Recombinant Scopolia japonica Ferredoxin

What is the primary structure of Scopolia japonica ferredoxin and how does it compare to other plant ferredoxins?

Scopolia japonica ferredoxin is a [2Fe-2S] iron-sulfur protein that functions as an electron carrier in photosynthetic electron transport. Its complete amino acid sequence has been determined through automated Edman degradation of the Cm-protein and peptides from enzymatic digestions. Comparative analysis reveals that S. japonica ferredoxin exhibits remarkable sequence similarity to Datura ferredoxins, with only 2-7 amino acid differences compared to Datura stramonium, D. metel, and D. arborea ferredoxins. Particularly noteworthy is its close relationship with D. arborea ferredoxin, showing only 2-3 amino acid differences .

Unlike these close relationships, S. japonica ferredoxin displays 8-19 amino acid differences when compared with other solanaceous ferredoxins, indicating significant evolutionary divergence within the family . This pattern of sequence conservation and variation provides valuable insights for protein chemotaxonomy studies.

What are the spectroscopic characteristics that indicate properly folded recombinant S. japonica ferredoxin?

Properly folded recombinant S. japonica ferredoxin with intact [2Fe-2S] clusters exhibits characteristic spectroscopic features that can be monitored to assess protein quality:

• UV-Visible spectroscopy: Absorption maxima at approximately 330, 420, and 460 nm, which are distinctive features of [2Fe-2S] cluster-containing ferredoxins

• A420/A280 ratio: Values above 0.45 typically indicate high purity and proper [2Fe-2S] cluster incorporation

• Circular dichroism: Distinctive peaks in both far-UV (reporting on secondary structure) and visible regions (reporting on the environment of the [2Fe-2S] cluster)

• EPR spectroscopy: The reduced form should display a characteristic signal at g ≈ 1.96

Deviations from these spectroscopic properties may indicate incomplete cluster incorporation, oxidative damage, or protein misfolding.

How does the electron transfer function of S. japonica ferredoxin compare with other plant ferredoxins in experimental systems?

This requires systematic investigation using standardized conditions and methods:

While specific data for S. japonica ferredoxin is limited, its high sequence similarity to Datura ferredoxins suggests comparable functional parameters. Differences in surface residues may affect partner protein interactions while maintaining core electron transfer capabilities .

What expression systems yield optimal quantities of functional recombinant S. japonica ferredoxin?

The optimal expression of recombinant S. japonica ferredoxin requires careful system selection and optimization:

Expression systems comparison:

When designing expression constructs, researchers should exclude the transit peptide sequence (approximately 52 amino acids) and focus on the mature protein sequence (about 97 amino acids) . To enhance proper [2Fe-2S] cluster incorporation, consider co-expression with iron-sulfur cluster assembly proteins or supplementation of the growth medium with iron sources and sulfur-containing amino acids.

What purification strategy provides the highest yield of active recombinant S. japonica ferredoxin?

Based on established protocols for plant ferredoxins, the following optimized purification strategy is recommended:

• Cell lysis: Sonication or pressure homogenization in buffer containing 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, and 5 mM β-mercaptoethanol

• Initial fractionation: Ammonium sulfate precipitation (typically 40-80% saturation)

• Ion-exchange chromatography: DEAE-Sepharose using a 0-500 mM NaCl gradient in 20 mM Tris-HCl pH 7.5

• Size exclusion chromatography: Superdex 75 column for final polishing

Throughout purification, monitor the characteristic absorption spectrum (330, 420, and 460 nm) to track functional protein. Scopolia japonica ferredoxin has been successfully isolated from fresh leaves (0.6 kg) yielding approximately 1.5 mg of purified protein, suggesting similar yields might be achievable with recombinant systems under optimal conditions .

What strategies overcome common challenges in expressing active recombinant S. japonica ferredoxin?

Researchers frequently encounter specific challenges when expressing plant ferredoxins like S. japonica ferredoxin:

Implementing these strategies can significantly improve the yield of active recombinant S. japonica ferredoxin.

How can researchers determine the three-dimensional structure of recombinant S. japonica ferredoxin?

