Recombinant Taeniopygia guttata LYR motif-containing protein 4 (LYRM4)

How does the NFS1-ISD11 complex form and what is its stoichiometry?

The NFS1-ISD11 complex typically forms with a stoichiometry of approximately 1:1 or 1:2 (NFS1:ISD11) . Size exclusion chromatography studies show that the complex elutes at approximately 145 kDa, corresponding to an NFS1 dimer with 1-2 ISD11 subunits . This complex is essential for NFS1 stability and function. Although NFS1 may retain its desulfurase activity without ISD11 in some experimental conditions, it becomes significantly more prone to aggregation and degradation . When designing experiments to study this interaction, co-expression systems are generally more successful than attempting to reconstitute the complex from separately purified components.

What is the impact of LYRM4 mutations on protein function and clinical presentation?

Mutations in LYRM4 can significantly impair iron-sulfur cluster assembly, leading to combined deficiencies in multiple metabolic pathways. Two documented mutations provide insights into structure-function relationships:

• p.R68L mutation (c.203G>T):

  • Found in patients with combined OXPHOS deficiency

  • Creates an NFS1-ISD11 complex more prone to aggregation

  • Shows impaired function in yeast complementation assays

  • Associated with variable clinical severity between patients

• p.Y31C mutation (c.92A>G):

  • Novel pathogenic variant identified in a patient with episodic cardiorespiratory arrest

  • Associated with 3-methylglutaconic aciduria, not previously reported in Fe-S cluster deficiencies

  • Affects a highly conserved amino acid across species

The clinical spectrum ranges from severe neonatal presentations with early mortality to episodic decompensation with normal development between crises .

How conserved is LYRM4 across species and what are its key structural elements?

LYRM4/ISD11 shows significant conservation across vertebrate species, particularly in regions critical for interaction with NFS1. The protein belongs to the LYR-motif family, characterized by a conserved leucine-tyrosine-arginine sequence. Key features include:

• Relatively small size (~11 kDa in most species)

• High alpha-helical content

• Conserved functional residues like R68, mutation of which causes disease in humans

• Typical LYR motif near the N-terminus

• Y31 position is highly conserved across species as demonstrated by comparative analysis

When working with Taeniopygia guttata LYRM4, researchers should account for these conserved elements in experimental design, as they likely represent critical functional domains.

What expression systems yield optimal results for recombinant LYRM4?

For successful expression of functional recombinant LYRM4, consider these approaches:

Most critical for any system is the co-expression with NFS1, which significantly enhances ISD11 stability and solubility . The complex should be purified together rather than attempting to purify ISD11 alone, as isolated ISD11 tends to be unstable or improperly folded.

What purification strategies minimize aggregation and maximize activity?

Purification of recombinant LYRM4 requires careful attention to protein stability:

• Buffer composition:

  • Include reducing agents (1-5 mM DTT or β-mercaptoethanol)

  • Add stabilizers (5-10% glycerol)

  • Maintain moderate ionic strength (150-300 mM NaCl)

  • Optimal pH range: 7.2-8.0

• Chromatography sequence:

  • Initial capture: Affinity chromatography (Ni-NTA for His-tagged constructs)

  • Intermediate purification: Ion exchange chromatography

  • Polishing: Size exclusion chromatography to remove aggregates and assess complex formation

• Handling precautions:

  • Maintain samples at 4°C throughout purification

  • Process quickly to minimize degradation

  • Consider on-column tag cleavage to reduce manipulation steps

  • Avoid freeze-thaw cycles by preparing single-use aliquots

The NFS1-ISD11 complex with the disease-causing R68L mutation shows increased aggregation compared to wild-type , highlighting the importance of monitoring the aggregation state throughout purification.

How should I optimize solubility when working with different LYRM4 constructs?

Optimization of LYRM4 solubility requires systematic testing of multiple factors:

• Fusion partner selection:

  • SUMO tag enhances solubility while allowing post-cleavage native N-terminus

  • MBP (maltose-binding protein) significantly improves solubility

  • Thioredoxin provides good solubility enhancement with smaller size

  • Avoid C-terminal tags that may interfere with NFS1 interaction

• Expression conditions:

  • Reduced temperature (16°C) with extended expression time

  • Co-expression with molecular chaperones (GroEL/ES)

  • Critical factor: Co-expression with NFS1 substantially improves ISD11 solubility

• Solubilizing additives:

  • Low concentrations of non-ionic detergents (0.05% Tween-20)

  • Stabilizing osmolytes (trehalose, sucrose, arginine)

  • Specific ions that may enhance stability (Mg²⁺, Zn²⁺)

• Construct optimization:

  • N-terminal methionine processing consideration

  • Potential removal of flexible regions prone to aggregation

  • Codon optimization for expression host

Systematic testing with a solubility screening approach will identify optimal conditions for your specific LYRM4 construct.

