Recombinant Gossypium barbadense Cytochrome c

What is Gossypium barbadense cytochrome c and what is its significance in plant biology?

Gossypium barbadense (Sea Island cotton) cytochrome c is a small heme-containing protein involved in electron transport processes within plant mitochondria. This protein plays a crucial role in cellular respiration and energy production through the mitochondrial electron transport chain. In G. barbadense, cytochrome c is encoded by the mitochondrial genome, which has been fully sequenced as a 677,434 bp circular molecule .

The protein is significant in plant biology for several reasons:

• It functions as an essential component of the respiratory electron transport chain

• It may contribute to stress response mechanisms in cotton plants

• It serves as a model system for studying plant mitochondrial proteins

• Its sequence and structure provide insights into the evolutionary divergence of cotton species

Methodologically, researchers studying G. barbadense cytochrome c should isolate mitochondria using discontinuous sucrose density gradient methods. The mitochondrial fraction can be collected from the interface between 52% and 36% sucrose, washed with 0.3 mol·L⁻¹ sucrose buffer, and then lysed in cetyltrimethyl ammonium bromide (CTAB) for DNA/protein extraction .

How does cytochrome c from Gossypium barbadense differ from other plant cytochrome proteins?

While the search results don't provide direct comparative data specific to G. barbadense cytochrome c, we can infer differences based on related research. Cytochrome proteins in plants display functional diversity, with cytochrome P450s being particularly diverse. The G. barbadense mitochondrial genome shows notable evolutionary divergence from other cotton species such as G. hirsutum .

Key differences include:

• Sequence variations: G. barbadense mitochondrial proteins often show specific sequence adaptations, potentially affecting the structure and function of cytochrome c

• Redox properties: Species-specific differences in heme environment can alter redox potential

• Post-translational modifications: These may vary between species, affecting protein stability and activity

• Expression patterns: G. barbadense shows tissue-specific and stress-responsive gene expression profiles

For research applications, these differences necessitate species-specific analytical approaches. Spectroscopic methods such as resonance Raman spectroscopy can be employed to detect structural differences in heme proteins like cytochrome c . When comparing cytochrome proteins across species, researchers should standardize their extraction and analytical methods to ensure valid comparisons.

What expression systems are most effective for producing recombinant Gossypium barbadense cytochrome c?

While the search results don't directly address expression systems specifically for G. barbadense cytochrome c, we can draw insights from related recombinant protein expression protocols described in the search results.

For bacterial expression systems, Escherichia coli strain BL21(DE3) has been successfully used for expressing recombinant terpene synthase from G. barbadense . This system can be adapted for cytochrome c expression using the following methodology:

• Clone the complete open reading frame (ORF) of G. barbadense cytochrome c into a suitable expression vector (e.g., pET-30a(+))

• Transform the plasmid into E. coli BL21(DE3) cells

• Culture transformed cells in LB medium with appropriate antibiotics at 37°C until OD₆₀₀ reaches ~0.6

• Induce protein expression with 1 mM IPTG

• Lower the temperature to 18°C and incubate for 20 hours to optimize protein folding

• Harvest cells by centrifugation (8,000 × g, 10 min, 4°C)

• Resuspend in extraction buffer (50 mM MOPS [pH 7.0], 5 mM MgCl₂, 5 mM sodium ascorbate, 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, and 10% [v/v] glycerol)

• Disrupt cells by sonication and centrifuge at 14,000 × g

For co-expression systems involving cytochrome proteins, the pETDUET-1 vector system has proven effective for cotton cytochrome P450 reductases, allowing simultaneous expression of multiple proteins . This approach would be valuable when cytochrome c needs to be studied in conjunction with its interaction partners.

What spectroscopic techniques are most informative for characterizing recombinant Gossypium barbadense cytochrome c?

Based on the search results, particularly regarding heme protein analysis, several spectroscopic techniques can be effectively applied to characterize recombinant G. barbadense cytochrome c:

• Resonance Raman Spectroscopy: This technique is particularly powerful for heme proteins, providing detailed information about the heme environment, spin state, and coordination. The resonance enhancement effect makes it highly sensitive to the heme prosthetic group .

