Recombinant Human Ankyrin repeat domain-containing protein 37 (ANKRD37)

What is the structural architecture of ANKRD37's ankyrin repeat domains?

ANKRD37 contains multiple ankyrin repeat motifs that fold into characteristic L-shaped domains. Each repeat consists of two antiparallel α-helices followed by a β-hairpin or extended loop. These repeats stack together to form a slightly curved structure resembling a cupped hand, where the β-hairpins represent the "fingers" and the α-helices form the "palm" . This architectural arrangement creates a specific protein-binding interface that mediates ANKRD37's interactions with partner proteins. Unlike globular proteins, ankyrin repeat domains are dominated by local, short-range interactions, which represents a distinct paradigm for protein stability and folding mechanisms .

How many ankyrin repeats are typically found in ANKRD37?

While the search results don't specifically mention the number of repeats in ANKRD37, analysis of ankyrin repeat proteins shows that they typically contain between 1-33 repeats, with most proteins having six or fewer repeats . The detection of terminal repeats can be challenging due to sequence divergence from the consensus, particularly as terminal repeats often contain polar residues that facilitate interactions with solvent, replacing the well-conserved hydrophobic residues found in internal repeats .

What cellular functions is ANKRD37 likely involved in?

Based on the known functions of ankyrin repeat proteins, ANKRD37 likely participates in cellular processes such as transcriptional regulation, signal transduction, or protein complex assembly. Ankyrin repeat proteins generally function as scaffolds for protein-protein interactions without enzymatic activity . Other ankyrin repeat proteins participate in cell-cell signaling, cytoskeleton integrity, transcription and cell-cycle regulation, inflammatory response, development, and various transport phenomena .

What techniques are most effective for analyzing ANKRD37 expression patterns across different tissues?

For analyzing ANKRD37 expression patterns, researchers should consider complementary approaches including:

• RNA-based methods: qRT-PCR for quantitative mRNA expression analysis, RNA-seq for comprehensive transcriptomic profiling, and in situ hybridization for spatial localization in tissue sections.

• Protein-based methods: Western blotting with ANKRD37-specific antibodies for protein quantification, immunohistochemistry/immunofluorescence for visualization in tissues, and mass spectrometry for proteomic analysis.

• Reporter systems: Creation of ANKRD37 promoter-reporter constructs to monitor transcriptional regulation in different cell types or in response to various stimuli.

The combination of these techniques provides a comprehensive view of ANKRD37 expression patterns, essential for understanding its physiological roles.

What factors regulate ANKRD37 expression and how can these be experimentally manipulated?

To investigate ANKRD37 regulation, researchers could explore:

• Transcription factor binding: Chromatin immunoprecipitation (ChIP) to identify transcription factors binding to the ANKRD37 promoter.

• Epigenetic regulation: Bisulfite sequencing to analyze DNA methylation patterns and ChIP for histone modifications associated with the ANKRD37 gene.

• Experimental manipulation strategies:

  • Gene silencing using siRNA or CRISPR/Cas9 systems

  • Overexpression using recombinant expression vectors

  • Treatment with chemical modulators of signaling pathways potentially regulating ANKRD37

Integrating these approaches provides a comprehensive understanding of ANKRD37 regulation mechanisms.

What expression systems are optimal for producing functional recombinant ANKRD37?

The choice of expression system depends on research objectives:

• Bacterial expression (E. coli):

  • Advantages: High yield, simplicity, cost-effectiveness

  • Limitations: Potential for improper folding of ankyrin repeats due to lack of eukaryotic chaperones

  • Optimization: Use specialized strains with enhanced disulfide bond formation capabilities; expression at lower temperatures (16-18°C); fusion with solubility-enhancing tags (MBP, SUMO)

• Insect cell systems:

  • Advantages: Eukaryotic folding machinery, post-translational modifications

  • Optimization: Baculovirus expression vector systems with optimized signal sequences and purification tags

• Mammalian expression systems:

  • Advantages: Native folding environment, appropriate post-translational modifications

  • Systems: HEK293 or CHO cells for transient or stable expression

The selection should be guided by whether native conformation and post-translational modifications are critical for the planned experiments.

