Recombinant Neurospora crassa Phosphatidylinositol transfer protein sfh-5 (sfh-5)

What is Neurospora crassa phosphatidylinositol transfer protein Sfh-5 and how does it differ from other PITPs?

Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Unlike typical PITPs that transfer phosphatidylinositol between membranes in vitro and stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Instead, it functions as a redox-active penta-coordinate high spin Fe(III) hemoprotein with an unusual heme-binding arrangement involving a co-axial tyrosine/histidine coordination strategy . This unique property establishes Sfh5 as the prototype of a new class of fungal hemoproteins, demonstrating the versatility of the Sec14-fold as a scaffold for binding chemically distinct ligands .

Methodology for characterization:

• Spectroscopic analysis reveals a Soret peak with an absorption maximum at 404 nm, characteristic of Fe(III) heme

• Sodium dithionite treatment causes a red-shift of the Soret peak to 427 nm, indicating reduction to Fe(II)

• Pyridine hemochromagen assays show formation of additional spectral peaks at 525 nm and 557 nm, diagnostic of β- and α-bands of non-covalently bound heme b

What is the genetic basis of Sfh-5 in Neurospora crassa?

In Neurospora crassa, Sfh-5 is encoded by the NCU02200 gene located on chromosome 1. The gene produces a phosphatidylinositol transfer protein that shares homology with SFH5 in Saccharomyces cerevisiae and similar proteins in other fungi including Magnaporthe oryzae and Kluyveromyces lactis . The protein sequence indicates conservation of certain features of the Sec14-fold while exhibiting unique properties related to its heme-binding capacity.

What are the recommended methods for expressing and purifying recombinant Neurospora crassa Sfh-5?

For optimal expression and purification of recombinant Sfh-5, researchers should consider the following methodological approach:

• Expression system: E. coli is commonly used with a His6-tag for ease of purification.

• Expression conditions: Induction at OD600 of 0.5-0.7 with IPTG (0.5 mM), followed by growth at 30°C for 3-4 hours.

• Purification steps:

  • Affinity chromatography using Ni-NTA columns

  • Further purification via gel filtration to separate the functional protein

The purified His6-Sfh5 protein exhibits an intense reddish-brown color resistant to dialysis or gel filtration, indicating a tightly-bound transition metal (Fe). Inductively coupled plasma mass spectrometry (ICP-MS) analysis can be used to identify the bound metal as Fe, with quantification indicating an Fe/Sfh5 molar ratio of approximately 0.33 .

How can researchers effectively analyze the heme-binding properties of Sfh-5?

To analyze the heme-binding properties of Sfh-5, researchers should implement the following approaches:

• Spectroscopic characterization:

  • UV-visible spectroscopy to identify characteristic Soret peaks (404 nm for Fe(III), 427 nm for Fe(II))

  • Pyridine hemochromagen assays to confirm non-covalent heme b binding

• Heme exchange assays:

  • Use apo-myoglobin as a heme acceptor

  • Monitor pseudo-peroxidase activities to track potential transfer

  • Results indicate Sfh5 binds heme with high affinity and the bound heme is not readily exchangeable

• Structural analysis:

  • X-ray crystallography reveals a penta-coordinate high spin Fe(III) heme with co-axial tyrosine/histidine coordination

  • Analysis of the complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems

What experimental designs are most appropriate for studying Sfh-5 function in vivo?

For in vivo studies of Sfh-5 function, split-plot experimental designs are particularly valuable. These designs enable researchers to analyze multiple factors simultaneously and account for variations in experimental unit levels.

Table 1: Split-Plot Design Structure for Sfh-5 Functional Studies

Where a = levels of Sfh-5 expression, b = levels of cellular conditions, r = replications, n = observations per treatment combination .

This experimental structure allows for assessment of:

• Main effects of Sfh-5 expression level

• Effects of various cellular conditions

• Interaction between Sfh-5 expression and cellular environment

• Appropriate error terms for each level of the experimental design

What is the relationship between Sfh-5 and phosphatidylinositol signaling in Neurospora crassa?

Unlike typical PITPs, Sfh-5 does not actively participate in phosphatidylinositol transfer between membranes. Research demonstrates an antagonistic relationship between heme-binding and phosphatidylinositol-binding in Sfh-5:

• Wild-type Sfh-5 exhibits minimal PtdIns transfer activity in vitro and does not effectively stimulate PtdIns 4-OH kinase activity in vivo.

