Recombinant Fritillaria agrestis Cytochrome b6-f complex iron-sulfur subunit, chloroplastic (petC)

What is the Fritillaria agrestis Cytochrome b6-f complex iron-sulfur subunit and what is its role in photosynthesis?

The cytochrome b6-f complex iron-sulfur subunit, chloroplastic (petC) from Fritillaria agrestis is a critical component of the photosynthetic electron transport chain. It functions as the Rieske iron-sulfur protein (also known as ISP or RISP) within the cytochrome b6-f complex . This protein plays a crucial role in transferring electrons from plastoquinol to plastocyanin during the light-dependent reactions of photosynthesis, contributing to the generation of a proton gradient across the thylakoid membrane that drives ATP synthesis . The mature protein spans amino acids 57-230 of the full sequence and contains the characteristic iron-sulfur cluster that facilitates electron transfer .

What are the structural characteristics of the recombinant Fritillaria agrestis petC protein?

The recombinant Fritillaria agrestis petC protein consists of 174 amino acids (positions 57-230 of the full protein) with the following amino acid sequence:

ADRVPDMGKRQTMNLLLLGALSLPTAGMLIPYGAFFVPPSSGGGGGGIVAKDAVGNDIVAAAWLKTHGPGDRTLAQGLRGDPTYLVVENDRSLATYGINAVCTHLGCVVPWNKAENKFLCPCHGSQYNNQGKVVRGPAPLSLALSHCDISEEGKVVFVPWVETDFRTGENPWWS

This recombinant protein typically includes an N-terminal His-tag to facilitate purification . The protein contains the characteristic iron-sulfur cluster binding domain typical of Rieske proteins, which is essential for its electron transfer function in the cytochrome b6-f complex . The cysteine and histidine residues in the sequence are particularly important as they coordinate the iron-sulfur cluster .

What expression systems are most effective for producing recombinant Fritillaria agrestis petC protein?

The most effective expression system for recombinant Fritillaria agrestis petC protein is the pET expression system in Escherichia coli . This system offers several advantages:

• High-level expression: The T7 promoter system can dedicate nearly all of the cell's resources to expressing the target protein, potentially comprising up to 50% of total cellular protein after just a few hours of induction .

• Tight regulation: The pET vector system incorporates the T7lac promoter system, which includes a lac operator sequence downstream of the T7 promoter, allowing for stringent control of expression to minimize leaky expression that might be toxic to host cells .

• Methodology for optimal expression:

  • Initial cloning in a non-T7 RNA polymerase-containing host to prevent plasmid instability

  • Transfer to an expression host containing the T7 RNA polymerase gene under the LacUV5 promoter

  • Induction with IPTG to trigger high-level expression

For optimal results, use BL21(DE3) or similar E. coli strains designed for recombinant protein expression, and culture at 18-25°C after induction to enhance proper folding of the iron-sulfur protein .

What purification strategies yield the highest purity for recombinant petC protein?

The most effective purification strategy for His-tagged recombinant petC protein involves:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Use Ni-NTA or similar resin with a binding buffer containing 20-50 mM Tris-HCl (pH 8.0), 300-500 mM NaCl, and 10-20 mM imidazole

  • Elute with a gradient or step-wise increase of imidazole (100-500 mM)

• Secondary purification:

  • Size exclusion chromatography to remove aggregates and improve homogeneity

  • Ion exchange chromatography to separate different charged species

• Quality assessment:

  • SDS-PAGE analysis to confirm purity (>90% purity is typically achievable)

  • Western blotting with anti-His antibodies to confirm identity

  • Spectroscopic analysis to confirm presence of the iron-sulfur cluster

For optimal results, perform all purification steps at 4°C and include protease inhibitors to prevent degradation. The final purified protein is typically stored in a Tris/PBS-based buffer with 6% trehalose at pH 8.0 for lyophilization, or with 50% glycerol for liquid storage at -20°C or -80°C .

What are the optimal storage conditions for maintaining recombinant petC protein activity?

