Recombinant Emericella nidulans Uncharacterized protein AN2204 (AN2204)

What is the basic characterization of Recombinant Emericella nidulans Uncharacterized Protein AN2204?

Recombinant Emericella nidulans Uncharacterized Protein AN2204 (Q5BB76) is a full-length protein (1-291 amino acids) that can be expressed with an N-terminal His-tag in E. coli expression systems . The protein remains largely uncharacterized in terms of its biological function, which presents significant research opportunities for functional investigation. The recombinant format allows researchers to conduct various in vitro studies to elucidate its properties and potential roles within fungal systems.

The protein is available in lyophilized powder form and requires reconstitution before experimental use . While categorized as "uncharacterized," this classification indicates that its precise biological function has not been definitively established, making it a candidate for comprehensive functional genomics and proteomics studies.

What are the recommended storage and handling conditions for recombinant AN2204?

For optimal stability and activity preservation, recombinant AN2204 protein should be stored at -20°C/-80°C upon receipt . The manufacturer recommends aliquoting the protein to avoid repeated freeze-thaw cycles, which can cause protein degradation and loss of biological activity. Working aliquots may be stored at 4°C for up to one week .

The lyophilized protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the manufacturer's default recommendation) before aliquoting and storing at -20°C/-80°C . The storage buffer consists of Tris/PBS-based buffer with 6% Trehalose at pH 8.0, which helps maintain protein stability during freeze-thaw cycles .

Before opening the vial, it should be briefly centrifuged to ensure all content is at the bottom of the tube . Proper handling and storage practices are crucial for maintaining protein integrity for experimental applications.

What experimental designs are most appropriate for investigating the function of uncharacterized proteins like AN2204?

When investigating uncharacterized proteins like AN2204, selecting the appropriate experimental design is crucial and should align with the specific research question. For functional characterization, multiple experimental design approaches can be employed:

• Non-parametric analysis designs: A basic ABA reversal design comparing cellular behavior or processes with and without AN2204 protein can establish whether the protein affects specific biological processes4. This approach measures the difference between treatment (protein present) and non-treatment (protein absent) conditions.

• Parametric analysis designs: When investigating optimal concentration or conditions for AN2204 activity, parametric analysis would determine what specific values of the independent variable (e.g., protein concentration) produce the most significant effects4. Multi-element designs allow testing of varying concentrations simultaneously.

• Component analysis designs: For investigating whether specific domains of AN2204 are responsible for its function, component analysis can isolate and test individual protein regions4. This approach is particularly valuable for proteins with distinct structural motifs.

• Comparative analysis designs: When comparing AN2204 with homologous proteins or mutated versions, multi-element treatment designs or multiple treatment reversal designs would be appropriate4. These designs allow comparison of different treatments (protein variants) to determine which is most effective.

The selection between these experimental designs should consider factors such as irreversibility of effects, potential sequence learning effects, and the specific mechanisms being investigated4.

How might AN2204 relate to known biosynthetic pathways in Emericella nidulans?

Emerging research suggests potential connections between uncharacterized proteins in Emericella nidulans and biosynthetic pathways producing bioactive compounds. While direct evidence linking AN2204 to specific pathways is not yet established, the discovery of the emericellamide biosynthetic pathway in A. nidulans provides an instructive model for investigating uncharacterized proteins .

Emericella nidulans (the sexual form of Aspergillus nidulans) produces emericellamide A and related compounds (C-F), which are mixed cyclic polyketide-nonribosomal peptides with antibiotic properties active against methicillin-resistant Staphylococcus aureus . The biosynthetic gene cluster for emericellamide contains one polyketide synthase and one nonribosomal peptide synthetase .

To investigate whether AN2204 might participate in similar biosynthetic pathways, researchers could:

• Conduct gene co-expression analyses to determine if AN2204 is co-regulated with known biosynthetic gene clusters

• Perform targeted gene disruption of AN2204 and analyze changes in the metabolome

• Use yeast two-hybrid or co-immunoprecipitation techniques to identify protein-protein interactions between AN2204 and known biosynthetic enzymes

• Apply comparative genomics to identify conserved domains shared with proteins involved in secondary metabolite biosynthesis

These approaches could reveal whether AN2204 contributes to the production of novel bioactive compounds in Emericella nidulans, potentially opening new avenues for antibiotic discovery .

What bioinformatic approaches would help predict the function of AN2204?