What are the most reliable assays for assessing LYRM4-NFS1 complex formation?

Multiple complementary techniques provide robust assessment of LYRM4-NFS1 complex formation:

• Size Exclusion Chromatography (SEC):

  • Monitors complex formation via shift in elution volume

  • Can detect complex stability under different conditions

  • Research shows properly formed complexes elute at approximately 145 kDa

  • Disease-associated mutants show altered elution profiles or increased aggregate peaks

• Thermal Shift Assays:

  • Measure stabilization of NFS1 by wild-type versus mutant ISD11

  • High-throughput screening of buffer conditions

  • Quantitative comparison of melting temperatures

• Analytical Ultracentrifugation:

  • Determines precise stoichiometry of complex

  • Distinguishes between different oligomeric states

  • Provides binding affinity data in solution

• Co-immunoprecipitation:

  • Confirms interaction in cell-based systems

  • Can detect additional components of the complex

  • Allows comparison of wild-type and mutant interactions

When designing these experiments, include both positive controls (known functional complex) and negative controls (non-interacting proteins) to validate results.

How can I assess the functional impact of LYRM4 mutations?

A comprehensive approach to evaluating LYRM4 mutations includes:

• Biochemical characterization:

  • Compare wild-type and mutant protein stability and folding

  • Assess NFS1 binding affinity and complex formation

  • Measure cysteine desulfurase activity of the reconstituted complex

  • Evaluate complex stability using size exclusion chromatography

• Cellular models:

  • Complementation studies in LYRM4-deficient cells

  • Measure activity of Fe-S dependent enzymes (complexes I, II, III, aconitases)

  • Monitor iron homeostasis and mitochondrial function

  • Assess response to oxidative stress

• In vivo functional validation:

  • Yeast complementation assays (documented approach for R68L mutation)

  • Competitive growth assays comparing wild-type and mutant strains

  • Quantitative fitness measurements under different growth conditions

• Patient-derived evidence:

  • Biochemical analysis of patient tissues (muscle, liver)

  • Western blot analysis of OXPHOS complexes and other Fe-S proteins

  • Enzyme activity measurements of respiratory chain complexes

This multi-level approach provides strong evidence for pathogenicity and mechanistic insights.

How can I differentiate between primary and secondary effects of LYRM4 dysfunction?

Distinguishing primary from secondary consequences requires systematic experimental design:

• Temporal analysis:

  • Time-course experiments following LYRM4 depletion or inactivation

  • Identify earliest affected pathways and proteins

  • Documented primary effects include decreased levels of complexes I, II, III, aconitases, and ferrochelatase

  • Secondary effects include iron accumulation and oxidative stress responses

• Direct target validation:

  • In vitro reconstitution with purified components

  • Rescue experiments with targeted supplementation

  • Systematic analysis of Fe-S cluster-containing proteins

• Comparative approaches:

  • Compare phenotypes with other Fe-S assembly defects

  • Analyze common vs. distinct features across different LYRM4 mutations

  • Example: Complex IV deficiency was variable between patients with the same LYRM4 mutation

• Multi-omics integration:

  • Correlate transcriptomics, proteomics, and metabolomics data

  • Pathway analysis to identify convergent mechanisms

  • Network modeling to predict causal relationships

This approach enables discrimination between direct consequences of impaired Fe-S cluster assembly and downstream metabolic adaptations.

How can CRISPR-Cas9 be effectively employed to study LYRM4 function?