• UV-Visible Absorption Spectroscopy: Absorbance peaks around 410 nm (Soret band) and 530-570 nm (α/β bands) provide information about the heme oxidation and coordination state.

• Circular Dichroism (CD): Useful for assessing protein secondary structure and folding.

• Nuclear Magnetic Resonance (NMR): Can provide atomic-level structural information for small proteins like cytochrome c.

For resonance Raman analysis specifically, researchers should:

• Use excitation wavelengths in the Soret band region (~410 nm) for maximum enhancement

• Prepare concentrated samples (typically 50-100 μM)

• Use low laser power to prevent sample photodegradation

• Compare spectra in both oxidized and reduced states to obtain comprehensive vibrational information

• Focus on specific marker bands that indicate coordination state (ν₂, ν₃, ν₄) and spin state (ν₁₀, ν₁₉)

The combination of these techniques provides complementary information about protein structure and function, creating a comprehensive characterization profile.

How can researchers assess the electron transfer activity of recombinant Gossypium barbadense cytochrome c?

To assess electron transfer activity of recombinant G. barbadense cytochrome c, researchers can adapt methods described for related cytochrome proteins in the search results:

Standard Reduction Assays:
Electron transfer capacity can be assessed using various electron acceptors:

• Cytochrome c reduction assay: Using mammalian cytochrome c as electron acceptor

• Potassium ferricyanide (K₃Fe(CN)₆) reduction: Measuring the decrease in absorbance at 420 nm

• Dichlorophenolindophenol (DCPIP) reduction: Monitoring absorbance changes at 600 nm

Methodology for Electron Transfer Assessment:

• Prepare reaction mixture containing:

  • 50-100 mM potassium phosphate buffer (pH 7.4)

  • 0.1-1 μM recombinant G. barbadense cytochrome c

  • Electron donor (typically NADPH at 50-200 μM)

  • Electron acceptor (cytochrome c, K₃Fe(CN)₆, or DCPIP)

• Initiate reaction by adding NADPH and monitor spectrophotometrically:

  • For cytochrome c: increase in absorbance at 550 nm

  • For K₃Fe(CN)₆: decrease in absorbance at 420 nm

  • For DCPIP: decrease in absorbance at 600 nm

• Calculate enzyme activity using extinction coefficients:

  • Reduced cytochrome c: ε₅₅₀ = 21 mM⁻¹cm⁻¹

  • K₃Fe(CN)₆: ε₄₂₀ = 1.02 mM⁻¹cm⁻¹

  • DCPIP: ε₆₀₀ = 21 mM⁻¹cm⁻¹

This methodological approach provides quantitative data on electron transfer capabilities, which can be used to compare wild-type and recombinant proteins or assess the effects of experimental conditions on electron transfer efficiency.

What purification strategies yield the highest purity and activity for recombinant Gossypium barbadense cytochrome c?

Based on the search results and standard practices for recombinant heme proteins, a multi-step purification strategy is recommended for obtaining high-purity, active recombinant G. barbadense cytochrome c:

Purification Protocol:

• Initial Extraction:

  • Resuspend bacterial pellet in extraction buffer (50 mM MOPS [pH 7.0], 5 mM MgCl₂, 5 mM sodium ascorbate, 5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride, and 10% [v/v] glycerol)

  • Disrupt cells by sonication

  • Centrifuge at 14,000 × g to remove cell debris

• Affinity Chromatography:

  • If His-tagged: Use Ni-NTA column with imidazole gradient elution

  • If non-tagged: Consider heme-affinity chromatography using modified sepharose

• Ion Exchange Chromatography:

  • Apply sample to cation exchange column (e.g., CM-Sepharose)

  • Elute with NaCl gradient (0-500 mM)

• Size Exclusion Chromatography:

  • Final polishing step using Superdex 75 or equivalent

  • Helps remove aggregates and dimers

• Quality Control Assessment:

  • SDS-PAGE analysis (expect band at ~12-14 kDa)

  • UV-visible spectroscopy (A₄₁₀/A₂₈₀ ratio >4 indicates good heme incorporation)

  • Heme content determination (pyridine hemochromogen assay)

Critical Factors for Activity Retention:

This comprehensive purification strategy should yield recombinant G. barbadense cytochrome c with >95% purity and high electron transfer activity, suitable for detailed biochemical and biophysical characterization.