Given the uncharacterized nature of AN2204, comprehensive bioinformatic analysis represents a cost-effective first step in functional prediction. A multi-tiered bioinformatic approach would include:

• Sequence-based homology searching: Using tools like BLAST, PSI-BLAST, and HMMER to identify distant homologs across species. Even modest sequence similarity might suggest functional relationships.

• Domain and motif prediction: Tools such as InterPro, SMART, and Pfam can identify conserved domains or motifs that might suggest biochemical functions. The presence of repeated YE motifs in AN2204 sequence merits special investigation for potential protein-protein interaction sites.

• Secondary and tertiary structure prediction: AlphaFold2, I-TASSER, or Phyre2 can generate structural models that may reveal structural similarities to proteins of known function, even in the absence of sequence homology.

• Gene neighborhood analysis: Examining genes adjacent to AN2204 in the Emericella nidulans genome may reveal functional associations, especially if they belong to a common operon or biosynthetic cluster.

• Phylogenetic profiling: Identifying organisms that contain or lack AN2204 homologs can suggest functional contexts based on shared ecological niches or metabolic capabilities.

• Gene expression correlation analysis: Using publicly available transcriptomic data to identify genes whose expression patterns correlate with AN2204, suggesting functional relationships.

• Protein-protein interaction prediction: Tools like STRING can predict potential interaction partners based on various evidence types, providing functional context.

These computational approaches should be used to generate testable hypotheses that guide subsequent experimental validation rather than as definitive functional assignments.

What analytical methods are recommended for confirming the purity and identity of recombinant AN2204?

Comprehensive characterization of recombinant AN2204 requires a multi-method analytical approach to confirm both identity and purity:

• SDS-PAGE: The primary method for assessing protein purity, with greater than 90% purity expected for recombinant AN2204 preparations . Coomassie or silver staining can visualize the protein band, with expected molecular weight calculated from the 291 amino acid sequence plus the His-tag.

• Western blotting: Using anti-His antibodies confirms the presence of the His-tagged recombinant protein and provides specificity beyond simple SDS-PAGE.

• Mass spectrometry:

  • MALDI-TOF MS verifies the molecular weight of the intact protein

  • LC-MS/MS peptide mapping after tryptic digestion confirms sequence identity and can identify post-translational modifications

  • Top-down proteomics approaches can analyze the intact protein

• Size exclusion chromatography (SEC): Evaluates protein homogeneity and detects aggregates or oligomeric states.

• Dynamic light scattering (DLS): Provides complementary data on protein homogeneity and hydrodynamic radius.

• Circular dichroism (CD) spectroscopy: Assesses secondary structure content and proper folding.

• Functional assays: If binding partners or enzymatic activities are identified, specific activity assays provide functional verification.

For recombinant AN2204, the combination of SDS-PAGE for purity assessment and mass spectrometry for identity confirmation represents the minimum analytical package required before proceeding with functional studies.

How can researchers design component analysis experiments to determine specific domains within AN2204?

• Domain prediction and truncation design:

  • Bioinformatic analysis to predict domain boundaries

  • Creation of a systematic series of N-terminal and C-terminal truncations

  • Generation of internal deletion constructs removing specific predicted domains

  • Site-directed mutagenesis of conserved residues within identified motifs

• Expression and purification of domain variants:

  • Parallel expression of full-length protein and domain variants

  • Optimization of expression conditions for each construct

  • Standardized purification protocols to ensure comparable preparations

• Functional comparison workflow:

  • Development of quantitative assays measuring specific activities

  • Direct comparison of full-length protein vs. domain variants

  • Statistical analysis determining significance of functional differences

• Structural studies of individual domains:

  • X-ray crystallography or NMR studies of isolated domains

  • Comparison of domain structures in isolation vs. full protein context

This component analysis approach can be particularly valuable for investigating the repeated tyrosine-glutamate (YE) motifs observed in the AN2204 sequence, potentially revealing their contribution to protein-protein interactions or other functions.

The experimental design should incorporate both positive and negative controls, with appropriate statistical methods for analyzing differences between full-length protein and domain variants. This systematic approach allows researchers to map function to specific regions of the protein structure.

What techniques can identify potential protein-protein interactions involving AN2204?

