Recombinant Nematostella vectensis Adenosine monophosphate-protein transferase FICD homolog (v1g194069)

What are the optimal storage and handling conditions for maintaining protein stability?

For optimal stability of the Recombinant Nematostella vectensis FICD homolog:

To preserve protein activity, repeated freeze-thaw cycles should be strictly avoided . Prior to opening, brief centrifugation is recommended to ensure all contents are at the bottom of the vial .

What is the biochemical function of Nematostella vectensis FICD homolog in cellular processes?

Nematostella vectensis FICD homolog functions as an adenosine monophosphate-protein transferase . This protein belongs to the Fic (Filamentation induced by cAMP) family and has two primary functional roles:

• Protein adenylyltransferase activity: FICD can catalyze the transfer of AMP from ATP to target proteins, a post-translational modification known as AMPylation .

• De-AMPylase activity: The protein also exhibits the reverse function as a de-AMPylase, removing AMP from modified proteins .

This bidirectional enzymatic activity suggests a regulatory role in protein function through reversible post-translational modification, which may be critical for cellular stress responses and homeostasis mechanisms in Nematostella vectensis.

How does the FICD homolog relate to other protein families in Nematostella vectensis?

Research on Nematostella vectensis has revealed interesting evolutionary connections between seemingly unrelated protein families. While specific interaction data for FICD homolog is limited, studies on other Nematostella proteins provide context for understanding potential relationships:

The Nematostella genome contains a homolog of Hyponastic Leaves1 (HYL1), previously thought to be plant-specific, which plays a crucial role in microRNA (miRNA) biogenesis . This discovery challenges the conventional view that miRNA systems evolved independently in plants and animals.

The HYL1 homolog Hyl1-like a (Hyl1La) has been shown to be essential for:

• Nematostella development and metamorphosis

• Efficient miRNA processing

• Interaction with precursor miRNAs (pre-miRNAs)

Through homology assessment approaches, researchers can identify evolutionarily conserved domains and functions between FICD and other protein families, providing insights into their shared ancestry and divergent specialization .

What expression systems are most effective for producing functional Recombinant Nematostella vectensis FICD homolog?

The recombinant FICD homolog is successfully expressed in E. coli expression systems with an N-terminal His tag . This prokaryotic expression system offers several advantages for this protein:

For research requiring post-translational modifications or investigating protein interactions in a more native-like environment, alternative expression systems could be considered:

When selecting an expression system, researchers should consider the experimental questions being addressed and whether native post-translational modifications are essential for the study.

What are the recommended reconstitution protocols for lyophilized FICD homolog?

For optimal reconstitution of lyophilized Recombinant Nematostella vectensis FICD homolog:

• Pre-reconstitution preparation:

  • Allow the vial to equilibrate to room temperature

  • Briefly centrifuge to collect all material at the bottom of the vial

• Reconstitution procedure:

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

  • Gently mix by inversion or slow rotation (avoid vortexing to prevent denaturation)

  • Allow protein to completely dissolve (5-15 minutes at room temperature)

• Post-reconstitution stabilization:

  • Add glycerol to 5-50% final concentration (50% is recommended for long-term storage)

  • Aliquot into working volumes to avoid repeated freeze-thaw cycles

  • Flash-freeze aliquots not immediately used

• Quality control verification:

  • Verify protein activity using appropriate functional assays

  • Check purity via SDS-PAGE (should be >90% as specified)

How can homology detection methods be optimized to identify functional relationships between FICD homologs across species?

Advanced homology detection for FICD homologs requires sophisticated computational approaches that go beyond simple sequence similarity:

• Deep learning language model approaches:
  Recent advancements in protein representation using deep learning language models have significantly improved homolog detection, especially for proteins with <30% sequence identity . These models can:

  • Capture complex evolutionary relationships

  • Identify functional homologs where traditional tools fail

  • Detect remote homology relationships with greater accuracy

• Multi-domain protein analysis strategies:
  For proteins like FICD homologs that may contain multiple domains, benchmarking against established datasets is essential:

  • Pfam-based domain identification

  • Gene3d structural domain recognition

  • SUPERFAMILY classification

• Homology detection validation metrics:

  Benchmark Type	Homolog Pairs	Application
  Pfam dataset	5,245 pairs	Domain-based homology
  Gene3d dataset	5,047 pairs	Structure-based homology
  SUPERFAMILY	5,656 pairs	Superfamily classification

• Integration with experimental validation:
  Computational predictions should be validated using:

  • Functional complementation assays

  • Structural analysis

  • Biochemical characterization of enzymatic activity

These enhanced homology detection methods can reveal evolutionary relationships between FICD homologs across diverse species, potentially identifying new model organisms for studying FICD function .