How can recombinant Gossypium barbadense cytochrome c be used to study plant stress responses?

Recombinant G. barbadense cytochrome c can serve as a valuable tool for investigating plant stress responses, particularly in the context of biotic stress. The search results provide insights into how cotton responds to herbivore attack, which can inform experimental approaches using recombinant cytochrome c:

Experimental Applications:

• Oxidative Stress Studies:

  • Cytochrome c release from mitochondria is a key marker of stress-induced programmed cell death

  • Recombinant protein can be used to quantify interaction with stress-responsive proteins

  • In vitro assays can assess how oxidative modifications affect cytochrome c function

• Herbivore Defense Response Analysis:

  • G. barbadense activates complex defense mechanisms against herbivores like H. armigera

  • Transcriptome analysis reveals 5,629 differentially expressed genes (DEGs) after herbivore infestation

  • Recombinant cytochrome c can be used to study electron transport changes during defense responses

• Jasmonic Acid (JA) Pathway Interactions:

  • 88 out of 90 DEGs associated with the JA pathway were upregulated after herbivore infestation

  • Recombinant cytochrome c can be used to study interactions with JA pathway components

  • Binding assays and activity measurements can reveal functional relationships

Methodological Approach:

• Expose G. barbadense plants to stress conditions (e.g., herbivore infestation, drought, temperature extremes)

• Extract native cytochrome c from stressed and control plants

• Compare properties with recombinant protein using:

  • Spectroscopic analysis to detect structural changes

  • Activity assays to measure functional alterations

  • Interaction studies with defense-related proteins

• Use recombinant protein with specific mutations to identify critical residues involved in stress response

This systematic approach allows researchers to dissect the specific role of cytochrome c in plant stress responses, potentially identifying novel target sites for enhancing plant resistance to biotic and abiotic stresses.

What are the comparative kinetics of wild-type versus recombinant Gossypium barbadense cytochrome c?

While the search results don't provide direct kinetic data comparing wild-type and recombinant G. barbadense cytochrome c, a methodological framework can be established for such comparisons based on approaches used for related proteins:

Kinetic Parameters to Compare:

• Electron Transfer Rates:

  • Measure pseudo-first-order rate constants (k) for electron transfer with various redox partners

  • Determine reaction order and dependence on protein concentration

• Binding Affinities:

  • Calculate dissociation constants (Kd) for interactions with physiological partners

  • Use surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

• Redox Potential:

  • Determine midpoint potentials (Em) using spectroelectrochemical methods

  • Compare pH dependence of redox properties

Experimental Methodology:

For accurate kinetic comparisons, researchers should:

• Ensure both proteins are in identical buffer conditions

• Verify structural integrity through spectroscopic methods

• Use steady-state and pre-steady-state kinetic approaches

• Perform measurements under varying conditions (pH, ionic strength, temperature)

Typical Kinetic Comparison Table:

Note: The exact values would need to be determined experimentally for G. barbadense cytochrome c specifically, as these may vary from the estimated ranges provided.

These comparative analyses can reveal subtle differences between wild-type and recombinant proteins, helping researchers optimize expression and purification protocols to produce recombinant proteins with native-like properties.

How do post-translational modifications affect the function of recombinant Gossypium barbadense cytochrome c?

Post-translational modifications (PTMs) can significantly impact cytochrome c function, and differences in PTMs between native and recombinant proteins may explain functional variations. While the search results don't directly address PTMs in G. barbadense cytochrome c, we can infer their importance from related research:

Key Post-translational Modifications in Plant Cytochrome c:

• Heme Attachment:

  • Covalent attachment via thioether bonds to conserved CXXCH motif

  • Critical for electron transfer function

  • May be incomplete in recombinant systems without proper machinery

• N-terminal Processing:

  • Removal of transit peptides after mitochondrial import

  • Affects protein stability and folding

  • Often absent in recombinant proteins expressed in bacteria

• Phosphorylation:

  • Regulates interaction with partners and electron transfer rates

  • Primarily occurs on serine, threonine, and tyrosine residues

  • Expression systems may lack plant-specific kinases

• Oxidative Modifications:

