Recombinant Danio rerio N-alpha-acetyltransferase 35, NatC auxiliary subunit (naa35), partial

What is Recombinant Danio rerio N-alpha-acetyltransferase 35 and what is its biological function?

Recombinant Danio rerio N-alpha-acetyltransferase 35 (naa35) functions as an auxiliary subunit of the NatC complex, which is responsible for N-terminal acetylation of proteins. This post-translational modification alters the electrostatic properties of substrate N-termini, potentially affecting protein folding, stability, half-life, interactions, and subcellular targeting . The protein is also known as Embryonic growth-associated protein (zEGAP) or MAK10 homolog in zebrafish . As part of the heterotrimeric NatC complex, naa35 works alongside the catalytic Naa30 subunit and a second auxiliary subunit Naa38 to co-translationally acetylate the N-termini of numerous target proteins .

How does the NatC complex differ structurally from other N-terminal acetyltransferase complexes?

The NatC complex, which includes naa35 as an auxiliary subunit, exhibits a strikingly different architecture compared to previously described N-terminal acetyltransferase (NAT) complexes . While the catalytic mechanism of acetyl transfer is conserved across NAT complexes, the heterotrimeric NatC complex has evolved a unique structure-function relationship. Crystal structure analysis has revealed that the NatC complex recognizes the first four amino acids of cognate substrates at the Naa30–Naa35 interface through a sequence-specific recognition mechanism . This structural divergence reflects how NAT machineries have evolved distinct architectures to acetylate specific subsets of target proteins .

What experimental models benefit most from studies using Recombinant Danio rerio naa35?

Zebrafish (Danio rerio) models benefit significantly from studies utilizing recombinant naa35, particularly in developmental biology and neuroscience research. Since naa35 is also known as Embryonic growth-associated protein (zEGAP) , it likely plays important roles in early development. The methodological approach to studying naa35 function typically involves:

• Loss-of-function studies using morpholinos or CRISPR-Cas9

• Rescue experiments with recombinant naa35 protein

• Comparative analysis with other vertebrate models

The recombinant protein allows researchers to conduct in vitro acetylation assays to characterize substrate preferences and compare enzymatic activities across developmental stages or tissue types. When designing such experiments, researchers should consider the proper controls and validation strategies to confirm specificity of the recombinant protein's activity.

What are the optimal storage and reconstitution protocols for Recombinant Danio rerio naa35?

For optimal storage and reconstitution of Recombinant Danio rerio naa35, researchers should follow these methodological guidelines:

Storage Protocol:

• Store lyophilized form at -20°C/-80°C for up to 12 months

• Store liquid form at -20°C/-80°C for up to 6 months

• Avoid repeated freeze-thaw cycles, as this can compromise protein activity

• Working aliquots can be stored at 4°C for up to one week

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

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

• Add 5-50% glycerol (with 50% as the recommended final concentration)

• Prepare small working aliquots to minimize freeze-thaw cycles

These guidelines are critical for maintaining protein stability and activity. The shelf life depends on multiple factors including storage state, buffer ingredients, storage temperature, and the intrinsic stability of the protein itself .

How can researchers effectively validate the enzymatic activity of Recombinant Danio rerio naa35 in experimental setups?

Validating the enzymatic activity of Recombinant Danio rerio naa35 requires a multifaceted approach, as naa35 is an auxiliary subunit rather than the catalytic component of the NatC complex. A comprehensive validation methodology includes:

• Complex Reconstitution: Express and purify all three components of the NatC complex (naa35, Naa30, and Naa38) and reconstitute the complex in vitro.

• Acetylation Assay: Monitor the transfer of an acetyl group from acetyl-CoA to specific peptide substrates using:

  • Radiometric assays with [14C]-acetyl-CoA

  • HPLC-based assays measuring CoA production

  • Colorimetric assays using DTNB (5,5'-dithiobis-(2-nitrobenzoic acid))

• Substrate Specificity Analysis: Test various peptide substrates with different N-terminal sequences to confirm the expected substrate specificity of the NatC complex.