CRISPR-Cas9 technology offers powerful approaches for LYRM4 research:

• Gene knockout strategies:

  • Complete LYRM4 elimination to study essentiality

  • Conditional knockout systems for temporal control

  • Tissue-specific inactivation to study organ-specific phenotypes

  • Careful design of sgRNAs to minimize off-target effects

• Knock-in of specific mutations:

  • Introduction of patient-derived mutations (e.g., R68L, Y31C)

  • Creation of isogenic cell lines for controlled comparisons

  • Introduction of conservative vs. non-conservative substitutions

  • Tagged versions for localization and interaction studies

• Transcriptional modulation:

  • CRISPRi for partial suppression without complete elimination

  • Tunable repression to establish dose-dependency

  • Combinatorial targeting with other Fe-S assembly components

• Screening applications:

  • CRISPR screens for synthetic lethal interactions

  • Identification of rescue factors for LYRM4 deficiency

  • Multiplexed mutagenesis of key residues

Consider including appropriate controls and validation strategies, including rescue experiments with wild-type LYRM4 to confirm specificity of observed phenotypes.

What structural biology approaches can elucidate LYRM4-NFS1 complex architecture?

Multiple structural biology techniques provide complementary insights:

• X-ray crystallography:

  • High-resolution structure of the complex

  • Identification of critical interaction interfaces

  • Challenge: Obtaining diffraction-quality crystals of the complex

• Cryo-electron microscopy:

  • Structure determination without crystallization

  • Visualization of conformational heterogeneity

  • Potential to capture different functional states

  • Integration into larger Fe-S assembly machinery

• Cross-linking mass spectrometry:

  • Identification of residues in close proximity

  • Constraint generation for computational modeling

  • Detection of conformational changes

• Hydrogen-deuterium exchange MS:

  • Mapping binding interfaces via altered solvent accessibility

  • Determining regions undergoing conformational changes

  • Complementary to high-resolution structural techniques

• Integrative modeling:

  • Combining multiple experimental data sources

  • Molecular dynamics simulations to explore dynamics

  • Computational mutagenesis to predict effects of variants

These approaches can help understand how disease-causing mutations like R68L disrupt complex formation and stability .

How can tissue-specific effects of LYRM4 deficiency be systematically studied?

Understanding tissue-specific manifestations requires integrated approaches:

• Expression and interactome profiling:

  • Tissue-specific LYRM4 expression patterns

  • Identification of tissue-specific interaction partners

  • Analysis of Fe-S protein expression across tissues

• Tissue-specific metabolic analysis:

  • Metabolomic profiling of affected tissues

  • Characterization of tissue-specific Fe-S dependent pathways

  • Clinical evidence shows differential effects on muscle, liver, and heart tissues

• Patient-derived models:

  • iPSC generation from patient cells

  • Differentiation into tissue-specific cell types

  • Organoid development for 3D tissue architecture

• Tissue-specific knockout models:

  • Cre-lox systems targeting specific tissues

  • Phenotypic characterization of tissue-specific knockouts

  • Rescue experiments with tissue-specific expression

• Systems biology integration:

  • Multi-omics data integration across tissues

  • Network modeling of tissue-specific vulnerabilities

  • Correlation with clinical phenotypes

This comprehensive approach can explain the tissue specificity observed in patients, where skeletal muscle, liver, and cardiac function are prominently affected .

What are the most common technical challenges when working with recombinant LYRM4?

Researchers frequently encounter several challenges when working with LYRM4:

• Protein instability and aggregation:

  • NFS1-ISD11-R68L complex shows increased aggregation compared to wild-type

  • Recommended solution: Co-express with NFS1; include stabilizing agents (glycerol, reducing agents)

  • Monitor aggregation state using dynamic light scattering

• Low expression yield:

  • Common issue with small mitochondrial proteins

  • Solution: Optimize codon usage; test multiple fusion tags; use specialized expression strains

• Activity assessment difficulties:

  • ISD11 function is primarily assessed indirectly via NFS1 activity

  • Solution: Develop multiple complementary assays; include appropriate controls

• Consistency between batches:

  • Variability in activity between preparations

  • Solution: Standardize purification protocols; implement rigorous quality control metrics

• Reconstitution of full activity:

  • Difficulty achieving native-like activity levels in vitro

  • Solution: Ensure complete complex formation; add all necessary cofactors; optimize buffer conditions

Maintaining detailed records of expression and purification conditions helps identify variables contributing to successful preparations.

How can I verify the quality and integrity of purified recombinant LYRM4?