• Heme-binding deficient Sfh-5 mutants show resuscitated PtdIns-transfer activity in vitro and enhanced stimulation of PtdIns 4-OH kinase activity in vivo.

• Structural analysis suggests heme-binding locks Sfh-5 into a substantially "closed" conformer, which appears to be the functional configuration .

This evidence indicates that Sfh-5's primary function may not be related to phosphoinositide signaling despite its structural relationship to other PITPs.

How does Sfh-5 contribute to oxidative stress responses in fungi?

Chemogenomic analyses have identified a specific "heme-requiring" signature for Sfh-5-deficient yeast when challenged with the organic oxidant oltipraz. This compound induces superoxide radical formation and triggers a general "heme-requiring" response in yeast .

The evidence for Sfh-5's role in oxidative stress responses includes:

• Enhanced sensitivity of Sfh-5-deficient yeast cells to oltipraz challenge, similar to strains with reduced heme-biosynthetic capacity (heterozygous diploid yeast strains +/hem1Δ, +/hem2Δ, +/hem3Δ, and +/hem12Δ).

• Parallels between Sfh-5's potential function and the role of hemophores in binding and releasing heme on demand.

• The identification of Sfh-5 orthologs in pathogenic fungi like Cryptococcus neoformans suggests potential importance in stress responses encountered by fungi in host contexts .

These findings suggest Sfh-5 may participate in stress responses to organic oxidants, particularly in pathogenic and commensal fungi interacting with host environments.

How is Sfh-5 evolutionarily conserved across fungal species?

Sfh-5 orthologs have been identified across diverse fungal species, indicating evolutionary conservation of this novel hemoprotein. Comparative analysis reveals:

• Orthologs in various fungi including:

  • Saccharomyces cerevisiae (SFH5)

  • Magnaporthe oryzae (rice blast fungus)

  • Kluyveromyces lactis

  • Eremothecium gossypii

  • Cryptococcus neoformans

• Conservation patterns:

  • The basic Sec14-fold structure is preserved across species

  • Heme-binding motifs show high conservation

  • Surface electrostatics vary, with Sfh-5 exhibiting distinctive electropositive character throughout its lipid-binding domain surface

• Functional diversification:

  • While the PtdIns headgroup-binding barcode residues are conserved in Sec14-like Sfh PITPs

  • Clear departure from the PtdCho-binding barcode in Sfh proteins

  • Distinct cavity microenvironments for each member, suggesting different ligand specificities

This conservation pattern establishes Sfh-5 as the prototype of a new class of fungal hemoproteins and demonstrates the versatility of the Sec14-fold as a scaffold for binding different ligands.

How might Neurospora crassa Sfh-5 relate to genome defense mechanisms like RIP?

While direct evidence linking Sfh-5 to genome defense mechanisms is limited, Neurospora crassa employs several distinctive epigenetic phenomena, including Repeat-Induced Point mutation (RIP), which could potentially intersect with Sfh-5 function.

The RIP mechanism in Neurospora crassa:

• Functions as a genome defense system that targets duplicated sequences with C→T mutations

• Operates during the sexual cycle

• Results in an unusually high mutation rate (136.6 ± 21.5 mutations per genome per generation)

Potential relationships between Sfh-5 and genome defense mechanisms could be explored through:

• Examination of Sfh-5 expression during sexual development

• Analysis of RIP efficiency in Sfh-5 mutants

• Investigation of potential regulatory roles in oxidative stress responses that might indirectly affect genome stability

How can site-directed mutagenesis help elucidate the structure-function relationship of Sfh-5?

Site-directed mutagenesis represents a powerful approach for investigating the structure-function relationship of Sfh-5, particularly regarding its heme-binding properties versus potential phosphatidylinositol transfer capabilities.

Table 2: Key Residues for Site-Directed Mutagenesis in Sfh-5

Successful research approaches include:

• Creation of heme binding-deficient Sfh-5 mutants to test if PtdIns-exchange activity can be resuscitated

• Structural analysis of mutants using X-ray crystallography to determine conformational changes

• Biochemical assays to measure heme and lipid binding/transfer activities in parallel

What advanced approaches can be used to identify potential interaction partners and biological pathways involving Sfh-5?