• Controls:

  • Positive control: Use a known functional NatC complex

  • Negative control: Omit one component of the complex or use a catalytically inactive mutant

  • Substrate control: Include non-cognate substrates that should not be acetylated

When analyzing enzymatic data, researchers should be aware that some NATs are prone to substrate or product inhibition, which may affect kinetic measurements .

What are the considerations for co-expressing the complete functional NatC complex including naa35?

Co-expressing the complete functional NatC complex requires careful consideration of multiple factors to ensure proper complex formation and enzymatic activity. The methodological approach should include:

• Construct Design:

  • Engineer the naa35 gene with an N-terminal 6xHis tag and TEV recognition site

  • Include untagged Naa30 (catalytic subunit) and Naa38 (second auxiliary subunit) in appropriate expression vectors

  • Consider using a pET-Duet vector system for co-expression of multiple proteins

• Expression System Selection:

  • For zebrafish NatC, E. coli expression systems are typically suitable

  • For human and other metazoan NAT complexes, insect cells (Sf9) may be preferable due to the potential role of inositol hexaphosphate (IP6) as a scaffolding molecule

• Purification Strategy:

  • Use affinity chromatography targeting the His-tagged naa35

  • Follow with size exclusion chromatography to isolate the intact complex

  • Verify complex formation by SDS-PAGE, noting that larger auxiliary units like naa35 tend to stain more strongly with Coomassie than the smaller catalytic subunits

• Quality Control:

  • Assess complex integrity by dynamic light scattering or native PAGE

  • Confirm enzymatic activity using acetylation assays

  • Verify proper subunit stoichiometry using quantitative mass spectrometry

These considerations ensure the production of a functionally active NatC complex suitable for subsequent biochemical and structural studies.

How can researchers distinguish between NatA, NatC, and other NAT complex substrates in proteomic studies?

Distinguishing between substrates of different NAT complexes in proteomic studies requires a sophisticated methodological approach that leverages the unique substrate preferences of each complex. For effective differentiation:

• N-terminal Peptide Enrichment Techniques:

  • Use Strong Cation Exchange (SCX) chromatography to enrich for N-terminal peptides

  • Apply Terminal Amine Isotopic Labeling of Substrates (TAILS) methodology

  • Implement Combined FRActional Diagonal Chromatography (COFRADIC)

• Differential Knockdown/Knockout Analysis:

  • Perform targeted knockdown of specific NAT complex components (e.g., siRNA against naa35 for NatC complex)

  • Compare N-terminal acetylomes between control and knockdown conditions

  • Identify differentially acetylated peptides as potential substrates

• Substrate Sequence Analysis:

  • NatA typically acetylates N-termini with small amino acids (Ala, Ser, Thr, Gly, Val) after initiator methionine removal

  • NatC preferentially acetylates proteins with hydrophobic N-terminal residues (Met-Leu, Met-Ile, Met-Phe, Met-Trp)

  • Create position-specific scoring matrices for each NAT complex based on confirmed substrates

• Quantitative Proteomics Workflow:

  Step	Methodology	Output
  Sample preparation	siRNA knockdown of specific NAT complex components	Cells with reduced expression of target NAT
  Protein extraction and digestion	Tryptic digestion with optimized protocols for N-terminal peptides	Peptide mixture
  N-terminal enrichment	TAILS or COFRADIC	Enriched N-terminal peptides
  LC-MS/MS analysis	High-resolution mass spectrometry	Raw spectral data
  Data analysis	Database searching with variable N-terminal acetylation	Identified acetylated and non-acetylated N-termini
  Statistical analysis	Comparison of acetylation levels between conditions	Statistically significant differences in acetylation

By systematically applying these approaches, researchers can confidently assign proteins as substrates of specific NAT complexes and understand the complex interplay between different acetylation machineries .

What structural features of naa35 are critical for substrate recognition in the NatC complex?