What purification strategy yields the highest purity and stability for recombinant ANKRD37?

A multi-step purification strategy is recommended:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Glutathione affinity chromatography for GST-fusion proteins

• Intermediate purification:

  • Ion exchange chromatography based on ANKRD37's predicted isoelectric point

  • Heparin affinity chromatography if DNA/RNA binding properties are suspected

• Polishing step:

  • Size exclusion chromatography to separate monomeric protein from aggregates and remove remaining impurities

• Stability optimization:

  • Buffer screening using differential scanning fluorimetry

  • Addition of stabilizing agents (glycerol, specific salts, reducing agents)

The purification protocol should be validated using SDS-PAGE, Western blotting, and mass spectrometry to confirm identity and purity.

How can the proper folding of ANKRD37's ankyrin repeats be verified?

Multiple complementary techniques should be employed:

• Circular dichroism (CD) spectroscopy: To assess secondary structure content and confirm the expected α-helical signature of ankyrin repeats

• Thermal denaturation studies: To analyze the cooperative unfolding behavior characteristic of properly folded ankyrin domains. Previous studies on ankyrin repeat domains have shown they typically exhibit two-state folding transitions despite their modular structure

• Limited proteolysis: Well-folded ankyrin repeat domains show characteristic resistance patterns to proteolytic digestion

• Analytical ultracentrifugation: To confirm homogeneity and expected hydrodynamic properties

• Functional assays: Verification of expected protein-protein interactions as the ultimate test of proper folding

What are the most effective techniques for identifying ANKRD37 binding partners?

Researchers should consider a multi-layered approach:

• Screening methods:

  • Yeast two-hybrid screening

  • Mammalian two-hybrid systems

  • Affinity purification coupled with mass spectrometry (AP-MS)

  • Proximity-based labeling techniques (BioID, APEX)

• Validation methods:

  • Co-immunoprecipitation in relevant cell types

  • GST pull-down assays with recombinant proteins

  • Surface plasmon resonance (SPR) for quantitative binding kinetics

  • Fluorescence resonance energy transfer (FRET) for interactions in living cells

The search results indicate numerous ankyrin repeat proteins have binding partners identified through these techniques, as shown in Table 1 from the literature .

How does the number of ankyrin repeats in ANKRD37 influence its binding specificity and affinity?

This question addresses a fundamental property of ankyrin repeat domains:

• Surface area effects: The number of ankyrin repeats directly impacts the available binding surface area. More repeats typically provide larger interaction interfaces, potentially increasing binding affinity and enabling multivalent interactions

• Conformational considerations: The natural curvature of stacked ankyrin repeats creates a specific three-dimensional binding surface. This curvature becomes more pronounced with increasing repeat numbers, potentially affecting binding partner specificity

• Experimental approaches to investigate this question:

  • Construct a series of ANKRD37 truncation mutants with varying numbers of repeats

  • Measure binding affinities (KD values) using techniques like isothermal titration calorimetry (ITC) or SPR

  • Analyze complex stability through thermal denaturation studies

  • Perform molecular dynamics simulations to model conformational effects

Studies on other ankyrin repeat proteins have demonstrated that deletion of C-terminal repeats can significantly impact stability, as shown in the Notch ankyrin domain where full seven-repeat constructs (Nank1-7*) had substantially higher stability than versions with fewer repeats .

What crystallization strategies are most successful for ankyrin repeat proteins like ANKRD37?

Crystallization of ankyrin repeat proteins presents unique challenges and opportunities:

• Construct optimization:

  • Systematic truncation series to identify stable, crystallizable fragments

  • Surface entropy reduction through mutation of high entropy residues (Lys, Glu) to alanine

  • Consideration of co-crystallization with binding partners to stabilize flexible regions

• Crystallization conditions:

  • Initial broad screening followed by focused optimization

  • Attention to pH ranges 6.0-8.0, where ankyrin repeat proteins typically maintain stability

  • Inclusion of additives that promote crystal contacts without destabilizing the protein

• Alternative approaches:

  • Fusion with crystallization chaperones (e.g., T4 lysozyme)

  • Synthetic ankyrin repeat proteins (DARPins) as crystallization aids

Successful crystallization of 13 natural and 3 designed ankyrin repeat proteins has been reported , providing precedent for structural studies of ANKRD37.

