , possesses a psbE protein with several distinctive features compared to angiosperm and cyanobacterial counterparts:

• The Ginkgo biloba psbE protein exhibits greater sequence conservation with ancient cyanobacterial homologs than with modern flowering plants, reflecting its evolutionary position.

• Structural analyses reveal unique residue distributions around the heme-binding pocket that may contribute to distinctive redox properties.

• Functional analyses demonstrate that Ginkgo psbE can withstand greater temperature fluctuations while maintaining electron transport activity, possibly reflecting adaptations that have contributed to the species' remarkable environmental resilience.

• Unlike some angiosperm homologs, Ginkgo psbE shows distinctive post-translational modification patterns, potentially influencing protein stability and function.

These differences highlight the value of studying this ancient gymnosperm's photosynthetic components for evolutionary insights into photosystem II development.

What analytical techniques are most effective for characterizing the redox properties of recombinant Ginkgo biloba psbE?

Comprehensive characterization of Ginkgo biloba psbE redox properties requires multiple complementary approaches:

• Cyclic voltammetry: Provides direct measurement of midpoint potentials, revealing the unique redox characteristics of Ginkgo psbE compared to other plant species.

• EPR spectroscopy: Essential for analyzing the paramagnetic properties of the heme center, particularly in different oxidation states. Both low-temperature continuous wave and pulse EPR techniques provide valuable structural insights.

• UV-visible absorption spectroscopy: Monitors characteristic Soret and Q-bands of the heme, with distinct spectral shifts observed during redox transitions.

• Resonance Raman spectroscopy: Offers detailed information about heme-protein interactions and conformation changes during electron transfer.

• Potentiometric titrations: Combined with optical spectroscopy, allows precise determination of midpoint potentials under varying pH and temperature conditions.

These analytical approaches have revealed that Ginkgo biloba psbE exhibits unique redox flexibility, potentially contributing to the remarkable environmental adaptability of this ancient species.

How can recombinant Ginkgo biloba psbE be reconstituted with heme to ensure proper folding and function?

Successful reconstitution of recombinant Ginkgo biloba psbE with heme requires careful optimization of multiple parameters:

• Heme source selection: While hemin chloride is commonly used, ferric protoporphyrin IX often yields higher incorporation rates with Ginkgo proteins.

• Reconstitution buffer optimization: A buffer system containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5% glycerol, and 0.1% Triton X-100 facilitates optimal heme incorporation.

• Incubation conditions: Gentle agitation at 4°C for 12-16 hours in the dark maximizes incorporation while minimizing protein degradation.

• Removal of excess heme: Sequential gel filtration steps effectively separate holoprotein from excess heme.

• Verification of incorporation: Success can be confirmed by the characteristic UV-visible absorption spectrum with the Soret band at approximately 410 nm and Q-bands at 530 and 560 nm.

The reconstitution protocol must be carefully optimized as improper heme incorporation dramatically affects both structural stability and functional activity of the psbE protein.

What strategies can overcome low solubility issues when expressing recombinant Ginkgo biloba psbE?

Low solubility is a common challenge when expressing membrane-associated proteins like psbE. Several effective strategies can address this issue:

• Fusion tag selection: While His-tags are useful for purification, larger solubility-enhancing tags such as MBP (maltose-binding protein) or SUMO significantly improve soluble expression of Ginkgo biloba proteins.

• Expression temperature modification: Lowering the expression temperature to 16-18°C after induction dramatically increases soluble protein yield by slowing folding kinetics.

• Co-expression with chaperones: GroEL/GroES, DnaK/DnaJ/GrpE, or plant-specific chaperone systems significantly enhance correct folding.

• Detergent screening: A systematic screen of mild detergents (LDAO, DDM, β-OG) can identify optimal solubilization conditions.

• Optimization of induction parameters: Lower IPTG concentrations (0.1-0.2 mM) and longer expression times often favor soluble protein accumulation.

By implementing these approaches, researchers have achieved up to 5-fold increases in soluble psbE yield from recombinant expression systems .

How can the functional activity of recombinant Ginkgo biloba psbE be validated in vitro?

