Recombinant Guillardia theta Cytochrome c biogenesis protein ccs1 (ccs1)

What is Guillardia theta Cytochrome c biogenesis protein CCS1 and what is its basic function?

Guillardia theta Cytochrome c biogenesis protein CCS1 (ccs1) is a protein involved in the biogenesis pathway of cytochrome c in the cryptophyte alga Guillardia theta. The protein plays a crucial role in the assembly and maturation of cytochrome c, which is essential for electron transport in cellular respiration and photosynthesis. In its recombinant form, it typically consists of 414 amino acids (full-length) and can be produced with tags such as His-tag to facilitate purification and experimental manipulation .

Similar to other CCS proteins across species, G. theta CCS1 likely functions in heme attachment and/or protein folding during cytochrome c maturation. Comparative analysis with the better-studied Arabidopsis thaliana CCS1 suggests it may be localized to chloroplasts or plastids in cryptophytes and participate in photosynthetic electron transport system assembly .

How does G. theta CCS1 compare structurally to CCS1 proteins in other organisms?

The protein shows evolutionary relationships with other cryptophyte proteins due to the unique evolutionary history of cryptophyte plastid genomes, which range from 121 Kbp (as in Guillardia theta) to larger sizes in other cryptophyte species . Unlike the CCS1 in yeast, which functions primarily as a copper chaperone for copper-zinc superoxide dismutase (SOD1), the G. theta CCS1 likely has more specialized functions related to photosynthetic machinery assembly in plastids.

What are the key conserved domains and motifs in G. theta CCS1 protein?

The G. theta CCS1 protein contains several important conserved regions that are crucial for its function:

• Transmembrane domains: Analysis of the amino acid sequence (MNYIIKFLNKLNNLTVAIIILLAIALASALGTVIEQNKNTDFYLKNYPLTKPLFNFVTSD LILKFGLDHVYTSWWFIFLIILLLLSLTLCTITRQLPALKLARLWQFYTNFNTKAKFQIR...) reveals hydrophobic segments consistent with membrane-spanning regions .

• Potential metal-binding motifs: By comparison with better-characterized CCS proteins from other organisms, G. theta CCS1 likely contains conserved cysteine-rich motifs involved in coordinating metal ions (possibly copper) during cytochrome c maturation.

• Substrate recognition domains: Regions that interact with the apocytochrome c substrate during the maturation process.

These domains work in concert to enable the proper folding and metalation of cytochrome c during its biogenesis.

What are the optimal conditions for expressing recombinant G. theta CCS1 in E. coli?

Successful expression of recombinant G. theta CCS1 in E. coli requires careful optimization of several parameters:

• Expression system: The protein is typically expressed with an N-terminal His tag to facilitate purification. E. coli is the preferred expression system due to its simplicity and high yield potential .

• Temperature: Lower induction temperatures (16-25°C) are often preferred to enhance proper folding of membrane-associated proteins like CCS1.

• Induction parameters: IPTG concentration typically between 0.1-1.0 mM, with induction periods ranging from 4-16 hours depending on temperature.

• Media composition: Enriched media such as Terrific Broth (TB) or Super Broth (SB) often yield better results than standard LB media, especially for membrane-associated proteins.

• Codon optimization: Since G. theta uses different codon preferences than E. coli, codon optimization of the expression construct may significantly improve yields.

The expressed protein can then be extracted and purified using nickel affinity chromatography targeting the His tag, followed by size exclusion chromatography if higher purity is required.

What are the best methods for reconstitution and storage of purified G. theta CCS1?

For optimal handling of purified G. theta CCS1:

• Reconstitution: The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Add glycerol to a final concentration of 5-50% (with 50% being standard) to stabilize the protein structure .

• Storage buffer: A Tris/PBS-based buffer with pH 8.0 containing 6% trehalose is recommended for maintaining protein stability .

• Aliquoting: Create multiple small working aliquots to avoid repeated freeze-thaw cycles, which can significantly decrease protein activity.

• Storage temperature: For long-term storage, maintain at -20°C/-80°C. Working aliquots can be stored at 4°C for up to one week .