How can NMR spectroscopy be optimally applied to study ANKRD37 structure and dynamics?

NMR approaches for ANKRD37 should consider:

• Sample preparation considerations:

  • Expression of isotopically labeled protein (15N, 13C, 2H as needed)

  • Optimization of buffer conditions for NMR (avoidance of paramagnetic ions, pH control)

  • Potential segmental labeling for larger constructs

• Experimental strategies:

  • Backbone assignment using TROSY-based experiments if molecular weight exceeds 20 kDa

  • Residual dipolar coupling measurements to define relative orientation of ankyrin repeats

  • Relaxation measurements to characterize dynamics of individual repeats

• Data analysis approaches:

  • Automated assignment software combined with manual verification

  • Integration with previously solved ankyrin repeat structures as templates

  • Modeling of repeat-repeat interfaces

What cellular pathways is ANKRD37 likely involved in and how can these be experimentally mapped?

To investigate ANKRD37's role in cellular pathways:

• Pathway analysis approaches:

  • Proteomics analysis following ANKRD37 knockdown/overexpression

  • Phosphoproteomics to identify signaling changes

  • Transcriptomics to identify genes regulated directly or indirectly by ANKRD37

• Functional screening methods:

  • CRISPR/Cas9 screening to identify synthetic lethal interactions

  • High-content imaging following ANKRD37 perturbation

  • Protein-fragment complementation assays to map physical interaction networks

• Integration with known ankyrin repeat protein functions:

  • Assessment of roles in established ankyrin repeat protein pathways such as:

    • Cell-cycle regulation (similar to INK4 proteins)

    • Transcriptional regulation (similar to IκB proteins)

    • Signal transduction (similar to Notch)

    • Cytoskeletal organization

The search results indicate ankyrin repeat proteins function in diverse cellular processes including cell-fate decisions, inflammatory responses, and cytoskeletal organization .

How can ANKRD37 binding interfaces be mapped at amino acid resolution?

Detailed mapping of ANKRD37 binding interfaces requires:

• Mutagenesis approaches:

  • Alanine scanning mutagenesis of surface residues

  • Charge reversal mutations at potential interaction sites

  • Creation of chimeric proteins by swapping repeats with other ankyrin repeat proteins

• Structural analysis of complexes:

  • X-ray crystallography of ANKRD37 with binding partners

  • Cryo-EM for larger complexes

  • Crosslinking mass spectrometry to identify residues in proximity at binding interfaces

• Computational methods:

  • Molecular docking guided by experimental constraints

  • Molecular dynamics simulations of protein-protein interactions

Previous co-crystal structures of ankyrin repeat proteins have shown that binding typically occurs at the concave inner surface comprising the β-hairpin/loop regions and inner short helices .

What methods are most effective for investigating ANKRD37 roles in disease processes?

To investigate ANKRD37 in disease contexts:

• Clinical correlation studies:

  • Analysis of ANKRD37 expression in patient samples using immunohistochemistry and qRT-PCR

  • Correlation of expression levels with disease progression and prognosis

  • Genotyping studies to identify disease-associated variants

• Functional disease modeling:

  • Gene editing in cell lines using CRISPR/Cas9 to introduce disease-associated mutations

  • Patient-derived iPSCs differentiated into relevant cell types

  • Animal models with modified ANKRD37 expression or function

• Mechanistic investigations:

  • Interactome analysis in disease vs. normal conditions

  • Signaling pathway perturbation studies

  • Drug screening to identify modulators of ANKRD37 function

Many ankyrin repeat proteins have been implicated in human diseases, with the INK4 family of tumor suppressors (p15, p16, p18, p19) being prominent examples .