To identify interaction partners and biological pathways involving Sfh-5, researchers should consider these advanced methodological approaches:

• Proximity-based labeling techniques:

  • BioID or TurboID fusion proteins to identify proximal interacting proteins in vivo

  • APEX2-based proximity labeling for temporal control of labeling reactions

• Genetic interaction screens:

  • Synthetic genetic array (SGA) analysis to identify genetic interactions

  • Chemogenomic profiling to identify conditions where Sfh-5 function becomes essential

• Organelle-specific localization and function:

  • Advanced imaging using split-GFP or other complementation-based techniques

  • Organelle-specific proximity labeling to identify compartment-specific interactions

• Systems biology approaches:

  • Integration of transcriptomics, proteomics, and metabolomics data

  • Network analysis to position Sfh-5 within cellular pathways, particularly oxidative stress response networks

• In vivo studies under specific stress conditions:

  • Challenge with oxidants like oltipraz that induce a "heme-requiring" response

  • Analysis of interactions with heme biosynthesis and distribution pathways

How can researchers address the challenges of studying low-expression proteins like Sfh-5 in Neurospora crassa?

Studying low-expression proteins like Sfh-5 presents significant technical challenges. Based on research indicating Sfh-5 is expressed at very low levels in yeast , similar challenges likely exist in Neurospora crassa. Researchers should consider:

• Enhanced expression strategies:

  • Use of strong promoters (e.g., ccg-1) for controlled overexpression

  • Inducible expression systems to minimize toxicity

• Improved detection methods:

  • Tandem affinity purification (TAP) tags for efficient isolation

  • Mass spectrometry approaches optimized for low-abundance proteins

  • Proximity-dependent biotin identification (BioID) to capture transient interactions

• Single-cell approaches:

  • Microfluidic-based single-cell analysis to account for expression heterogeneity

  • Single-molecule fluorescence approaches to detect low copy number events

• Functional genomics in defined conditions:

  • Study function under specific stress conditions (e.g., oxidative stress)

  • Create sensitized genetic backgrounds where Sfh-5 function becomes critical

• Tag selection considerations:

  • Utilize small epitope tags to minimize functional interference

  • Consider split tags for confirming proper protein folding

How can researchers address issues with Sfh-5 protein stability during purification and analysis?

Sfh-5's unique properties as a hemoprotein require specific considerations for maintaining stability during purification and analysis:

• Buffer optimization:

  • Include reducing agents (e.g., 1-2 mM DTT) to prevent oxidation of thiol groups

  • Maintain physiological pH (7.0-7.5) to prevent heme dissociation

  • Consider including glycerol (5-10%) to enhance protein stability

• Temperature control:

  • Perform all purification steps at 4°C

  • Store purified protein at -80°C with flash-freezing in liquid nitrogen

  • Avoid repeated freeze-thaw cycles

• Protection of heme coordination:

  • Avoid harsh reducing or oxidizing conditions that might affect the Fe redox state

  • Consider oxygen-free environments for handling reduced forms

  • Monitor heme status via spectroscopic analysis throughout purification

• Analytical considerations:

  • For functional assays, verify heme occupancy via spectroscopic analysis

  • When comparing mutants, normalize for heme content

  • Consider the potential for heterogeneity in heme loading (reported ~30% loading in recombinant protein)

What strategies can help resolve contradictory data in Sfh-5 functional studies?

When facing contradictory data in Sfh-5 functional studies, consider these methodological approaches:

• Reconciling in vitro versus in vivo observations:

  • Verify protein folding and heme incorporation in recombinant proteins

  • Consider the cellular context and potential interaction partners present in vivo but absent in vitro

  • Examine differences in post-translational modifications

• Addressing species-specific differences:

  • Perform careful comparative studies between Sfh-5 from different fungal species

  • Consider evolutionary differences in protein sequence and cellular context

  • Analyze strain backgrounds for genetic modifiers that might affect outcomes

• Resolving conflicting phenotypic data:

  • Implement rigorous statistical designs such as split-plot approaches

  • Control for environmental variables that might affect stress responses

  • Consider genetic background effects and potential compensatory mechanisms

• Technical approaches to resolve contradictions:

  • Utilize multiple complementary techniques to examine the same phenomenon

  • Implement controls for heme loading status when comparing protein activities

  • Consider temporal aspects of protein function, particularly in stress responses