Recombinant Neurospora crassa Mitochondria fission 1 protein (fis-1), partial

What is the predicted structure of Neurospora crassa fis-1 protein based on homology with other organisms?

The Neurospora crassa fis-1 protein likely shares structural similarities with Fis1 proteins from other organisms. Based on structural studies of homologous proteins, N. crassa fis-1 likely contains tetratricopeptide repeat (TPR) domains that form a six-helix bundle. The central four helices likely consist of two tandem TPR-like motifs that create both concave and convex surfaces for protein-protein interactions . Similar to yeast Fis1, the N. crassa fis-1 may possess an N-terminal arm that can regulate binding to adapter proteins involved in the mitochondrial fission machinery . To determine the actual structure, researchers should consider X-ray crystallography or NMR studies of the purified recombinant protein, as has been done with yeast and human FIS1.

What experimental approaches can distinguish between basic and specialized functions of fis-1?

To determine whether N. crassa fis-1 functions primarily in basal fission or in specialized processes like stress-induced fission:

• Generate fis-1 knockout strains and assess mitochondrial morphology under normal conditions

• Subject wild-type and fis-1 knockout strains to various stressors (oxidative, metabolic, heat)

• Use fluorescently-labeled mitochondria to quantify fission events under different conditions

• Perform complementation studies with wild-type and mutant versions of fis-1

• Assess mitophagy rates in wild-type versus knockout strains

Recent research in neurons has shown that Fis1 may have unexpected compartment-specific roles in maintaining mitochondrial dynamics , suggesting that researchers should examine mitochondrial morphology in different cellular regions of N. crassa hyphae.

What expression systems yield optimal results for producing functional recombinant N. crassa fis-1 protein?

For expressing recombinant N. crassa fis-1, consider the following systems:

For partial fis-1 protein, E. coli expression is often suitable if the transmembrane domain is excluded. To optimize expression:

• Use codon-optimized sequences for the expression host

• Include solubility tags (MBP, GST, SUMO) for improved folding

• Test multiple growth temperatures and induction conditions

• For the partial protein, ensure the construct maintains the TPR domain integrity, as this is crucial for function and protein-protein interactions

What are the critical considerations when designing constructs for partial fis-1 protein expression?

When designing constructs for partial fis-1 expression:

• Identify domain boundaries based on sequence alignment with characterized Fis1 proteins from yeast and humans

• Ensure retention of the TPR domains that are essential for protein-protein interactions

• Consider excluding the C-terminal transmembrane domain for improved solubility

• Preserve the N-terminal arm, which is crucial for function in yeast Fis1

• Incorporate affinity tags that won't interfere with protein folding or function

If mimicking studies done with yeast Fis1, researchers should note that both the concave and convex surfaces of the TPR domain are important for function, and mutations disrupting these interfaces abolish Fis1 activity . Domain truncation experiments should therefore preserve the structural integrity of both surfaces.

How can researchers verify that recombinant partial fis-1 maintains its native structural features?

To verify structural integrity of recombinant partial fis-1:

• Circular dichroism (CD) spectroscopy to assess secondary structure content and compare with predicted α-helical content

• Thermal shift assays to evaluate protein stability

• Size exclusion chromatography to confirm monomeric state and proper folding

• Nuclear magnetic resonance (NMR) for more detailed structural analysis

• Binding assays with known interaction partners predicted from homology (if available)

Intrinsic tryptophan fluorescence can also be used to probe conformational states, as has been done with human FIS1 to study the position of the N-terminal arm . Comparing experimental data with the known structural features of yeast and human Fis1 can provide insights into the structural conservation of the N. crassa protein.

What in vitro assays can demonstrate the functional activity of recombinant fis-1?

Several approaches can assess functional activity of recombinant fis-1:

• Protein-protein interaction assays:

  • Pull-down assays with predicted binding partners

  • Surface plasmon resonance to measure binding kinetics

  • Yeast two-hybrid screening to identify interactors

  • Isothermal titration calorimetry for thermodynamic parameters of binding

• Liposome binding and tubulation assays:

  • Reconstitution of fis-1 into liposomes

  • Microscopy analysis of liposome morphology changes

• GTPase activation assays:

  • If fis-1 interacts with dynamin-related GTPases, measure GTPase activity enhancement

For partial protein constructs, compare activity with full-length protein to determine if functional domains are preserved . Crosslinking experiments can also be used to identify protein-protein interactions, similar to studies with human FIS1 .

How does the absence of the transmembrane domain affect partial fis-1 protein function, and how can this be addressed experimentally?

The absence of the transmembrane domain in partial fis-1 will affect:

• Membrane localization, which is critical for in vivo function

• Proper orientation of the protein relative to binding partners

• Potential conformational constraints important for function

To address these limitations:

• Anchor the partial protein to model membranes using:

  • Liposome reconstitution systems

  • Supported lipid bilayers

  • Chemical tethering to synthetic membranes

• Compare partial and full-length protein activities using:

  • Cell-free expression systems with membrane fractions

  • Co-expression with binding partners

  • Complementation studies in fis-1 knockout N. crassa

• Use computational modeling to predict how membrane anchoring affects protein conformation and function

Studies with human FIS1 have shown that the N-terminal arm conformation can be sensitive to environmental changes , suggesting that membrane anchoring might influence protein activity in ways that are lost with partial constructs.