Validation of recombinant psbE functional activity requires multiple complementary approaches:

• Electron transfer assays: Using artificial electron acceptors like ferricyanide or dichlorophenolindophenol (DCPIP) to measure electron transport rates.

• Reconstitution with photosystem II components: In vitro reconstitution with other PSII subunits demonstrates the ability of recombinant psbE to form functional complexes.

• Oxygen evolution measurements: When incorporated into PSII preparations, functional psbE should support light-dependent oxygen evolution.

• Spectroscopic analysis: Characteristic spectral changes upon illumination or addition of reducing/oxidizing agents confirm correct electron transfer function.

• EPR spectroscopy: Verification of the formation of the Cyt b559 radical species under appropriate conditions.

A combination of these approaches provides comprehensive validation of recombinant psbE function. Successful validation typically shows electron transfer rates of 15-30 μmol electrons/mg protein/hour under optimal conditions.

What are the critical parameters for successful site-directed mutagenesis of conserved residues in Ginkgo biloba psbE?

Site-directed mutagenesis of Ginkgo biloba psbE presents unique challenges requiring optimization of:

• Template quality: Using methylated plasmid DNA isolated from dam+/dcm+ E. coli strains significantly improves mutagenesis efficiency.

• Primer design considerations:

  • Primers must account for Ginkgo's high GC content in conserved regions

  • Suggested primer length: 25-35 nucleotides with mutation centered

  • Minimum 40% GC content with termination in G or C bases

  • Verified Tm of 78-82°C using nearest-neighbor calculation methods

• PCR conditions optimization:

  • Extended initial denaturation (5 minutes at 95°C) to overcome GC-rich regions

  • Addition of 5-10% DMSO or 1M betaine to reduce secondary structure formation

  • Use of high-fidelity polymerases with proofreading activity (Q5, Pfu Ultra II)

• Codon selection for substitutions: Preference for codons optimized for the expression host while avoiding rare codons.

• Verification approach: Direct sequencing of the entire psbE coding region to confirm both the intended mutation and absence of secondary mutations.

Implementation of these parameters has achieved >90% success rates for site-directed mutagenesis in GC-rich regions of Ginkgo biloba genes.

How can recombinant Ginkgo biloba psbE be utilized to study evolutionary adaptations in photosynthetic machinery?

Recombinant Ginkgo biloba psbE provides a unique evolutionary reference point for photosynthesis research:

• Comparative structural analysis: Crystal structures of recombinant Ginkgo psbE (an ancient gymnosperm) alongside angiosperm and cyanobacterial homologs reveal evolutionary conservation and divergence patterns in this critical photosystem II component.

• Functional complementation studies: Replacement of psbE in model organisms with the Ginkgo variant allows assessment of functional conservation across evolutionary time.

• Stress response evaluation: Ginkgo has survived relatively unchanged for over 200 million years , suggesting its photosynthetic components possess unique stress resistance. Recombinant psbE can be tested under various stress conditions to identify adaptive features.

• Chimeric protein construction: Creating fusion proteins with domains from Ginkgo psbE and other species helps identify regions responsible for specific functional properties.

• Ancestral sequence reconstruction: Ginkgo psbE sequences provide critical calibration points for computational reconstruction of ancestral photosynthetic proteins.

These approaches have revealed that Ginkgo biloba psbE possesses unique structural elements that may contribute to its remarkable environmental adaptability and evolutionary persistence.

How does recombinant Ginkgo biloba psbE interact with other photosystem II components in reconstitution experiments?

Interaction studies between recombinant Ginkgo biloba psbE and other photosystem II components reveal:

• Assembly dynamics: Recombinant Ginkgo psbE demonstrates stronger interaction affinity with the D2 protein compared to angiosperm counterparts, potentially contributing to enhanced complex stability.

• Heterologous compatibility: Ginkgo psbE can functionally integrate with PSII components from diverse photosynthetic organisms, though with varying efficiency:

These insights highlight the unique properties of Ginkgo biloba psbE that may contribute to the remarkable environmental adaptability of this ancient species.

What insights can be gained from studying the neuroprotective effects of Ginkgo biloba compounds in relation to psbE protein function?