The structural features of naa35 that are critical for substrate recognition in the NatC complex have been elucidated through crystallographic studies. These structural elements create a unique molecular architecture that enables specific substrate binding:

• Interface Formation with Naa30:

  • naa35 forms a critical interface with the catalytic Naa30 subunit, creating a substrate binding pocket

  • This interface is responsible for recognizing the first four amino acids of cognate substrates

  • The binding pocket accommodates the hydrophobic N-terminal sequences typically acetylated by NatC (Met-Leu, Met-Ile, Met-Phe, Met-Trp)

• Conformational Changes:

  • Substrate binding induces specific conformational changes in the complex

  • A sequence-specific, ligand-induced conformational change in Naa30 enables efficient acetylation

  • This conformational flexibility is essential for proper catalytic function

• Ribosome Association:

  • naa35 contains an elongated tip region with a ribosome-binding patch

  • This structural feature facilitates co-translational acetylation by positioning the NatC complex near the ribosomal exit tunnel

  • The ribosome association ensures timely modification of nascent polypeptide chains

• Evolutionary Divergence:

  • Unlike catalytic subunits, the large auxiliary subunits like naa35 share low sequence identity with other NAT complex auxiliary subunits

  • This divergence reflects the specialized roles these subunits play in substrate selection

  • The unique structural features of naa35 contribute to the distinct substrate specificity of the NatC complex

Understanding these critical structural features provides insights into the molecular basis of substrate selectivity and catalytic efficiency of the NatC complex, which can inform the design of specific inhibitors or engineered variants with altered specificity.

How does the expression and function of naa35 in Danio rerio compare to its orthologs in other model organisms?

The expression and function of naa35 in Danio rerio show both conserved and species-specific characteristics when compared to its orthologs in other model organisms. This comparative analysis reveals important evolutionary insights:

• Cross-Species Comparison:

  Species	Gene Names	Alternative Names	Function	Species-Specific Features
  Danio rerio	naa35, mak10, zgc:64157, wu:fb21g10	Embryonic growth-associated protein (zEGAP)	NatC auxiliary subunit	Potential role in embryonic development
  Homo sapiens	NAA35, EGAP, MAK10, MAK10P, bA379P1.1	N-alpha-acetyltransferase 35	NatC auxiliary subunit	Implicated in disease processes
  Mus musculus	Naa35, Mak10, AI158944, A330021G12Rik, etc.	N-alpha-acetyltransferase 35	NatC auxiliary subunit	Multiple transcript variants
  Saccharomyces cerevisiae	MAK10, NAA35	Mak10p	NatC auxiliary subunit	Well-studied model for NatC structure
  Schizosaccharomyces pombe	mak10	NatC N-acetyltransferase complex subunit Mak10	NatC auxiliary subunit	Predicted function based on homology

• Developmental Expression Patterns:

  • In zebrafish, naa35 (as zEGAP) may have specific roles in embryonic development

  • Temporal and spatial expression analyses across developmental stages reveal tissue-specific patterns

  • Functional studies using morpholinos or CRISPR-based approaches can elucidate developmental roles

• Functional Conservation:

  • The core function as a NatC auxiliary subunit is conserved across species

  • All orthologs participate in the formation of the heterotrimeric NatC complex

  • The substrate specificity of NatC appears largely conserved, suggesting functional conservation of naa35

• Experimental Approaches for Comparative Studies:

  • Complementation studies in yeast to test functional conservation

  • Heterologous expression of orthologs to assess interchangeability

  • Cross-species rescue experiments to determine functional equivalence

  • Structural studies to compare binding interfaces and substrate recognition mechanisms

These comparative analyses are particularly valuable for translating findings from model organisms to human disease contexts and for understanding the evolutionary conservation of N-terminal acetylation machinery .

What are common challenges in achieving high purity and yield of recombinant naa35, and how can they be addressed?

Achieving high purity and yield of recombinant naa35 presents several challenges that researchers commonly encounter. These challenges and their methodological solutions include:

• Protein Solubility Issues:

  • Challenge: naa35 is a large protein that may form inclusion bodies when expressed alone.