For optimal storage of recombinant petC protein:

• Short-term storage (up to one week):

  • Store working aliquots at 4°C in Tris/PBS-based buffer

• Long-term storage:

  • Primary recommendation: Store at -80°C in aliquots containing 50% glycerol to prevent freeze-thaw damage

  • Alternative: Lyophilized powder can be stored at -20°C

• Critical considerations:

  • Avoid repeated freeze-thaw cycles as they significantly reduce protein activity

  • For reconstitution of lyophilized protein, use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 50% for samples that will be refrozen

  • Centrifuge vials briefly before opening to ensure all material is at the bottom

The stability studies indicate that the protein maintains >90% activity when stored according to these recommendations, but activity decreases significantly after multiple freeze-thaw cycles or when stored above 4°C for extended periods .

How can recombinant petC be used to study the assembly of the cytochrome b6f complex?

Recombinant petC serves as an excellent tool for studying cytochrome b6f complex assembly through several experimental approaches:

• Complementation studies:

  • Use the recombinant protein to rescue mutants deficient in petC expression

  • Research with Lemna perpusilla mutant no. 1073 demonstrated that absence of functional petC results in deficiency of all four protein subunits of the cytochrome b6f complex, highlighting petC's key role in complex assembly

• Protein-protein interaction studies:

  • Use pull-down assays with His-tagged petC to identify interaction partners

  • Study assembly intermediates by co-expressing petC with other subunits

• In vitro assembly assays:

  • Reconstitute partial or complete cytochrome b6f complexes using purified components

  • Monitor assembly kinetics and stability

• Structure-function relationship analysis:

  • Generate site-directed mutants of key residues to assess their impact on complex assembly

  • The data from Lemna perpusilla studies suggests that petC plays a critical regulatory role, as its absence results in increased turnover rates of other subunits like subunit IV (10-fold higher protein turnover in petC-deficient mutants)

This approach has revealed that the Rieske Fe-S protein (petC) has a crucial function beyond electron transport—it stabilizes the entire cytochrome b6f complex and prevents premature degradation of other subunits .

What experimental methods can be used to assess the electron transfer function of recombinant petC?

Several experimental methods can be employed to assess the electron transfer function of recombinant petC:

• Spectroscopic analysis:

  • UV-visible spectroscopy to monitor the characteristic absorption spectra of the iron-sulfur cluster

  • Electron Paramagnetic Resonance (EPR) spectroscopy to directly observe the redox state of the iron-sulfur cluster

  • Circular dichroism to evaluate structural integrity related to function

• Electrochemical methods:

  • Cyclic voltammetry to determine redox potentials

  • Protein film voltammetry on electrode surfaces to study electron transfer kinetics

• Reconstitution assays:

  • In vitro reconstitution of electron transfer using purified components

  • Measurement of electron transfer rates using spectrophotometric methods with artificial electron donors and acceptors

• pH-dependent activity assays:

  • Evaluation of electron transfer rates at different pH values

  • Studies have shown that mutations in petC, such as the P171L substitution, can alter the pH dependency of electron transfer, providing insights into mechanism

Note: These values are representative based on similar studies and may vary depending on experimental conditions .

How do mutations in key residues of petC affect the function of the cytochrome b6f complex?

Mutations in key residues of petC can significantly impact the function of the cytochrome b6f complex in several ways:

• Electron transfer efficiency:

  • Mutations in the conserved cysteine and histidine residues that coordinate the iron-sulfur cluster directly disrupt electron transfer

  • The P171L substitution alters the pH dependency of electron transfer, likely by affecting the structural dynamics around the iron-sulfur cluster

  • Mutations in the flexible linker region between the membrane anchor and the iron-sulfur domain can impact the range of movement necessary for efficient electron transfer

• Complex stability and assembly:

  • Studies with Lemna perpusilla mutant no. 1073 demonstrated that absence of functional petC leads to increased turnover rates of other complex subunits

  • Point mutations in the interface regions between petC and other subunits can destabilize the entire complex

  • Some mutations may not completely eliminate function but rather alter the kinetics or environmental sensitivity of the complex

• Interaction with electron transfer partners:

  • Mutations in the surface-exposed regions can affect docking with plastoquinone or plastocyanin

  • Changes in charge distribution can alter the efficiency of partner recognition

Experimental data suggests that even single amino acid substitutions can have profound effects on both the stability and function of the cytochrome b6f complex, highlighting the precisely evolved structure-function relationships in this protein .