Multiple complementary approaches can reveal the structure of recombinant S. japonica ferredoxin:

X-ray crystallography protocol:

• Concentrate purified ferredoxin to 10-15 mg/mL in 20 mM Tris-HCl pH 7.5, 50 mM NaCl

• Screen crystallization conditions using sparse matrix screening (typical conditions for plant ferredoxins include PEG 4000, ammonium sulfate, and pH 6.0-8.0)

• Optimize promising conditions to obtain diffraction-quality crystals

• Collect diffraction data at synchrotron radiation facilities

• Process data and solve structure by molecular replacement using related ferredoxin structures

Alternative structural approaches:

• NMR spectroscopy: For solution structure determination (requires ¹⁵N/¹³C labeling)

• Small-angle X-ray scattering (SAXS): For low-resolution envelope determination

• Homology modeling: Using the crystal structures of related plant ferredoxins as templates

While specific structural data for S. japonica ferredoxin is limited, its high sequence similarity to Datura ferredoxins suggests a conserved β-grasp fold with the [2Fe-2S] cluster coordinated by four conserved cysteine residues.

What methods are most effective for investigating the interaction between S. japonica ferredoxin and partner proteins?

Multiple complementary techniques can characterize protein-protein interactions:

Binding and kinetic studies:

• Surface plasmon resonance (SPR): Immobilize ferredoxin on a CM5 chip and flow partner proteins to determine kon, koff, and Kd values

• Isothermal titration calorimetry (ITC): Directly measure thermodynamic parameters (ΔH, ΔS, and Kd)

• Microscale thermophoresis (MST): Measure binding with minimal protein consumption

Structural studies:

• Co-crystallization: Attempt to crystallize S. japonica ferredoxin in complex with partners

• Cross-linking coupled with mass spectrometry: Identify interaction interfaces

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Map binding surfaces

Computational approaches:

• Molecular docking simulations

• Molecular dynamics of protein complexes

• Electrostatic surface potential analysis to identify complementary regions

The combination of these approaches provides a comprehensive view of the molecular basis for specificity in S. japonica ferredoxin interactions.

How can researchers identify the determinants of redox potential in S. japonica ferredoxin?

The redox potential of ferredoxins is finely tuned by their protein environment. To identify these determinants:

• Sequence-based analysis: Compare S. japonica ferredoxin with ferredoxins of known redox potentials, focusing on residues near the [2Fe-2S] cluster

• Site-directed mutagenesis: Create a systematic library of variants focusing on:

  • Residues within 5Å of the [2Fe-2S] cluster

  • Surface-exposed charged residues

  • Hydrogen-bonding network participants

• Structure-potential correlation: Measure redox potentials of variants using:

  • Cyclic voltammetry

  • Spectroelectrochemistry with redox mediators

  • Equilibrium with redox partners of known potential

• Computational electrostatics: Calculate the effect of specific residues on the electrostatic environment of the [2Fe-2S] cluster

These approaches can identify key amino acids responsible for the specific redox properties of S. japonica ferredoxin and guide rational design of variants with altered redox potentials.

What electron transfer pathways involve S. japonica ferredoxin in native and experimental systems?

In native plant systems, ferredoxins like the one from S. japonica participate in multiple electron transfer pathways:

In experimental systems, recombinant S. japonica ferredoxin can be coupled with:

• Non-native redox partners for biotechnological applications

• Artificial electron acceptors like cytochrome c or DCPIP for activity assays

• Electrode surfaces for bioelectrochemical applications

The ability of ferredoxin to participate in these diverse pathways makes it a versatile component for both natural and engineered electron transfer systems.

How can researchers use S. japonica ferredoxin sequence data for evolutionary and taxonomic studies?

S. japonica ferredoxin provides valuable data for investigating evolutionary relationships:

• Multiple sequence alignment: Align S. japonica ferredoxin with other plant ferredoxins, focusing on the 97-residue mature protein

• Phylogenetic analysis:

  • Generate trees using distance-based, maximum likelihood, or Bayesian methods

  • Apply appropriate evolutionary models (typically JTT or WAG for proteins)

  • Assess node support through bootstrap analysis (1000 replicates recommended)

• Taxonomic implications: The close relationship between S. japonica and Datura ferredoxins (particularly D. arborea) supports their taxonomic proximity within Solanaceae

• Rate analysis: Calculate evolutionary rates to identify conserved vs. variable regions

• Comparative analysis: Compare ferredoxin-based phylogenies with those derived from other markers (rbcL, matK, ITS) to identify potential incongruences

This approach has already revealed that S. japonica is more closely related to Datura species than to other Solanaceae members like Nicotiana or Capsicum, demonstrating the value of ferredoxin sequences in resolving taxonomic relationships .