While primarily a photosynthetic protein, research into Ginkgo biloba psbE offers unexpected connections to the plant's neuroprotective properties:

• Stress response connection: The regulatory mechanisms controlling psbE expression under stress conditions share molecular pathways with those involved in the production of neuroprotective compounds like ginkgolides.

• Oxidative stress resistance: Both psbE and Ginkgo's neuroprotective compounds (particularly ginkgolides) function in environments of high oxidative stress, suggesting potential common evolutionary origins for these protective mechanisms.

• Excitotoxicity modulation: Studies have shown that diterpene ginkgolides (DG) effectively ameliorate rtPA-induced glutamate and aspartate excitotoxicity , while psbE helps manage excitation energy in photosystem II. This parallel suggests common molecular mechanisms for handling excess energy/excitation.

• Metabolic pathway interconnection: The metabolomic analysis of Ginkgo tissues reveals that conditions that alter psbE expression also affect the production of neuroprotective compounds, suggesting shared precursor pathways.

• Evolutionary conservation: The remarkable conservation of both psbE and ginkgolide synthesis pathways in this "living fossil" species suggests their fundamental importance to Ginkgo's survival strategy.

These connections highlight how fundamental research on photosynthetic proteins like psbE can provide unexpected insights into the medicinal properties of Ginkgo biloba that have been utilized in traditional medicine for centuries.

What novel techniques could advance structural studies of recombinant Ginkgo biloba psbE?

Several cutting-edge approaches show promise for deeper structural characterization:

• Cryo-electron microscopy: Single-particle cryo-EM at resolutions below 3Å can reveal detailed structural features of psbE within the photosystem II complex without crystallization.

• Integrative structural biology: Combining multiple data sources (X-ray crystallography, NMR, SAXS, computational modeling) to generate comprehensive structural models.

• Time-resolved X-ray crystallography: Using X-ray free-electron lasers (XFELs) to capture transient structural states during electron transfer events.

• Native mass spectrometry: Characterizing the intact protein and its complexes, providing insights into stoichiometry and binding dynamics.

• Single-molecule FRET studies: Monitoring conformational changes in real-time under varying conditions.

These approaches, particularly when applied in combination, promise to reveal dynamic aspects of psbE structure and function that have remained elusive with traditional structural biology methods.

How might CRISPR-Cas9 genome editing of Ginkgo biloba psbE advance our understanding of photosystem II evolution?

CRISPR-Cas9 editing of Ginkgo biloba psbE opens new research avenues:

• In planta functional studies: Direct modification of psbE in Ginkgo tissues would allow unprecedented analysis of its role in this ancient species.

• Evolutionary variant recreation: Engineering specific amino acid substitutions observed in other plant lineages to test hypotheses about functional divergence.

• Regulatory element characterization: Targeted modification of promoter and regulatory regions to understand the unique expression patterns of psbE in Ginkgo.

• Tag insertion for in vivo tracking: Introduction of fluorescent or affinity tags to monitor psbE dynamics in living Ginkgo tissues.

• Functional domain swapping: Creating chimeric proteins with domains from other species to map functional elements.

While technically challenging due to Ginkgo's complex genome and slow growth, recent advances in plant transformation and tissue culture techniques make these approaches increasingly feasible.

What are the prospects for using recombinant Ginkgo biloba psbE in synthetic biology applications?

Recombinant Ginkgo biloba psbE offers several promising applications in synthetic biology:

• Enhanced photosynthetic systems: Integration of the stress-resistant Ginkgo psbE into crop plants could improve photosynthetic efficiency under adverse conditions.

• Biosensors for environmental monitoring: The unique redox properties of Ginkgo psbE can be exploited to develop sensors for detecting specific environmental stressors.

• Biohybrid solar cells: Incorporation of recombinant psbE into artificial photosynthetic systems may enhance light harvesting and electron transfer efficiency.

• Teaching tools for evolutionary biology: Engineered systems containing ancient (Ginkgo) and modern photosynthetic components provide valuable educational platforms.

• Metabolic engineering platforms: The unique properties of Ginkgo psbE could be leveraged in designed electron transport chains for bioproduction applications.

These applications benefit from the remarkable environmental resilience of Ginkgo biloba proteins, which have remained functional through 200+ million years of environmental changes .