Understanding the protein interaction network of AN2204 is crucial for functional characterization. A comprehensive protein interaction discovery workflow should include:

• In vitro interaction methods:

  • Pull-down assays: Using His-tagged AN2204 as bait to capture interacting proteins from cellular lysates, followed by mass spectrometry identification

  • Surface Plasmon Resonance (SPR): Quantitative measurement of binding kinetics with candidate interactors

  • Isothermal Titration Calorimetry (ITC): Thermodynamic characterization of binding interactions

  • Microscale Thermophoresis (MST): Detecting interactions based on changes in thermophoretic mobility

• Cellular interaction methods:

  • Yeast two-hybrid (Y2H) screening: Systematic screening against cDNA libraries from Emericella nidulans

  • Bimolecular Fluorescence Complementation (BiFC): Visualizing interactions in cellular contexts

  • Proximity-dependent biotin identification (BioID): Identifying proteins in close proximity in vivo

  • Co-immunoprecipitation (Co-IP): Validating interactions in native cellular environments

• High-throughput screening approaches:

  • Protein microarrays: Testing interactions against large panels of purified proteins

  • Next-generation sequencing coupled Y2H: Comprehensive interaction mapping

• Computational prediction methods:

  • Protein docking simulations: Predicting physical compatibility with candidate interactors

  • Co-evolution analysis: Identifying potential interactors based on correlated evolutionary patterns

The search results indicate that traditional methods such as yeast two-hybrid, co-IP, and pull-down assays have been successfully employed for interaction studies in Emericella nidulans proteins . A multi-method approach is recommended to overcome the limitations of individual techniques and to distinguish between direct and indirect interactions.

What parameters should be considered when designing comparative analyses between AN2204 and homologous proteins?

When designing comparative analyses between AN2204 and its homologs, researchers should implement a structured experimental framework addressing multiple parameters:

• Sequence-based comparative parameters:

  • Percent identity and similarity at full sequence level

  • Conservation specifically within predicted functional domains

  • Conservation of key residues identified through structural modeling

  • Evolutionary rate analysis to identify rapidly or slowly evolving regions

• Structural comparative parameters:

  • Secondary structure conservation despite sequence divergence

  • Surface electrostatic potential mapping

  • Conservation of binding pockets or catalytic sites

  • Comparison of protein dynamics through molecular dynamics simulations

• Functional comparative parameters:

  • Standardized biochemical assays applied identically across homologs

  • Complementation studies in knockout/knockdown models

  • Localization patterns in cellular contexts

  • Interactome comparison using standardized interaction discovery methods

• Experimental design considerations:

  • Multi-element designs allow direct comparison of multiple homologs simultaneously4

  • Control for expression level differences when comparing protein activities

  • Include taxonomically diverse homologs to separate general from species-specific functions

  • Incorporate both positive and negative controls in all assays

A robust comparative analysis would examine homologs from diverse fungal species, potentially including industrially or medically relevant Aspergillus species. This approach can identify conserved functions that may be essential across species versus specialized adaptations unique to Emericella nidulans.

How might researchers investigate the potential role of AN2204 in biosynthetic pathways using comparative analysis?

To investigate whether AN2204 contributes to biosynthetic pathways in Emericella nidulans, researchers can implement a comparative analysis framework focusing on secondary metabolite production:

• Metabolomic comparative analysis:

  • Compare metabolite profiles between wild-type strains and AN2204 deletion mutants

  • Conduct parallel analysis across multiple culture conditions to capture condition-specific effects

  • Perform targeted metabolomics focusing on known Emericella nidulans secondary metabolites, including emericellamides

  • Apply multivariate statistical analyses to identify significant differences in metabolite patterns

• Transcriptomic comparative analysis:

  • Compare gene expression profiles between wild-type and AN2204 mutants

  • Focus particularly on expression changes in known biosynthetic gene clusters

  • Analyze co-expression networks to identify genes consistently co-regulated with AN2204

• Biosynthetic pathway reconstruction:

  • Based on metabolomic changes, propose potential biochemical reactions that may involve AN2204

  • Compare with characterized biosynthetic pathways like the emericellamide pathway

  • Test complementation with known pathway enzymes to identify potential functional overlap

• Heterologous expression studies:

  • Express AN2204 in heterologous hosts alongside known biosynthetic pathways

  • Analyze changes in metabolite production to identify synthetic capabilities

Given that Emericella nidulans produces various bioactive compounds including emericellamides A and C-F (cyclic polyketide-nonribosomal peptides with antibiotic properties) , comparative analysis might reveal whether AN2204 contributes to these or related biosynthetic pathways. This approach could potentially uncover novel antibiotic compounds or biosynthetic mechanisms of pharmaceutical interest.

What methodological considerations are important when analyzing potentially contradictory data about AN2204 function?