How do environmental factors affect the expression and activity of native S. japonica ferredoxin?

While specific data for S. japonica is limited, research on plant ferredoxins suggests:

• Light regulation: Ferredoxin gene expression is typically light-regulated through:

  • Photoreceptor-mediated signaling pathways

  • Light-responsive elements in promoter regions

  • Nuclear protein binding to Fed-1 5'-flanking sequences, as demonstrated in pea

• Nutrient effects:

  • Iron availability directly affects ferredoxin levels due to [2Fe-2S] cluster requirements

  • Nitrogen status may modulate expression due to ferredoxin's role in nitrogen assimilation

• Developmental regulation:

  • Expression typically peaks during leaf development

  • Varies across plant tissues with highest levels in photosynthetically active tissues

• Stress responses:

  • Oxidative stress may lead to ferredoxin degradation

  • Some stress conditions trigger enhanced expression of specific ferredoxin isoforms

Understanding these regulatory mechanisms could inform optimization strategies for recombinant expression and in planta studies.

How can recombinant S. japonica ferredoxin be utilized in synthetic biology applications?

Recombinant S. japonica ferredoxin offers several biotechnological applications:

• Biocatalytic systems:

  • Coupling with P450 monooxygenases for regio- and stereoselective hydroxylations

  • Integration into multi-enzyme redox cascades for complex transformations

  • Mediating electron transfer between photosystems and engineered enzymes

• Photobiological hydrogen production:

  • Connecting photosynthetic electron transport to hydrogenases

  • Optimizing electron flux for improved hydrogen yields

• Biosensor development:

  • Creating electrochemical biosensors based on ferredoxin-electrode interactions

  • Developing optical biosensors that report on redox state changes

• Protein engineering platform:

  • Using S. japonica ferredoxin as a scaffold for developing novel electron carriers

  • Creating fusion proteins with catalytic domains for directed electron transfer

The small size, stability, and well-defined electron transfer properties make S. japonica ferredoxin a versatile component for diverse synthetic biology applications.

What methods determine if recombinant S. japonica ferredoxin retains native-like electron transfer properties?

Researchers can assess the functional integrity of recombinant S. japonica ferredoxin through:

• Spectroscopic characterization:

  • UV-visible absorption spectrum should match native ferredoxin

  • CD spectroscopy to confirm secondary structure

  • EPR spectroscopy to verify [2Fe-2S] cluster environment

• Redox potential determination:

  • Cyclic voltammetry to measure formal potential

  • Spectroelectrochemistry to generate potential curves

  • Comparison with native ferredoxin or closely related proteins

• Kinetic measurements:

  • Rate of reduction by photosystem I

  • Rate of oxidation by ferredoxin-dependent enzymes

  • Stopped-flow analysis of electron transfer kinetics

• Partner protein interactions:

  • Binding affinity measurements using SPR or ITC

  • Activity assays with natural electron transfer partners

  • Competition assays with native ferredoxin

These complementary approaches provide a comprehensive assessment of whether the recombinant protein faithfully reproduces the functional properties of native S. japonica ferredoxin.

How can researchers modify S. japonica ferredoxin to optimize specific electron transfer applications?

Strategic modifications can enhance ferredoxin performance for specific applications:

• Redox potential tuning:

  • Targeted mutations of residues near the [2Fe-2S] cluster

  • Modifying hydrogen bonding networks affecting cluster environment

  • Altering surface charge distribution

• Partner specificity engineering:

  • Surface residue modifications at interaction interfaces

  • Loop engineering to enhance or reduce specific interactions

  • Fusion with partner-specific binding domains

• Stability enhancement:

  • Introduction of disulfide bridges

  • Core packing optimization

  • Surface redesign to improve solubility

• Immobilization strategies:

  • Introduction of unique surface cysteines for directed coupling

  • Addition of affinity tags at non-interfering positions

  • Fusion with self-assembling domains for ordered arrays

Such modifications should be guided by detailed structural information and validated through the functional assays described above to ensure that beneficial properties are maintained while enhancing desired characteristics.