Comprehensive quality control should include:

• Purity assessment:

  • SDS-PAGE with Coomassie staining (>95% purity)

  • Western blotting with specific antibodies

  • Mass spectrometry confirmation of identity

• Structural integrity:

  • Circular dichroism to confirm secondary structure

  • Thermal shift assay to assess stability

  • Size exclusion chromatography to detect aggregation

  • Dynamic light scattering for homogeneity

• Functional validation:

  • NFS1 binding assay

  • Protection of NFS1 from aggregation (documented effect of wild-type ISD11)

  • Cysteine desulfurase activity of the reconstituted complex

• Storage stability:

  • Activity retention after freeze-thaw cycles

  • Time-course stability at different temperatures

  • Optimization of buffer components for long-term storage

• Batch consistency:

  • Lot-to-lot comparison using standardized assays

  • Reference standards for comparative analysis

  • Detailed documentation of preparation conditions

For critical experiments, multiple quality control criteria should be satisfied to ensure reliable results.

How should contradictory results in LYRM4 functional studies be interpreted?

When faced with contradictory results, consider:

• Methodological differences:

  • Expression systems (bacterial vs. eukaryotic)

  • Purification approaches and protein quality

  • Assay conditions (buffer composition, temperature, pH)

  • Detection methods and sensitivity thresholds

• Biological context variations:

  • Cell type or tissue specificity

  • Acute vs. chronic effects of LYRM4 deficiency

  • Variable clinical severity seen even with identical mutations

  • Different outcomes in related patients with same mutation

• Technical factors:

  • Sample size and statistical power

  • Reproducibility across laboratories

  • Reagent quality and specificity

  • Control selection and implementation

• Reconciliation strategies:

  • Direct side-by-side comparisons under identical conditions

  • Systematic variation of key parameters

  • Integration of multiple methodologies

  • Meta-analysis of published data

• Reporting considerations:

  • Transparent documentation of all methods

  • Publication of negative and conflicting results

  • Consideration of publication bias

The variability in clinical outcomes observed in patients with identical LYRM4 mutations suggests that modifier factors may influence the phenotypic expression of LYRM4 deficiency.

What new methodologies are emerging for studying Fe-S cluster biogenesis involving LYRM4?

The field is advancing with several innovative approaches:

• Advanced imaging techniques:

  • Super-resolution microscopy for subcellular localization

  • Single-molecule FRET to monitor conformational changes

  • Live-cell imaging with genetically encoded sensors

• High-throughput screening platforms:

  • Automated purification and activity assays

  • Small molecule screens for modulators of ISD11 function

  • CRISPR screens for genetic interactors

• Synthetic biology approaches:

  • Minimal reconstituted systems for Fe-S cluster assembly

  • Cell-free expression systems for rapid testing

  • Designer Fe-S proteins as functional reporters

• Chemical biology tools:

  • Activity-based probes for Fe-S transfer

  • Photocrosslinking to capture transient interactions

  • Chemically induced dimerization to control complex formation

• Computational approaches:

  • Machine learning for predicting mutation effects

  • Molecular dynamics simulations of complex assembly

  • Systems biology modeling of Fe-S cluster biogenesis

These emerging technologies promise to provide new insights into the fundamental biology of Fe-S cluster assembly and the pathogenic mechanisms of LYRM4 mutations.

How can therapeutic approaches for LYRM4 deficiency be rationally designed and tested?

Development of potential therapeutic strategies should consider:

• Molecular mechanism targeting:

  • Stabilization of mutant ISD11 protein

  • Enhancement of residual complex formation

  • Bypassing defective steps in Fe-S cluster assembly

• Metabolic support strategies:

  • The striking difference in clinical outcomes between patients with identical mutations suggests therapeutic windows

  • Sulfur donor availability (cysteine) may be limiting in neonatal period

  • Antioxidant supplementation to mitigate secondary oxidative stress

• Preclinical model development:

  • Patient-derived cells for drug screening

  • Animal models of LYRM4 deficiency

  • Organoid models for tissue-specific interventions

• Gene therapy approaches:

  • AAV-mediated gene delivery to affected tissues

  • Genome editing to correct pathogenic mutations

  • RNA-based therapies to modulate expression

• Drug repurposing opportunities:

  • Screening of approved drugs for beneficial effects

  • Compounds known to modulate iron metabolism

  • Mitochondrial targeted therapies

Studies suggest that vulnerability during the neonatal period may be related to limited availability of cysteine as a sulfur donor , pointing to potential therapeutic interventions focused on this metabolic pathway.