What methodologies are most effective for investigating FICD homolog interactions with target proteins?

Several complementary approaches can be employed to comprehensively characterize FICD homolog interactions:

• Immunoprecipitation-based approaches:

  • Similar to techniques used for Hyl1La studies in Nematostella, protein G SureBeads magnetic beads with monoclonal mouse anti-FLAG antibodies can be used for tagged FICD homolog

  • Optimal lysis buffer composition: 25 mM Tris-HCl (pH 7.4), 150 mM KCl, 25 mM EDTA, 0.5% NP-40, 1 mM DTT, supplemented with protease inhibitors

• In vitro binding assays:

  • Synthetic substrate preparation for assessing adenylylation activity

  • Quantitative PCR following immunoprecipitation to identify nucleic acid interactions

  • Fluorescence-based assays to measure binding kinetics

• Crosslinking mass spectrometry (XL-MS):

  • Allows identification of transient protein-protein interactions

  • Can provide structural constraints for modeling complex assemblies

  • Especially valuable for identifying AMPylation target proteins

• Advanced microscopy techniques:

  • Fluorescence resonance energy transfer (FRET) to visualize protein interactions in vivo

  • Implementation of plasmid constructs with fluorescent tags (e.g., memOrange2) under appropriate promoters (e.g., TBP promoter for ubiquitous expression)

• Functional validation in vivo:

  • Morpholino-based knockdown approaches as demonstrated with other Nematostella proteins

  • Analysis of developmental phenotypes following FICD manipulation

  • Rescue experiments with wild-type and mutant variants

What is the developmental significance of FICD homolog in Nematostella vectensis?

While specific developmental roles of FICD homolog in Nematostella remain to be fully characterized, insights can be gained from studies of other regulatory proteins in this model organism:

Knockdown studies of miRNA processing components in Nematostella have demonstrated profound developmental impacts, including:

• Arrested metamorphosis

• Failure to settle and form primary polyps

• Developmental abnormalities persisting through 9 days post-fertilization

These phenotypes were observed in morphants of several miRNA biogenesis components, including HEN1, Dicer1, AGO1, and AGO2 . Given that FICD homologs regulate protein function through post-translational modification, they may similarly influence developmental timing and tissue differentiation.

For experimental developmental studies, researchers typically:

• Inject morpholinos or expression constructs into Nematostella zygotes

• Monitor development through 9 days post-fertilization

• Compare developmental outcomes against control-injected animals

• Quantify settlement and metamorphosis rates

How does the evolutionary conservation of FICD homologs inform our understanding of post-translational regulation?

The presence of FICD homologs across diverse phyla provides valuable insights into the evolution of post-translational regulatory mechanisms:

• Evolutionary significance:

  • Similar to the discovery of "plant-specific" HYL1 homologs in Cnidaria , FICD conservation suggests ancient origins for this regulatory mechanism

  • May indicate that AMPylation as a regulatory mechanism predates the divergence of major metazoan lineages

• Conservation patterns:

  • Cross-species comparison can identify highly conserved catalytic residues essential for function

  • Variable regions may represent lineage-specific adaptations to different cellular environments

• Functional implications:

  • Conservation of FICD across species suggests fundamental roles in cellular homeostasis

  • May interact with ancient cellular stress response pathways

  • Could represent an early evolution of protein activity regulation through reversible post-translational modification

• Research applications:

  • Nematostella, as a basal metazoan, provides an excellent model for studying ancestral protein functions

  • Comparative studies between Nematostella FICD and homologs in other organisms can illuminate functional evolution

  • Understanding ancient regulatory mechanisms may provide insights into fundamental cellular processes conserved across eukaryotes