  • Methionine oxidation alters redox properties

  • Lysine carbonylation under stress conditions

  • May occur differently in recombinant systems

Methodological Approaches to Study PTMs:

• Mass Spectrometry-Based Analysis:

  • LC-MS/MS for comprehensive PTM identification

  • Compare native and recombinant protein profiles

  • Quantify modification stoichiometry

• Site-Directed Mutagenesis:

  • Generate variants mimicking or preventing specific PTMs

  • Assess functional impact through activity assays

  • Compare with native protein behavior

• Expression System Optimization:

  • Co-expression with modifying enzymes

  • Use of eukaryotic expression systems

  • Addition of heme precursors during expression

Impact of PTMs on Functional Properties:

Understanding these PTMs and their functional consequences is essential for producing recombinant G. barbadense cytochrome c with native-like properties and for interpreting experimental results correctly.

What are common challenges in heterologous expression of Gossypium barbadense cytochrome c and how can they be addressed?

Heterologous expression of G. barbadense cytochrome c presents several challenges that researchers should anticipate and address. Based on the search results and general principles of recombinant heme protein expression, the following challenges and solutions can be identified:

Common Challenges and Solutions:

• Incomplete Heme Incorporation

  • Challenge: Bacterial expression systems often lack sufficient heme or heme incorporation machinery

  • Solution: Supplement growth media with δ-aminolevulinic acid (0.5-1.0 mM) as a heme precursor and optimize growth conditions (18°C post-induction for 20h)

• Protein Misfolding and Inclusion Body Formation

  • Challenge: Rapid overexpression leads to aggregation and inclusion body formation

  • Solution: Lower induction temperature (18°C), reduce IPTG concentration (0.1-0.5 mM), and use solubility-enhancing fusion tags (SUMO, thioredoxin)

• Improper Post-translational Processing

  • Challenge: Bacterial systems lack eukaryotic PTM machinery

  • Solution: Consider eukaryotic expression systems (yeast, insect cells) for critical applications requiring native-like PTMs

• Low Expression Yields

  • Challenge: Plant proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for expression host, use strong promoters (T7), and screen multiple expression strains

• Protein Instability

  • Challenge: Recombinant cytochrome c may be unstable without native environment

  • Solution: Include stabilizing additives (glycerol 10-20%, reducing agents) in all buffers

Optimization Strategy Table:

By systematically addressing these challenges, researchers can significantly improve the yield and quality of recombinant G. barbadense cytochrome c, making it suitable for detailed structural and functional studies.

How can researchers ensure proper heme incorporation in recombinant Gossypium barbadense cytochrome c?

Proper heme incorporation is critical for the functionality of recombinant cytochrome c. Based on the search results and standard practices for heme protein expression, the following comprehensive approach can be implemented:

Strategies for Optimizing Heme Incorporation:

• Heme Precursor Supplementation:

  • Add δ-aminolevulinic acid (δ-ALA) at 0.5-1.0 mM to culture media

  • δ-ALA is the first committed precursor in heme biosynthesis

  • Add at the time of induction or 1-2 hours prior

• Expression Conditions Optimization:

  • Lower temperature (16-18°C) after induction slows protein synthesis, allowing time for heme incorporation

  • Extended expression time (20-24 hours) improves heme incorporation efficiency

  • Maintain aerobic conditions for proper heme synthesis

• Co-expression with Helper Proteins:

  • Co-express with bacterial cytochrome c maturation (Ccm) proteins

  • The pETDUET-1 vector system allows co-expression of multiple proteins

  • Consider adding specific lyases or chaperones that assist heme incorporation

• Expression Host Selection:

  • E. coli strains with enhanced heme synthesis capacity

  • Yeast systems for more native-like post-translational processing

Assessment of Heme Incorporation:

• UV-Visible Spectroscopy:

  • A₄₁₀/A₂₈₀ ratio >4 indicates good heme incorporation

  • Distinct α and β bands in the reduced spectrum (550-560 nm)

  • Soret band position (typically ~410 nm) reveals heme environment

• Pyridine Hemochromogen Assay:

  • Quantitative determination of heme content

  • Compare with theoretical 1:1 heme:protein ratio

• Resonance Raman Spectroscopy:

  • Provides detailed information about heme coordination and environment

  • Helps confirm native-like heme incorporation

Protocol for Heme Incorporation Assessment:

• Purify recombinant protein using standard methods

• Record UV-visible spectrum (250-700 nm)

• Calculate heme:protein ratio using extinction coefficients

• Perform activity assays to confirm functional heme incorporation

• Use resonance Raman to confirm proper heme environment

By systematically implementing these strategies and assessment methods, researchers can significantly improve heme incorporation in recombinant G. barbadense cytochrome c, leading to proteins with native-like spectroscopic and functional properties.