  • Solution: Co-express with partner proteins (Naa30 and Naa38) to promote proper folding and solubility.

  • Alternative: Optimize expression conditions (temperature, induction time, media composition) and consider fusion tags (SUMO, MBP) that enhance solubility .

• Degradation During Purification:

  • Challenge: Proteolytic degradation during cell lysis and purification.

  • Solution: Include protease inhibitor cocktails, maintain cold temperatures throughout purification, and minimize processing time.

  • Monitoring: Track protein integrity via SDS-PAGE at each purification step.

• Co-purification of Contaminants:

  • Challenge: Bacterial proteins that non-specifically bind to affinity resins.

  • Solution: Implement multi-step purification strategies (affinity chromatography followed by ion exchange and size exclusion chromatography).

  • Quality control: Verify final purity by SDS-PAGE (target >85% purity) .

• Low Expression Yield:

  • Challenge: Poor expression levels in heterologous systems.

  • Solution: Optimize codon usage for the expression host, test different expression systems (E. coli, yeast, baculovirus, mammalian cells) .

  • Strategy: Screen multiple constructs with different boundaries or tags in parallel.

• Complex Stability Issues:

  • Challenge: Dissociation of the NatC complex during purification.

  • Solution: Include stabilizing agents in buffers (glycerol, NaCl, reducing agents) and optimize pH and ionic strength.

  • Analysis: Use analytical size exclusion chromatography to monitor complex integrity.

Implementing these strategic approaches can significantly improve the yield and purity of recombinant naa35, facilitating downstream biochemical and structural studies.

How should researchers interpret unexpected changes in enzymatic activity of the NatC complex containing recombinant naa35?

Interpreting unexpected changes in enzymatic activity of the NatC complex containing recombinant naa35 requires systematic troubleshooting and analysis. Researchers should follow this methodological framework:

• Establish Baseline Activity:

  • Before investigating changes, establish reproducible baseline activity measurements using standardized substrates and conditions.

  • Document key parameters (temperature, pH, buffer composition, substrate and cofactor concentrations).

• Systematic Analysis of Activity Changes:

  Observation	Possible Causes	Diagnostic Approaches	Corrective Measures
  Complete loss of activity	Denaturation, proteolysis, cofactor depletion	SDS-PAGE analysis, mass spectrometry, cofactor supplementation	Optimize storage conditions, add stabilizing agents, refresh cofactors
  Reduced activity	Partial denaturation, inhibitor presence, suboptimal pH/temperature	Activity vs. pH/temperature profiles, inhibitor screening	Adjust reaction conditions, buffer exchange, add reducing agents
  Altered substrate specificity	Conformational changes, subunit stoichiometry issues	Compare kinetic parameters with various substrates, check complex composition	Reconstitute complex with defined stoichiometry, verify structural integrity
  Substrate/product inhibition	High substrate or CoA concentrations	Vary substrate concentrations, add CoA scavengers	Work at lower substrate concentrations (<100 μM acetyl-CoA)

• Investigate Structural Integrity:

  • Some NATs are prone to substrate or product inhibition, particularly human NatA which is sensitive to CoA .

  • Use biophysical methods (circular dichroism, thermal shift assays) to assess structural integrity.

  • Consider analytical size exclusion chromatography to verify complex formation.

• Control Experiments:

  • Compare activity with fresh preparations of the complex.

  • Test activity of the catalytic subunit alone versus the complete complex.

  • Introduce known mutations that affect activity as reference points.

• Advanced Troubleshooting:

  • For sequence-specific issues, consider a ligand-induced conformational change in Naa30 that enables efficient acetylation .

  • If working with human or metazoan complexes, consider the potential role of inositol hexaphosphate (IP6) as a scaffolding molecule .

This structured approach allows researchers to systematically identify and address factors affecting NatC complex activity, leading to more reliable and reproducible experimental results.

What are emerging techniques for studying the in vivo role of naa35 in zebrafish development and disease models?

