Recombinant Schizosaccharomyces pombe Probable cytochrome b5 1 (SPBC29A10.16c)

What is the structure and key functional domains of Schizosaccharomyces pombe Probable cytochrome b5 1?

Schizosaccharomyces pombe Probable cytochrome b5 1 (SPBC29A10.16c) is a small heme-binding protein of 124 amino acids with a molecular structure typical of the cytochrome b5 family. Like other cytochrome b5 proteins, it possesses:

• A highly conserved heme-binding motif (-HPGG-) located in the N-terminal domain (specifically at positions 43-46 in the amino acid sequence)

• A predominant N-terminal domain that likely protrudes into the cytosol

• A hydrophobic transmembrane domain near the C-terminus that anchors the protein to the endoplasmic reticulum and/or outer mitochondrial membrane

• A short C-terminal tail that influences intracellular localization

The full amino acid sequence as reported in the recombinant expression system is:
MSVKYFEPEEIVEHNNSKDMYMVINGKVYDVSNFADDHPGGLDIMLDYAGQDATKAYQDIGHSIAADELLEEMYIGDLKPGTEERLKELKKPRSFDNDTPPLPLLIALIVLPAIAVIVFVKLNK

The protein contains the signature cytochrome b5 family heme-binding domain with the conserved motif [FY]-[LIVMK]-(I)-(Q)-H-P-[GA]-G, which is essential for its electron transfer capabilities .

What expression systems are recommended for producing recombinant S. pombe cytochrome b5 1?

For effective expression of recombinant S. pombe cytochrome b5 1 (SPBC29A10.16c), E. coli is the preferred heterologous expression system based on available research data. The methodological approach includes:

• Vector selection: Utilize expression vectors with strong promoters (such as T7) compatible with E. coli expression systems and include an N-terminal His-tag for purification purposes .

• Expression conditions: When expressing similar cytochrome proteins, researchers have reported successful expression using the following approaches:

  • For unlabeled protein: Standard expression in LB media at 35-37°C

  • For isotopically labeled protein ([U-15N] or [U-13C,U-15N]):

    • Cells are typically harvested after approximately 20 hours of incubation at 35°C with shaking at 200 rpm

    • For triple-labeled protein ([U-13C,U-2H,U-15N]), longer incubation periods (up to 48 hours) are recommended

• Purification approach: Employ affinity chromatography using the N-terminal His-tag, followed by size exclusion chromatography if higher purity is required.

• Storage considerations: Store the purified protein as a lyophilized powder. After reconstitution, it's recommended to:

  • Reconstitute 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 the default)

  • Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles

How should researchers reconstitute and handle purified recombinant S. pombe cytochrome b5 1 protein?

Proper handling of recombinant S. pombe cytochrome b5 1 (SPBC29A10.16c) is critical for maintaining its structural integrity and functional activity. The recommended protocol includes:

• Initial preparation:

  • Briefly centrifuge the vial containing lyophilized protein to bring contents to the bottom

  • Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Stabilization:

  • Add glycerol to a final concentration of 5-50% (default recommendation is 50%)

  • This helps maintain protein stability during storage

• Storage conditions:

  • For long-term storage: Aliquot the reconstituted protein and store at -20°C/-80°C

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

  • Avoid repeated freeze-thaw cycles as this can compromise protein integrity

• Buffer considerations:

  • The protein is typically supplied in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

  • When designing experiments, consider this buffer composition and its potential influence on your assay systems

• Quality control:

  • Before use, verify protein integrity using SDS-PAGE

  • The product should show greater than 90% purity on SDS-PAGE analysis

What experimental approaches can be used to study the electron transfer functions of S. pombe cytochrome b5 1?

Investigating the electron transfer capabilities of S. pombe cytochrome b5 1 requires sophisticated biochemical and biophysical approaches:

• Cytochrome c reductase activity assays:

  • Isolate microsomal fractions containing the recombinant cytochrome b5

  • Measure NADH- and NADPH-dependent cytochrome c reductase activities

  • Compare activities using different electron donors (NADH vs. NADPH) to assess functional preferences

• Spectroscopic analysis:

  • UV-visible spectroscopy to monitor the redox state of the heme group

  • The reduced and oxidized forms of cytochrome b5 have characteristic absorption spectra that can be used to track electron transfer events

  • Circular dichroism (CD) spectroscopy to analyze protein secondary structure and conformational changes upon redox reactions

• Protein-protein interaction studies:

  • Split ubiquitin membrane yeast-two-hybrid (Y2H) assays to identify potential interaction partners

  • Biomolecular fluorescence complementation (BiFC) to confirm interactions in vivo

  • These methods have successfully identified interactions between cytochrome b5 proteins and their functional partners in other systems

• NMR spectroscopy:

  • For structural characterization and mapping interaction interfaces

  • Differential line broadening of NMR resonances can provide insights into the interaction epitope recognized by potential redox partners

  • This approach has been successfully used to characterize cytochrome b5 interactions with cytochrome P450 enzymes

• Redox potential measurements:

  • Determine the redox potential of the heme group (~20 mV in other cytochrome b5 proteins)

  • This information is crucial for understanding the protein's electron transfer capabilities

How can researchers investigate the potential interaction between S. pombe cytochrome b5 1 and cytochrome P450 enzymes?