What controls and validation methods are essential when working with recombinant Gossypium barbadense cytochrome c?

Robust controls and validation methods are critical for ensuring the reliability and reproducibility of research involving recombinant G. barbadense cytochrome c. Based on the search results and standard practices in protein biochemistry, the following comprehensive framework is recommended:

Essential Controls:

• Expression Controls:

  • Empty vector control (same expression conditions)

  • Non-induced transformed cells

  • Expression of well-characterized protein (e.g., GFP) to verify system function

• Purification Controls:

  • Protein-free buffer processed through all purification steps

  • Well-characterized protein purified using identical protocol

  • Commercial cytochrome c (e.g., from bovine heart) as reference standard

• Activity Assay Controls:

  • Reaction mixture without recombinant protein

  • Heat-inactivated recombinant protein

  • Commercial cytochrome c in parallel assays

  • Substrate-free and enzyme-free reactions

Validation Methods:

• Structural Validation:

  • SDS-PAGE for purity and molecular weight confirmation

  • Western blot with anti-cytochrome c antibodies

  • Mass spectrometry for accurate mass determination

  • Circular dichroism for secondary structure assessment

  • UV-visible spectroscopy for heme environment characterization

  • Resonance Raman spectroscopy for detailed heme coordination analysis

• Functional Validation:

  • Electron transfer activity with standard acceptors (cytochrome c, K₃Fe(CN)₆, DCPIP)

  • Redox potential determination through spectroelectrochemistry

  • Binding assays with physiological partners

  • Thermal stability assessment

Validation Checklist and Acceptance Criteria:

By implementing these controls and validation methods, researchers can ensure that their recombinant G. barbadense cytochrome c preparations are of high quality and suitable for reliable experimental applications in both basic and applied research contexts.

How can recombinant Gossypium barbadense cytochrome c contribute to understanding cotton evolution and speciation?

Recombinant G. barbadense cytochrome c represents a valuable tool for investigating the evolutionary relationships and speciation processes within the Gossypium genus. The search results highlight the evolutionary divergence between G. barbadense and other cotton species , providing a foundation for such studies:

Research Applications in Evolutionary Studies:

• Comparative Sequence and Structure Analysis:

  • G. barbadense shows specific evolutionary adaptations in its mitochondrial genome (677,434 bp circular molecule)

  • Recombinant cytochrome c can be used to assess functional consequences of sequence variations

  • Structural differences in cytochrome c may correlate with environmental adaptations

• Molecular Clock Applications:

  • Cytochrome c is relatively conserved, making it useful for dating evolutionary events

  • Recombinant proteins from different Gossypium species can be compared to establish divergence rates

  • Correlation with geological events can provide insights into cotton speciation timing

• Functional Evolution Studies:

  • Compare biochemical properties (redox potential, thermal stability) across Gossypium species

  • Assess adaptation-related functional differences in electron transfer efficiency

  • Identify species-specific post-translational modifications

Methodological Approach:

• Express recombinant cytochrome c from multiple Gossypium species (G. barbadense, G. hirsutum, G. arboreum, etc.)

• Perform comprehensive sequence and structural comparisons

• Measure functional parameters under identical conditions

• Correlate differences with evolutionary history and ecological adaptations

Potential Experimental Design:

By systematically comparing recombinant cytochrome c proteins across Gossypium species, researchers can gain valuable insights into the molecular basis of cotton evolution and speciation, potentially informing both fundamental evolutionary biology and applied cotton improvement efforts.

What role might Gossypium barbadense cytochrome c play in plant defense mechanisms against herbivores?