Emerging techniques for studying the in vivo role of naa35 in zebrafish development and disease models encompass several cutting-edge methodological approaches:

• CRISPR-Cas9 Genome Editing:

  • Generate precise point mutations in naa35 to study structure-function relationships

  • Create conditional knockouts using inducible systems (e.g., CreERT2/loxP)

  • Implement base editing or prime editing for subtle modifications

  • Establish tissue-specific knockouts to distinguish local versus global effects

• Live Imaging Techniques:

  • Use fluorescent protein fusions to track naa35 localization during development

  • Implement light-sheet microscopy for long-term, low-phototoxicity imaging

  • Apply FRET-based sensors to monitor protein-protein interactions in real-time

  • Utilize super-resolution microscopy to visualize subcellular localization

• Single-Cell Approaches:

  • Perform single-cell RNA-seq to identify cell populations affected by naa35 dysfunction

  • Apply spatial transcriptomics to map expression patterns in tissue context

  • Implement CyTOF or CODEX for protein-level single-cell analysis

  • Use lineage tracing to follow cell fate decisions influenced by naa35

• Chemical Biology Tools:

  • Develop small molecule inhibitors specific to the NatC complex

  • Implement photocrosslinking to capture transient interactions

  • Apply chemical genetics with engineered naa35 variants

  • Utilize targeted protein degradation (PROTACs, dTAGs) for rapid protein depletion

• Disease Modeling Applications:

  • Engineer zebrafish models mimicking human disease mutations

  • Implement high-throughput behavioral assays to detect subtle phenotypes

  • Apply metabolomics and proteomics to characterize systemic effects

  • Utilize drug screening platforms for therapeutic development

These emerging techniques provide powerful tools for dissecting the complex roles of naa35 in zebrafish development and disease, potentially revealing novel insights into N-terminal acetylation biology with translational implications .

How can structural studies of naa35 inform the development of specific inhibitors or modulators of the NatC complex?

Structural studies of naa35 provide crucial insights that can directly inform the rational design of specific inhibitors or modulators of the NatC complex. This structure-guided drug discovery approach involves:

• Target Site Identification:

  • Crystal structures reveal that naa35 forms a critical interface with Naa30 for substrate recognition

  • The substrate binding pocket at the Naa30-naa35 interface presents a druggable site

  • Sequence-specific, ligand-induced conformational changes in Naa30 enable efficient acetylation, suggesting potential allosteric sites

• Structure-Based Drug Design Strategies:

  • Virtual screening against the substrate binding pocket or protein-protein interfaces

  • Fragment-based approaches to identify chemical scaffolds with binding potential

  • Structure-activity relationship (SAR) studies to optimize lead compounds

  • Molecular dynamics simulations to identify cryptic pockets or transient states

• Targeting NatC-Specific Features:

  • The divergent architecture of NatC compared to other NAT complexes enables development of selective inhibitors

  • The ribosome-binding patch in the elongated tip region of NatC presents a unique target site

  • Species-specific structural differences can be exploited for selective targeting

• Methodological Framework for Inhibitor Development:

  Phase	Methodology	Key Considerations
  Initial screening	Structure-based virtual screening, biochemical assays	Target specific protein-protein interfaces or catalytic site
  Hit validation	Biophysical binding assays (SPR, ITC, NMR)	Confirm direct binding to target site
  Mechanism studies	Enzymatic assays, X-ray crystallography with bound inhibitors	Determine inhibition mechanism (competitive, non-competitive, etc.)
  Selectivity profiling	Counter-screening against other NAT complexes	Ensure specificity for NatC over NatA, NatB, etc.
  Cellular studies	Cell penetration, target engagement, phenotypic assays	Verify activity in cellular context
  In vivo validation	Zebrafish models with naa35 mutations, rescue experiments	Confirm efficacy and selectivity in vivo

• Therapeutic Applications:

  • Alterations in N-terminal acetylation are implicated in several diseases, including cancers and developmental disorders

  • Selective NatC inhibitors could provide new therapeutic approaches for conditions where NatC activity is dysregulated

  • Zebrafish models provide an excellent system for initial in vivo validation of compound efficacy and toxicity

This structure-guided approach leverages the unique architectural features of the NatC complex to develop selective modulators with potential research and therapeutic applications .