What is the role of post-translational modifications in petC function and how can they be analyzed?

Post-translational modifications (PTMs) of petC play critical roles in regulating its function within the cytochrome b6f complex:

• Types of PTMs in petC:

  • Iron-sulfur cluster insertion: The most critical modification for function

  • Disulfide bond formation: Contributes to structural stability

  • Phosphorylation: May regulate activity under different conditions

  • Oxidative modifications: Can occur during stress conditions and may affect function

• Analytical methods for studying PTMs:

  • Mass spectrometry (MS):

    • Liquid chromatography-tandem MS (LC-MS/MS) for comprehensive PTM mapping

    • Top-down proteomics for intact protein analysis

  • Spectroscopic methods:

    • EPR for iron-sulfur cluster analysis

    • Circular dichroism for secondary structure analysis

  • Activity assays under different redox conditions to assess functional impacts

• Iron-sulfur cluster analysis:

  • UV-visible spectroscopy to monitor characteristic absorbance peaks

  • EPR spectroscopy to characterize the paramagnetic properties of the cluster

  • Mössbauer spectroscopy for detailed iron oxidation state analysis

• Experimental approach for studying PTM effects:

Studies on related proteins suggest that PTMs can significantly alter the redox properties and stability of the iron-sulfur cluster, directly impacting electron transfer efficiency in the photosynthetic electron transport chain .

What are the common challenges in expressing active recombinant petC and how can they be addressed?

Expressing active recombinant petC presents several challenges that can be addressed through specific strategies:

• Inclusion body formation:

  • Challenge: Overexpression often leads to insoluble protein aggregates

  • Solutions:

    • Lower induction temperature (18-20°C)

    • Reduce IPTG concentration (0.1-0.5 mM)

    • Co-express with molecular chaperones (GroEL/GroES)

    • Use fusion tags that enhance solubility (SUMO, MBP)

• Iron-sulfur cluster incorporation:

  • Challenge: Recombinant expression may result in incomplete iron-sulfur cluster assembly

  • Solutions:

    • Supplement growth media with iron (FeCl₃ or Fe(NH₄)₂(SO₄)₂) and sulfur sources

    • Co-express iron-sulfur cluster assembly machinery proteins

    • Consider in vitro cluster reconstitution after purification

• Protein degradation:

  • Challenge: The iron-sulfur protein may be unstable during expression or purification

  • Solutions:

    • Include protease inhibitors throughout purification

    • Maintain reducing conditions with DTT or β-mercaptoethanol

    • Perform all steps at 4°C

    • Minimize time between lysis and final storage

• Low yield:

  • Challenge: Expression levels may be insufficient for experimental needs

  • Solutions:

    • Optimize codon usage for E. coli

    • Try different E. coli expression strains (BL21(DE3), Rosetta, Arctic Express)

    • Scale up culture volume or use high-density fermentation

• Activity loss during storage:

  • Challenge: Protein may lose activity during storage

  • Solutions:

    • Store in small aliquots to avoid freeze-thaw cycles

    • Add stabilizers like trehalose (6%) or glycerol (50%)

    • Consider lyophilization for long-term storage

Implementing these strategies has been shown to increase the yield of correctly folded, active recombinant petC protein by up to 3-5 fold compared to standard expression protocols .

How can researchers troubleshoot issues with the reconstitution of lyophilized recombinant petC?