The search results provide compelling evidence for complex defense mechanisms in G. barbadense against herbivores, particularly the cotton bollworm Helicoverpa armigera . While cytochrome c is not directly mentioned in this context, we can infer potential roles based on known functions of cytochrome proteins in plant defense:

Potential Roles in Defense Mechanisms:

• Oxidative Burst Signaling:

  • Herbivore infestation triggers transcriptional changes in G. barbadense (5,629 differentially expressed genes)

  • Cytochrome c release from mitochondria can serve as a signaling event in stress responses

  • May contribute to programmed cell death pathways that limit herbivore damage

• Integration with Hormone Signaling:

  • Jasmonic acid (JA) pathway plays a central role in G. barbadense defense (88 out of 90 JA-related genes upregulated)

  • Cytochrome c may interact with components of hormone signaling cascades

  • Could influence defense-related metabolic shifts

• Connection to Secondary Metabolite Production:

  • Herbivore-induced terpene production includes selinene, α-gurjunene, β-elemene, and limonene

  • Cytochrome P450s are involved in terpene biosynthesis pathways

  • Energy requirements for defense compound synthesis depend on efficient electron transport systems

Experimental Approaches to Investigate This Role:

• Gene Expression Analysis:

  • Monitor cytochrome c expression patterns during herbivore infestation

  • Compare with expression of defense-related genes

  • Use qPCR methods as described for G. barbadense

• Protein Localization Studies:

  • Track cytochrome c localization before and after herbivore attack

  • Assess potential release from mitochondria during defense responses

  • Use fluorescently tagged recombinant protein for visualization

• Functional Interaction Assays:

  • Identify defense-related proteins that interact with cytochrome c

  • Assess effects of recombinant cytochrome c on defense enzyme activities

  • Evaluate impact on ROS generation and signaling

• Genetic Modification Approaches:

  • Modulate cytochrome c expression levels and assess impact on herbivore resistance

  • Use recombinant protein to complement cytochrome c-deficient plants

  • Evaluate changes in defense compound production

By exploring these potential roles, researchers can develop a more comprehensive understanding of the molecular mechanisms underlying G. barbadense defense against herbivores, potentially identifying novel targets for enhancing crop protection strategies.

How can structural insights from recombinant Gossypium barbadense cytochrome c inform protein engineering for improved stress tolerance?

Structural characterization of recombinant G. barbadense cytochrome c can provide valuable insights for protein engineering approaches aimed at enhancing plant stress tolerance. While the search results don't directly address this application, we can develop a methodological framework based on the available information:

Structural Features Relevant to Stress Tolerance:

• Redox-Active Sites:

  • Heme environment determines redox potential

  • Conservative substitutions around heme pocket can alter electron transfer properties

  • Spectroscopic methods like resonance Raman can characterize these sites

• Protein Stability Determinants:

  • Regions affecting thermal and oxidative stability

  • Hydrogen bonding networks and salt bridges

  • Hydrophobic core packing

• Interaction Surfaces:

  • Domains mediating binding to partner proteins

  • Regions involved in membrane association during stress

  • Post-translational modification sites

Protein Engineering Strategies:

• Rational Design Approach:

  • Target specific residues based on structural analysis

  • Introduce stabilizing mutations identified from thermophilic organisms

  • Modify surface charges to enhance specific interactions

• Directed Evolution Methods:

  • Create libraries of cytochrome c variants

  • Screen for enhanced stability under stress conditions

  • Select for improved electron transfer under oxidative stress

• Chimeric Protein Design:

  • Combine stress-tolerant domains from different species

  • Engineer fusion proteins with enhanced functional properties

  • Create cytochrome c variants with novel functions

Methodological Workflow:

• Determine high-resolution structure of G. barbadense cytochrome c using X-ray crystallography or NMR

• Identify regions critical for stability and function using computational analysis

• Design variants with targeted modifications

• Express and characterize recombinant variants using spectroscopic and functional assays

• Test promising variants in plant systems for enhanced stress tolerance

Potential Engineering Targets and Expected Outcomes:

By systematically applying these protein engineering approaches to recombinant G. barbadense cytochrome c, researchers can develop novel variants with enhanced properties for agricultural applications, potentially contributing to the development of stress-tolerant cotton cultivars.