What are the recommended best practices for ensuring reproducibility in experiments using recombinant naa35?

Ensuring reproducibility in experiments using recombinant naa35 requires adherence to a comprehensive set of best practices that address multiple aspects of experimental design, execution, and reporting:

• Protein Production and Quality Control:

  • Document complete expression and purification protocols, including vector constructs, expression systems, and purification steps

  • Implement consistent quality control measures (SDS-PAGE, mass spectrometry, activity assays)

  • Establish minimum purity standards (>85% by SDS-PAGE)

  • Record and report protein concentration determination methods

• Storage and Handling Protocols:

  • Store lyophilized protein at -20°C/-80°C (shelf life up to 12 months)

  • Store liquid formulations at -20°C/-80°C (shelf life up to 6 months)

  • Avoid repeated freeze-thaw cycles

  • Prepare working aliquots for short-term use (up to one week at 4°C)

  • Document buffer composition and additives (glycerol percentage, salt concentration)

• Experimental Design Considerations:

  • Include positive and negative controls in all experiments

  • Perform technical and biological replicates (minimum triplicate measurements)

  • Blind analysis where applicable to reduce bias

  • Use statistical power calculations to determine appropriate sample sizes

• Assay Standardization:

  • For kinetic studies, standardize substrate concentration ranges (consider potential substrate/product inhibition)

  • Maintain consistent reaction conditions (temperature, pH, buffer composition)

  • Calibrate instruments regularly and include calibration standards

  • Document detailed assay protocols including all reagents and their sources

• Data Management and Reporting:

  • Maintain comprehensive laboratory records with raw data

  • Report all experimental conditions, including those that yielded negative results

  • Share complete datasets through appropriate repositories

  • Document all statistical analyses and data transformations

By implementing these best practices, researchers can significantly enhance the reproducibility of experiments using recombinant naa35, facilitating comparison of results across different studies and advancing our understanding of NatC complex function .

What key considerations should researchers keep in mind when interpreting results from studies using recombinant naa35?

When interpreting results from studies using recombinant naa35, researchers should consider several key factors that may influence experimental outcomes and their biological relevance:

• Protein Context Dependencies:

  • naa35 functions as part of the heterotrimeric NatC complex, and its activity depends on proper complex formation with Naa30 and Naa38

  • Results from studies using isolated naa35 may not reflect its native biological function

  • Consider whether experiments with the complete NatC complex would provide more physiologically relevant insights

• Species-Specific Variations:

  • Subtle differences exist between naa35 orthologs from different species

  • When extrapolating results across species (e.g., from zebrafish to humans), account for these variations

  • For translational studies, consider validating key findings in the target species

• Technical Limitations and Artifacts:

  • Recombinant protein may lack post-translational modifications present in vivo

  • Expression tags (His, GST, etc.) may influence protein function or interactions

  • E. coli-expressed proteins lack eukaryotic-specific co-factors that might be important for function

  • The purity of the preparation (target >85%) can affect experimental outcomes

• Physiological Relevance Assessment:

  • Consider whether experimental conditions (substrate concentrations, buffer composition, etc.) reflect the cellular environment

  • Evaluate whether observed in vitro effects would occur at physiological concentrations

  • Validate key findings in cellular or in vivo models when possible

• Data Integration Framework:

  • Integrate results from multiple experimental approaches (biochemical, structural, cellular, in vivo)

  • Consider alternative explanations for unexpected results

  • Place findings in the context of the broader literature on N-terminal acetylation

  • Recognize the limitations of current models and methodologies

By carefully considering these factors, researchers can avoid misinterpretation of experimental results and develop more accurate models of naa35 function in the context of the NatC complex and N-terminal acetylation biology .