• Freeze-thaw handling: Centrifuge vials briefly before opening to bring contents to the bottom. Avoid repeated freeze-thaw cycles as they can dramatically reduce protein activity and integrity .

What assays can be used to verify the functional activity of recombinant G. theta CCS1?

Several complementary approaches can be employed to assess the functional activity of recombinant G. theta CCS1:

• Metal binding assays: Given the likely role in metal ion handling (by comparison to other CCS proteins), copper binding can be assessed using direct competition assays with copper chelators such as BCA (bicinchoninic acid) or BCS (bathocuproine sulfonate), which allow colorimetric quantification of copper-bound species .

• Interaction studies: Co-immunoprecipitation or pull-down assays to detect interaction with cytochrome c or other components of the cytochrome c maturation pathway.

• Reconstitution experiments: In vitro reconstitution of cytochrome c maturation using purified components including the recombinant CCS1, apocytochrome c, heme, and other necessary factors.

• Complementation assays: Testing whether the G. theta CCS1 can functionally complement CCS1-deficient mutants in model organisms such as Chlamydomonas or Arabidopsis.

• Spectroscopic analysis: Monitoring changes in spectroscopic properties during interactions with substrate proteins or cofactors as indicators of functional activity.

How does the phosphorylation state of CCS1 affect its function in cytochrome c biogenesis?

While specific phosphorylation data for G. theta CCS1 is not directly reported in the search results, insights can be drawn from related CCS1 proteins such as Arabidopsis thaliana CCS1, which undergoes phosphorylation at multiple sites:

• Phosphorylation sites: In A. thaliana CCS1, phosphorylation occurs at residues T58, S60, S62, S64, T69, and T72, suggesting a regulated cluster of phosphorylation events .

• Regulatory implications: These phosphorylation events likely represent regulatory mechanisms that could:

  • Modulate protein-protein interactions during cytochrome c maturation

  • Regulate subcellular localization or membrane association

  • Control protein stability or turnover

  • Serve as molecular switches that activate or deactivate specific functions of CCS1

• Experimental approaches: Researchers studying G. theta CCS1 should consider site-directed mutagenesis of potential phosphorylation sites to create phosphomimetic (e.g., Ser/Thr → Asp) or phosphodeficient (e.g., Ser/Thr → Ala) variants to assess functional consequences.

What is known about metal binding capabilities of G. theta CCS1 and how can they be characterized?

The metal binding properties of G. theta CCS1 can be inferred from studies of related proteins and characterized through several methods:

• Metal binding motifs: By analogy with other CCS proteins, G. theta CCS1 likely contains conserved cysteine-rich motifs that coordinate metal ions, particularly copper.

• Affinity measurements: Direct competition assays with metal chelators can determine binding affinities. In related copper chaperones, Cu(I) dissociation constants have been measured in the range of 10^-17 to 10^-21 M, indicating very high affinity binding .

• Spectroscopic characterization: Techniques such as X-ray absorption spectroscopy (XAS), electron paramagnetic resonance (EPR), or UV-visible spectroscopy can provide information about the coordination environment and oxidation state of bound metals.

• Mutagenesis studies: Site-directed mutagenesis of putative metal-binding residues coupled with functional assays can identify critical residues for metal coordination.

• Thermodynamic analysis: Isothermal titration calorimetry (ITC) can provide detailed thermodynamic parameters of metal binding events.

Understanding the metal binding properties is essential for elucidating the molecular mechanism of G. theta CCS1 in cytochrome c biogenesis, particularly if it functions in a manner similar to other metallochaperones.

How has the CCS1 protein evolved across cryptophyte species, and what does this reveal about functional specialization?

The evolution of CCS1 proteins across cryptophyte species reflects the unique evolutionary history of these organisms and their plastids:

• Plastid genome context: Cryptophyte plastid genomes range in size from approximately 121 Kbp in Guillardia theta to larger sizes in other species . The genomic context of ccs1 within these plastid genomes provides insights into evolutionary constraints and selection pressures.