How can molecular dynamics simulations enhance our understanding of N. crassa fis-1 structure-function relationships?

Molecular dynamics (MD) simulations offer powerful insights into fis-1 function:

• Predict conformational dynamics of the N-terminal arm and TPR domains

• Model interactions with binding partners based on homology to yeast and human systems

• Simulate the effects of mutations on protein stability and binding interfaces

• Explore the impact of membrane association on protein orientation and dynamics

• Identify potential sites for post-translational modifications or allosteric regulation

Recent MD simulations with human FIS1 revealed that its N-terminal arm can adopt an intramolecular conformation similar to yeast Fis1p, which was supported by experimental data from intrinsic tryptophan fluorescence and NMR experiments . Similar approaches could be applied to N. crassa fis-1 to predict how its structure relates to function and to guide experimental design.

What evolutionary insights can be gained by comparing fis-1 functional mechanisms across fungal species?

Comparative analysis of fis-1 across fungal species offers several research opportunities:

• Identify conserved structural features essential for function

• Discover species-specific adaptations in fis-1-mediated processes

• Trace the evolution of the mitochondrial fission machinery

• Understand how different fungi regulate mitochondrial dynamics

Research approaches should include:

• Phylogenetic analysis of fis-1 sequences across diverse fungi

• Structure prediction and comparison across species

• Complementation studies using fis-1 from different fungi

• Identification of lineage-specific interaction partners

Studies in yeast have shown that Fis1 interacts with adaptor proteins Mdv1 and Caf4 , while mammalian FIS1 has different binding partners. Investigating whether N. crassa fis-1 follows the yeast paradigm or has evolved distinct interactions would provide valuable evolutionary insights.

How might fis-1 function in non-canonical pathways beyond mitochondrial fission in N. crassa?

Recent research suggests that Fis1 proteins may have broader functions beyond canonical mitochondrial fission:

• Stress response pathways: Human FIS1 appears to have roles in stress-induced mitochondrial fission and mitophagy rather than basal "housekeeping" fission

• Development and differentiation: In Neurospora crassa, mutations in other mitochondrial proteins like fmf-1 affect sexual development , suggesting potential developmental roles for mitochondrial dynamics proteins

• Specialized compartment functions: In neurons, Fis1 unexpectedly regulates dendritic mitochondrial networks through effects on both fission and fusion balance

• Quality control mechanisms: Potential roles in mitophagy and cellular adaptation to changing metabolic conditions

To investigate these possibilities in N. crassa, researchers should:

• Examine fis-1 expression patterns during different developmental stages

• Assess phenotypes of fis-1 mutants under various stress conditions

• Investigate interactions with proteins involved in stress response, development, and quality control

• Perform transcriptomic and proteomic analyses to identify pathways affected by fis-1 disruption

What strategies can address difficulties in distinguishing between direct and indirect effects of fis-1 manipulation?

Distinguishing direct from indirect effects of fis-1 manipulation requires:

• Acute vs. chronic manipulation:

  • Use inducible expression systems for temporal control

  • Apply optogenetic tools for rapid activation/inactivation

  • Develop chemical-genetic approaches for specific inhibition

• Domain-specific mutants:

  • Create point mutations in specific functional domains

  • Use structure-guided mutagenesis targeting key residues in binding interfaces

  • Develop chimeric proteins with domains from related organisms

• Proximal detection methods:

  • Implement BioID or APEX2 proximity labeling to identify direct interactors

  • Use FRET sensors to detect immediate conformational changes

  • Apply split-GFP complementation to visualize direct interactions

• Rescue experiments:

  • Perform complementation with wild-type and mutant variants

  • Use orthogonal systems from other organisms for functional rescue

Yeast studies have shown that mutations in specific residues of Fis1 (I24A/L25A, F43A/N44A, and W47A) disrupt binding to adaptor proteins and abolish mitochondrial fission activity . Similar structure-guided mutagenesis approaches could be applied to N. crassa fis-1.

How can researchers resolve contradictory findings about fis-1 function in different experimental systems?

To address contradictory findings about fis-1 function:

• Standardize experimental conditions:

  • Define growth conditions, cell types, and developmental stages

  • Establish common assays and quantification methods

  • Create standard operating procedures for mitochondrial analyses

• Multifaceted functional assessment:

  • Combine morphological analysis with functional measurements

  • Assess mitochondrial membrane potential, as loss of Fis1 in neurons reduced membrane potential

  • Measure both fission and fusion events, as Fis1 may affect both processes

  • Evaluate calcium handling, which was affected by Fis1 knockdown in neurons

• Genetic background considerations:

  • Test effects in multiple strain backgrounds

  • Control for compensatory mechanisms and genetic modifiers

  • Create double knockouts with related proteins to uncover redundancy

• Integrative analysis:

  • Combine in vitro, in vivo, and in silico approaches

  • Apply systems biology methods to model complex interactions

  • Use multi-omics to capture broader cellular responses

Recent findings in neurons showed that Fis1 knockdown unexpectedly led to shorter dendritic mitochondria rather than the elongated mitochondria predicted by its canonical role as a fission protein . This highlights the importance of thoroughly characterizing fis-1 function in N. crassa rather than assuming conserved functions from other systems.