What essential resources and tools are available for researchers working with recombinant naa35?

Researchers working with recombinant naa35 can access several essential resources and tools that facilitate experimental design, data analysis, and interpretation:

• Protein Information Resources:

  • UniProt entry Q7T322 for Danio rerio naa35 - provides curated protein information, sequence data, and functional annotations

  • Protein Data Bank (PDB) - contains structural information for NAT complexes

  • The Zebrafish Information Network (ZFIN) - offers genetic and expression data for zebrafish naa35

• Commercial Sources for Recombinant Protein:

  • Cusabio offers Recombinant Danio rerio naa35 (Product Code: CSB-EP759697DIL)

  • MyBioSource provides recombinant naa35 from various species including Danio rerio

• Sequence Analysis Tools:

  • Phyre2 or PsiPred servers - generate secondary structure predictions useful for construct design

  • Multiple sequence alignment tools (Clustal Omega, MUSCLE) - help identify conserved regions across species

• Expression and Purification Resources:

  • pET-Duet vector systems - facilitate co-expression of multiple proteins

  • Detailed protocols for expression and purification of NAT complexes

• Functional Assay Methodologies:

  • Established protocols for acetylation assays using radiometric, HPLC-based, or colorimetric detection

  • Mass spectrometry-based N-terminal proteomics workflows

• Zebrafish Research Tools:

  • Established zebrafish lines for developmental studies

  • CRISPR-Cas9 protocols optimized for zebrafish

  • Phenotypic analysis pipelines for developmental studies

• Data Analysis Software:

  • Kinetics software for enzyme activity analysis

  • Proteomics data analysis tools for N-terminal acetylation studies

  • Structural visualization software for analyzing protein-protein interfaces

These resources provide researchers with the necessary tools to conduct comprehensive studies on recombinant naa35 and its role in the NatC complex, facilitating advances in our understanding of N-terminal acetylation biology .

How can researchers effectively collaborate across disciplines to advance understanding of naa35 function?

Effective interdisciplinary collaboration to advance understanding of naa35 function requires strategic approaches that bridge different research domains and methodologies. A comprehensive framework includes:

• Establishing Cross-Disciplinary Teams:

  • Integrate structural biologists, biochemists, developmental biologists, and computational scientists

  • Create collaborative networks that span basic and translational research

  • Develop shared terminology and conceptual frameworks to facilitate communication

• Complementary Methodological Approaches:

  Discipline	Contribution	Methodologies	Integration Points
  Structural Biology	Elucidation of protein structure and interactions	X-ray crystallography, cryo-EM, NMR	Inform biochemical assay design and molecular modeling
  Biochemistry	Characterization of enzymatic activity and specificity	Enzyme kinetics, substrate profiling	Connect structure to function, identify key residues
  Cell Biology	Cellular context and regulation	Microscopy, protein interaction studies	Link biochemical findings to cellular functions
  Developmental Biology	In vivo function in zebrafish	Genetic manipulations, phenotypic analysis	Provide physiological relevance to molecular findings
  Computational Biology	Data integration and prediction	Molecular dynamics, systems biology	Generate testable hypotheses, integrate diverse datasets

• Data Sharing and Integration Strategies:

  • Implement FAIR principles (Findable, Accessible, Interoperable, Reusable) for data management

  • Utilize common data repositories and standardized formats

  • Develop integrated analytical pipelines that combine diverse data types

  • Create visualization tools that communicate complex findings across disciplines

• Collaborative Research Models:

  • Establish regular interdisciplinary meetings or workshops focused on N-terminal acetylation

  • Create shared resource centers for specialized techniques or equipment

  • Develop joint funding proposals that explicitly value interdisciplinary approaches

  • Implement team science training to enhance collaborative effectiveness

• Translational Research Connections:

  • Partner with clinical researchers to explore disease relevance

  • Connect basic findings to potential therapeutic applications

  • Collaborate with pharmaceutical researchers on drug development

  • Engage with patient advocacy groups when disease connections are established