How can disease-associated mutations in ANKRD37 be functionally characterized?

For comprehensive characterization of disease-associated mutations:

• Structural impact assessment:

  • In silico modeling of mutation effects on protein stability and conformation

  • Thermal denaturation studies comparing wild-type and mutant proteins

  • Limited proteolysis to detect conformational changes

• Functional consequences evaluation:

  • Quantitative binding assays to assess effects on protein-protein interactions

  • Cellular localization studies using fluorescently tagged constructs

  • Functional complementation assays in knockout cell lines

• Integrated approaches:

  • Creation of isogenic cell lines differing only in the mutation of interest

  • Multi-omics profiling to comprehensively characterize downstream effects

  • Phenotypic screening to identify cellular processes affected by mutations

How can protein engineering approaches be applied to study ANKRD37 function?

Innovative protein engineering strategies include:

• Designer ankyrin repeat proteins:

  • Creation of consensus-designed ankyrin repeat domains as scaffolds

  • Development of ANKRD37-based protein binders through directed evolution

  • Construction of chimeric proteins with repeats from different ankyrin proteins

• Optogenetic and chemogenetic control:

  • Integration of light-sensitive domains to enable spatiotemporal control of ANKRD37 interactions

  • Creation of rapamycin-inducible dimerization systems to control ANKRD37 localization and function

• Biosensor development:

  • Creation of FRET-based biosensors using ANKRD37 to monitor protein interactions in real-time

  • Development of split-protein complementation reporters based on ANKRD37 binding properties

Recent consensus-based protein design strategies have been successfully applied to ankyrin repeat proteins, leveraging the large number of available sequences .

What computational approaches can predict novel functions and interactions of ANKRD37?

Advanced computational methods include:

• Sequence-based predictions:

  • Machine learning algorithms trained on known ankyrin repeat protein functions

  • Evolutionary analysis to identify conserved functional residues

  • Co-evolution analysis to predict protein-protein interactions

• Structure-based approaches:

  • Molecular dynamics simulations to identify conformational states

  • Virtual screening for potential binding partners or small molecule modulators

  • Fragment-based computational design of interaction surfaces

• Network-based methods:

  • Integration of protein-protein interaction networks, gene expression data, and phenotypic information

  • Graph theory approaches to predict functional associations

  • System-level modeling of ANKRD37 in cellular pathways

How has ANKRD37 evolved across species and what does this reveal about its function?

Evolutionary analysis of ANKRD37 can provide functional insights through:

• Phylogenetic analysis methods:

  • Construction of phylogenetic trees to map ANKRD37 evolution

  • Identification of orthologs across diverse species

  • Analysis of repeat number variation across evolutionary lineages

• Sequence conservation patterns:

  • Calculation of sequence conservation scores at binding interfaces versus non-interface regions

  • Identification of species-specific insertions or deletions

  • Detection of signatures of positive or negative selection

• Functional divergence assessment:

  • Comparative expression pattern analysis across species

  • Cross-species complementation experiments

  • Comparison of binding partner networks in different organisms

The analysis of ankyrin repeat domains across species has revealed remarkable conservation of structure despite sequence variation, highlighting the evolutionary importance of this protein interaction module .

What experimental approaches can validate cross-species functional conservation of ANKRD37?

To assess functional conservation across species:

• Heterologous expression studies:

  • Expression of ANKRD37 orthologs from different species in human cell lines

  • Assessment of subcellular localization patterns

  • Evaluation of protein-protein interaction profiles

• Domain swapping experiments:

  • Creation of chimeric proteins with domains from ANKRD37 orthologs

  • Functional complementation testing in knockout systems

  • Binding affinity measurements with predicted conserved partners

• Model organism studies:

  • CRISPR/Cas9 modification to humanize ANKRD37 in model organisms

  • Phenotypic comparison of mutants across species

  • Cross-species rescue experiments

Note: This table presents typical patterns observed in ankyrin repeat proteins; specific values for ANKRD37 would require detailed sequence analysis.