When confronted with contradictory data regarding AN2204 function, researchers should implement a structured approach to resolve discrepancies:

• Experimental variable identification:

  • Systematically catalog differences in experimental conditions between contradictory studies

  • Focus particularly on protein preparation methods, buffer compositions, and assay conditions

  • Evaluate whether the recombinant protein tag (His-tag) might influence function differently across studies

• Replication strategy:

  • Design factorial experiments that systematically vary the identified experimental variables

  • Include side-by-side replication of contradictory protocols within the same laboratory

  • Implement blinded analysis procedures to minimize expectation bias

• Method triangulation:

  • Apply multiple independent methodologies to address the same functional question

  • For each function being investigated, implement at least three different assay types

  • Evaluate concordance across different methodological approaches

• Statistical analysis framework:

  • Apply rigorous statistical tests appropriate for the experimental design (parametric or non-parametric)4

  • Calculate effect sizes to quantify the magnitude of observed effects

  • Implement meta-analysis techniques to integrate results across studies

  • Consider Bayesian approaches to update confidence in specific hypotheses given new data

• Biological context consideration:

  • Investigate whether contradictory results might reflect genuine biological complexity

  • Consider whether AN2204 might have different functions under different conditions

  • Evaluate whether post-translational modifications might alter function in different contexts

This methodological framework allows researchers to determine whether contradictory results reflect technical artifacts, biological complexity, or statistical anomalies, ultimately leading to a more nuanced understanding of AN2204 function.

How can researchers leverage structural biology approaches to study AN2204?

A comprehensive structural biology investigation of AN2204 would involve a multi-technique approach progressing from computational prediction to experimental structure determination:

• Computational structure prediction:

  • AlphaFold2 or RoseTTAFold prediction of full-length structure

  • Domain-specific modeling focusing on regions of predicted functional importance

  • Molecular dynamics simulations to assess conformational flexibility

  • Prediction of potential ligand-binding pockets or catalytic sites

• X-ray crystallography workflow:

  • Construct optimization (including surface entropy reduction)

  • Crystallization condition screening (sparse matrix and targeted approaches)

  • Data collection strategy optimization

  • Structure solution and refinement

  • Validation against biochemical and functional data

• NMR spectroscopy approaches:

  • 2D and 3D heteronuclear experiments for backbone and side-chain assignments

  • Relaxation measurements to identify dynamic regions

  • Chemical shift perturbation studies to map interaction interfaces

  • Residual dipolar coupling measurements for improved structural accuracy

• Cryo-electron microscopy:

  • Particularly valuable if AN2204 forms larger complexes with interaction partners

  • Single-particle analysis for high-resolution structure determination

  • Tomography for cellular contextualization

• Integrative structural biology:

  • Combining data from multiple structural techniques

  • Cross-validation between computational predictions and experimental structures

  • Correlating structural features with functional assay results

The structural analysis should pay particular attention to the repeated tyrosine-glutamate (YE) motifs identified in the AN2204 sequence, as these may form important structural or functional elements. Additionally, structural comparison with proteins from characterized biosynthetic pathways in Emericella nidulans could provide insights into potential functional roles .

What strategies can identify the biological significance of AN2204 in Emericella nidulans?

To comprehensively determine the biological significance of AN2204 within Emericella nidulans, researchers should implement a multi-faceted approach combining genetic, cellular, and systems biology techniques:

• Genetic manipulation strategies:

  • CRISPR/Cas9-mediated gene knockout to create ΔAN2204 strains

  • Conditional expression systems (inducible promoters) to control AN2204 levels

  • Gene replacement with tagged versions for localization studies

  • Point mutations of conserved residues to create separation-of-function alleles

• Phenotypic characterization matrix:

  • Growth rate analysis across diverse carbon and nitrogen sources

  • Stress response profiling (oxidative, osmotic, temperature, pH)

  • Secondary metabolite production analysis

  • Cell morphology and development assessment

  • Competitive fitness assays with wild-type strains

• Omics integration approach:

  • Transcriptomics: RNA-seq comparing wild-type vs. ΔAN2204 strains

  • Proteomics: Quantitative proteome analysis focusing on pathway components

  • Metabolomics: Targeted and untargeted metabolite profiling

  • Interactomics: Comprehensive protein interaction network mapping

  • Multi-omics data integration to identify regulated pathways

• Comparative biology framework:

  • Cross-species complementation studies

  • Phenotypic comparison with homologous gene knockouts in related species

  • Evolutionary rate analysis to assess selective pressure

This multi-dimensional approach allows researchers to distinguish between direct and indirect effects of AN2204 deletion, identify condition-specific functions, and place the protein within its biological context. Given the known production of bioactive compounds like emericellamides in Emericella nidulans , particular attention should be paid to changes in secondary metabolite production in ΔAN2204 strains.