Troubleshooting reconstitution issues with lyophilized recombinant petC requires a systematic approach:

• Incomplete dissolution:

  • Problem: Protein forms visible aggregates or precipitates during reconstitution

  • Solutions:

    • Ensure proper centrifugation of the vial before opening to collect all material at the bottom

    • Reconstitute using deionized sterile water to a concentration of 0.1-1.0 mg/mL

    • Allow longer dissolution time at 4°C with gentle agitation

    • Avoid vortexing, which can cause denaturation; instead, use gentle inversion or rotation

• Activity loss after reconstitution:

  • Problem: Protein shows reduced or no activity after reconstitution

  • Solutions:

    • Verify pH of reconstitution buffer (optimal pH is 8.0)

    • Add reducing agents (1-5 mM DTT) to protect the iron-sulfur cluster

    • Consider iron-sulfur cluster reconstitution if activity is severely compromised

• Protein concentration determination issues:

  • Problem: Difficulty in accurately determining protein concentration

  • Solutions:

    • Use multiple methods to cross-validate (Bradford, BCA, A280)

    • Account for the contribution of the iron-sulfur cluster to absorbance measurements

    • Prepare a standard curve using known quantities of similar proteins

• Storage after reconstitution:

  • Problem: Uncertainty about how to store reconstituted protein

  • Solutions:

    • For extended storage, add glycerol to a final concentration of 50%

    • Aliquot immediately after reconstitution to avoid freeze-thaw cycles

    • Store working aliquots at 4°C for up to one week

    • For longer storage, keep at -20°C/-80°C

• Systematic troubleshooting approach:

Following these guidelines can improve reconstitution success rates from approximately 60% to over 90% while maintaining protein activity .

How can recombinant petC be used to study the evolution of photosynthetic electron transport chains?

Recombinant petC provides valuable tools for evolutionary studies of photosynthetic electron transport chains:

• Comparative structural analysis:

  • Express and purify petC from diverse photosynthetic organisms (cyanobacteria, algae, various plant species)

  • Perform structural comparisons using X-ray crystallography, cryo-EM, or computational modeling

  • Analyze conservation patterns of key functional domains across evolutionary lineages

• Functional conservation and divergence:

  • Conduct cross-species complementation studies

  • Test if petC from Fritillaria agrestis can functionally replace the protein in other species

  • Measure electron transfer kinetics of petC from different evolutionary sources under standardized conditions

• Experimental approaches:

  • Create chimeric proteins combining domains from different species to identify species-specific functional elements

  • Perform site-directed mutagenesis to convert residues to those found in other species

  • Test adaptation to different environmental conditions (temperature, pH, light intensity)

• Phylogenetic analysis combined with functional data:

  • Correlate sequence variations with functional differences

  • Identify evolutionary adaptations in response to different ecological niches

  • Reconstruct ancestral sequences and express them to study the evolution of function

This approach has revealed that while the core iron-sulfur cluster binding domain is highly conserved across species, variations in other regions reflect adaptations to specific environmental conditions and interactions with other components of the photosynthetic machinery .

What are the implications of petC research for improving photosynthetic efficiency in crop plants?

Research on petC has significant implications for agricultural biotechnology and crop improvement:

• Engineering electron transport for enhanced photosynthesis:

  • Targeted modifications of petC could optimize electron flow through the cytochrome b6f complex

  • Studies suggest that the cytochrome b6f complex is often a rate-limiting step in photosynthetic electron transport

  • Modifications aimed at reducing susceptibility to photoinhibition could improve plant performance under fluctuating light conditions

• Stress tolerance enhancement:

  • Research on pH dependency of electron transfer in petC variants provides insights for developing crops with better tolerance to pH fluctuations

  • Understanding how specific amino acid changes (such as the P171L substitution) affect function under different conditions can guide precision engineering

  • Modifications that enhance stability of the cytochrome b6f complex under heat stress could improve crop resilience to climate change

• Experimental evidence from model systems:

  • Studies in Lemna perpusilla demonstrate that petC is essential for cytochrome b6f complex assembly and stability

  • This provides a foundation for identifying critical residues that could be targets for improvement

• Potential agricultural impacts:

The application of fundamental petC research to agriculture represents a promising frontier in crop improvement, with potential to enhance photosynthetic efficiency by 5-15% under certain conditions, which could translate to significant yield improvements .