• Secondary endosymbiosis: Cryptophytes acquired their plastids through secondary endosymbiosis, creating a complex evolutionary history that has shaped the structure and function of plastid proteins including CCS1.

• Functional specialization: Comparative analysis of CCS1 sequences across cryptophyte species can reveal conserved domains essential for core functions versus variable regions that may contribute to species-specific adaptations.

• Phylogenetic analysis: Constructing phylogenetic trees based on CCS1 sequences from different cryptophytes can elucidate evolutionary relationships and patterns of molecular evolution.

These evolutionary analyses provide a framework for understanding the functional specialization of G. theta CCS1 within the context of cryptophyte biology and photosynthetic adaptation.

How can recombinant G. theta CCS1 be used to study copper trafficking pathways in photosynthetic organisms?

Recombinant G. theta CCS1 represents a valuable tool for investigating copper trafficking in photosynthetic systems:

• In vitro reconstitution: Purified recombinant G. theta CCS1 can be used to reconstitute copper transfer pathways in vitro, allowing detailed kinetic and thermodynamic characterization of metal transfer steps.

• Affinity gradient analysis: By determining Cu(I) binding affinities at different sites within CCS1 and its target proteins, researchers can establish whether copper transfer proceeds via a thermodynamically driven affinity gradient, as demonstrated in other copper chaperone systems where transfer proceeds from sites with lower affinity to sites with higher affinity .

• Interaction network mapping: Combining recombinant G. theta CCS1 with techniques such as cross-linking mass spectrometry or proximity labeling can map the copper trafficking interactome in cryptophyte plastids.

• Biosensor development: Engineered variants of G. theta CCS1 incorporating fluorescent sensors could be developed to monitor copper trafficking in vivo.

These approaches can reveal fundamental principles of metal homeostasis in photosynthetic organisms and potentially identify novel targets for biotechnological applications.

What experimental approaches can resolve the dual chaperoning roles of CCS1 in both copper delivery and protein folding?

Investigating the proposed dual chaperoning functions of CCS1 proteins requires sophisticated experimental strategies:

• Domain-specific mutations: Creating variants with mutations in putative copper-binding versus protein-interaction domains can separate metal chaperoning from protein folding functions.

• Real-time monitoring: Developing assays that simultaneously track copper transfer and conformational changes in substrate proteins can provide direct evidence for dual chaperoning.

• Structural studies: Obtaining high-resolution structures of CCS1-substrate complexes at different stages of the maturation process can reveal the molecular basis of dual chaperoning.

• In vitro reconstitution with defined components: Systematic analysis of cytochrome c maturation with purified components can dissect the specific contributions of CCS1 to different aspects of the process.

By analogy with other systems, CCS proteins may function as "dual chaperones" mediating both copper delivery and protein folding/disulfide bond formation. In yeast CCS1, studies have shown it possesses both copper binding and molecular chaperoning roles in the activation of superoxide dismutase (SOD1) .

How can studies of G. theta CCS1 inform our understanding of metalloprotein assembly in diverse biological systems?

Research on G. theta CCS1 can provide broader insights into fundamental principles of metalloprotein assembly:

• Affinity gradient principles: Studies of copper transfer from CCS1 to its targets can test whether the "thermodynamically driven affinity gradient" model observed in other metal transfer pathways is conserved across diverse biological systems .

• Coordination chemistry in biological context: Detailed characterization of metal binding sites in G. theta CCS1 can reveal how protein environments tune metal coordination geometry and reactivity for specific biological functions.

• Evolution of metal homeostasis systems: Comparative analysis of CCS1 across cryptophytes and other photosynthetic lineages can illuminate how metal trafficking systems have evolved to accommodate different cellular environments and physiological demands.

• Integration of metal delivery and protein folding: The potential dual chaperone role of CCS1 provides a model for studying how metal insertion is coordinated with protein folding to ensure proper metalloprotein assembly.

These fundamental insights extend beyond cryptophyte biology and can inform our understanding of metalloprotein assembly in diverse systems from bacteria to humans, with potential applications in biotechnology and medicine.