Investigating interactions between S. pombe cytochrome b5 1 and cytochrome P450 enzymes requires a multi-technique approach:

• In vitro reconstitution experiments:

  • Express and purify both the recombinant cytochrome b5 1 and target cytochrome P450 enzymes

  • Reconstitute them in appropriate membrane mimetics (detergent micelles or lipid bicelles)

  • Measure cytochrome P450 activity in the presence and absence of cytochrome b5 to assess functional modulation

• Structural characterization of the complex:

  • NMR spectroscopy is particularly valuable for characterizing membrane protein interactions

  • For optimal NMR studies of the complex, isotopically labeled cytochrome b5 should be prepared:

    • [U-15N]-labeled for simple 2D experiments

    • [U-13C,U-15N]-labeled for backbone assignments

    • [U-13C,U-2H,U-15N]-labeled for more complex 3D experiments

• Computational modeling and docking:

  • Use data-driven docking algorithms like HADDOCK (High Ambiguity Driven Biomolecular Docking)

  • Integrate experimental restraints from NMR, mutagenesis, and biochemical data

  • Generate and validate structural models of the complex

• Mutagenesis studies:

  • Create strategic mutations in the heme-binding motif (-HPGG-) and assess their impact on:

    • Protein stability

    • Heme incorporation

    • Interaction with cytochrome P450 enzymes

    • Electron transfer efficiency

• Kinetic analysis:

  • Perform steady-state and pre-steady-state kinetic measurements

  • Determine how cytochrome b5 affects the rate-limiting steps in cytochrome P450 catalysis

  • Assess whether cytochrome b5 alters substrate binding, product release, or electron transfer steps

What roles might S. pombe cytochrome b5 1 play in cellular metabolism based on studies of homologous proteins?

Based on research on homologous cytochrome b5 proteins in other organisms, S. pombe cytochrome b5 1 likely participates in multiple metabolic pathways:

• Fatty acid metabolism:

  • Cytochrome b5 proteins provide electrons for fatty acid desaturation, hydroxylation, and elongation reactions

  • The interaction with fatty acid elongase components suggests involvement in very-long-chain fatty acid (VLCFA) synthesis

  • Studies in plants have shown that cytochrome b5 interacts with elongase components like ELO1 and ELO2, forming part of a larger protein complex

• Sterol biosynthesis:

  • Cytochrome b5 may modulate the activity of cytochrome P450 enzymes involved in ergosterol biosynthesis in fungi

  • In X. dendrorhous, researchers have demonstrated connections between cytochrome b5 reductase (CBR) genes and sterol biosynthesis

  • The presence of differential NADH- and NADPH-dependent cytochrome c reductase activities suggests regulatory roles in redox metabolism

• Specialized metabolite formation:

  • In plants, cytochrome b5 participates in forming specialized metabolites like flavonoids and phenolic esters

  • The S. pombe homolog might similarly participate in secondary metabolite formation

• Regulatory functions beyond electron transfer:

  • Cytochrome b5 proteins interact with non-catalytic proteins involved in various cellular processes

  • These interactions suggest roles in coordinating metabolic and cellular processes based on redox status or carbon availability

  • The protein may serve as a sensor linking cellular redox state to specific metabolic pathways

How can comparative studies with cytochrome b5 proteins from other organisms enhance our understanding of the S. pombe protein?

Comparative studies offer valuable insights into the function and evolution of S. pombe cytochrome b5 1:

• Sequence alignment and phylogenetic analysis:

  • Compare the S. pombe cytochrome b5 1 sequence with homologs from diverse organisms

  • Identify conserved regions beyond the heme-binding motif

  • This approach can reveal evolutionary relationships and functional constraints

• Structural comparisons:

  • The first structure of full-length rabbit ferric microsomal cytochrome b5 (16 kDa) has been determined by NMR in different membrane mimetics

  • Similar NMR studies on the S. pombe protein could reveal structural similarities and differences

  • Comparing membrane-binding domains may provide insights into subcellular localization and membrane interactions

• Functional complementation:

  • Express S. pombe cytochrome b5 1 in other organisms with cytochrome b5 deletions

  • Assess whether the S. pombe protein can restore normal function

  • This approach has been valuable in studying the X. dendrorhous CBR and CYB5 genes

• Comparative protein-protein interaction networks:

  • Map interaction partners of cytochrome b5 across different species

  • Identify conserved and species-specific interactions

  • This data can reveal functional adaptations and evolutionary innovations

The table below summarizes key features of cytochrome b5 proteins from different organisms for comparative analysis:

What methodological challenges exist in studying membrane-associated proteins like S. pombe cytochrome b5 1?

Membrane protein research presents several technical challenges that researchers should consider when working with S. pombe cytochrome b5 1:

• Expression and purification challenges:

  • Maintaining proper folding and heme incorporation during heterologous expression

  • Obtaining sufficient yields for structural and functional studies

  • Developing efficient purification protocols that preserve the native conformation

• Membrane mimetic selection:

  • Different membrane mimetics (detergents, bicelles, nanodiscs) can influence protein structure and function

  • For NMR studies of cytochrome b5, both DPC (dodecylphosphocholine) micelles and lipid bicelles have been successfully used

  • The choice of membrane mimetic should be experimentally validated for the S. pombe protein

• Structural characterization methods:

  • X-ray crystallography is challenging due to the presence of the hydrophobic transmembrane domain

  • NMR spectroscopy requires isotopic labeling and careful optimization of experimental conditions

  • For triple-labeled ([U-13C,U-2H,U-15N]) cytochrome b5, cells must be gradually adapted to grow in 100% D2O media

• Functional assays in membrane environments:

  • Reconstituting electron transfer chains in artificial membrane systems

  • Ensuring proper orientation of the protein in the membrane

  • Maintaining native-like protein-protein interactions

• In vivo studies:

  • Generating viable deletion mutants in S. pombe

  • Developing appropriate phenotypic assays to detect subtle metabolic changes

  • Using techniques like the ones employed for X. dendrorhous, where researchers generated targeted gene deletions (cbr.1::hph and cbr.2::zeo) to study gene function