Recombinant Nautilus macromphalus Uncharacterized protein IMPP17

What is known about the structure and function of IMPP17 in Nautilus macromphalus?

IMPP17 likely belongs to the Shell Matrix Protein (SMP) family, which plays essential roles in shell formation and structural maintenance. While specific information about IMPP17 is limited, studies on related Nautilus species have identified numerous shell-specific sequences through multiomics approaches. These proteins typically function in calcium carbonate nucleation, crystal growth regulation, and polymorph selection during shell biomineralization . As with many shell proteins, IMPP17 may contain domains that interact directly with calcium carbonate crystals or other shell matrix components to orchestrate the precise arrangement of mineral components in the nautilus shell.

How is IMPP17 identified and classified within cephalopod shell proteomes?

Identification of shell matrix proteins like IMPP17 typically follows a multiomics approach combining transcriptomics and proteomics:

• Transcriptomic analysis of mantle tissue (the shell-secreting organ) using next-generation sequencing platforms such as Ion Torrent PGM

• Extraction of shell matrix proteins using demineralization protocols

• LC-MS/MS analysis of shell matrix proteins

• Matching of spectra to translated transcriptome data to identify shell-specific proteins

• Sequence annotation through BLASTp/BLASTx searches against databases like GenBank

• Domain identification using tools such as SMART, PROSITE, InterProScan, and Pfam

Proteins are considered shell-specific when they are matched by at least two LC-MS/MS polypeptides and show higher expression in the mantle compared to other tissues.

What evolutionary relationships exist between IMPP17 and other molluscan shell proteins?

Shell matrix proteins in cephalopods like Nautilus show complex evolutionary relationships with proteins from other molluscan lineages. Studies on Nautilus pompilius have revealed that some shell proteins and protein domains are conserved across all Conchiferans (shelled mollusks), while others are specific to certain lineages . Phylogenetic analyses typically indicate that many shell matrix protein families were present in the ancestral Conchiferan but were independently recruited for shell formation in different molluscan lineages. For uncharacterized proteins like IMPP17, comparative analysis with other molluscan species can provide valuable evolutionary context and potential functional insights.

What expression systems are most effective for producing recombinant Nautilus shell proteins?

Multiple expression systems can be utilized for recombinant shell protein production, each with distinct advantages:

For shell proteins like IMPP17, which may require specific folding conditions or post-translational modifications, testing multiple expression systems is often necessary to identify optimal production conditions.

What purification strategies are recommended for recombinant IMPP17?

Purification of recombinant shell matrix proteins like IMPP17 typically involves multi-step strategies:

• Initial capture:

  • Affinity chromatography using fusion tags (His, GST, MBP)

  • Ion exchange chromatography based on predicted isoelectric point

• Intermediate purification:

  • Size exclusion chromatography to separate monomeric protein from aggregates

  • Hydrophobic interaction chromatography

• Final polishing:

  • Reversed-phase HPLC for highest purity

  • Tag removal using specific proteases

Shell proteins often require specific considerations including calcium-affinity chromatography for calcium-binding proteins, inclusion of stabilizing agents to prevent aggregation, and testing both native and denaturing conditions as some shell proteins may be intrinsically disordered .

How can researchers verify the functional activity of recombinant IMPP17?

Verifying functional activity of recombinant shell proteins requires specialized assays:

• Calcium binding assays:

  • Isothermal titration calorimetry (ITC)

  • Calcium overlay assays

  • Fluorescence-based calcium binding assays

• Mineral formation assays:

  • In vitro crystallization assays with ammonium carbonate diffusion method

  • Analysis of crystal morphology and polymorph selection

  • Atomic force microscopy to observe crystal growth modification

• Structural characterization:

  • Circular dichroism (CD) spectroscopy to verify proper folding

  • Small-angle X-ray scattering (SAXS) for solution structure

• Interaction studies:

  • Surface plasmon resonance (SPR) to study interactions with other shell matrix components

  • Co-immunoprecipitation with shell extracts

Each assay provides complementary information about the protein's functional properties in the context of shell formation.

What methodological approaches can resolve contradictory data in IMPP17 functional studies?

When facing contradictory results in IMPP17 functional studies, researchers should implement a systematic troubleshooting approach:

• Expression system validation:

  • Compare protein from multiple expression systems (bacterial, yeast, insect, mammalian)

  • Analyze post-translational modifications using mass spectrometry

  • Verify protein folding using biophysical techniques (CD, fluorescence spectroscopy)

• Experimental condition optimization:

  • Test protein activity across a range of pH, temperature, and ionic strength conditions

  • Evaluate the impact of different calcium concentrations and other divalent cations

  • Assess the effect of potential cofactors or interacting proteins

• Complementary technique application:

  • Use multiple independent methods to measure the same parameter

  • Combine in vitro studies with in vivo approaches when possible

  • Implement both structural and functional analyses

• Statistical rigor enhancement:

  • Increase biological and technical replicates

  • Apply appropriate statistical tests for significance

  • Conduct power analyses to ensure adequate sample sizes

When contradictions persist, consider that the protein may have multiple functions or context-dependent activities, which is common for shell matrix proteins.

How do environmental factors influence IMPP17 expression and function in shell formation?

The expression and function of shell matrix proteins like IMPP17 are likely influenced by various environmental factors:

Research methodologies to study these effects include:

• qPCR analysis of gene expression under controlled environmental conditions

• Proteomics of shell matrices from specimens exposed to different environments

• In vitro assays of protein function under varying physicochemical conditions

• Structural analysis of proteins in different environmental contexts

Understanding these relationships is crucial for predicting how changing marine environments may impact shell formation in nautilus populations.

What computational approaches best predict structure-function relationships in uncharacterized shell proteins?

Computational approaches for predicting structure-function relationships in uncharacterized shell proteins like IMPP17 include:

• Sequence-based predictions:

  • Hidden Markov Models for domain identification

  • Disorder prediction (PONDR, IUPred) for intrinsically disordered regions

  • Evolutionary coupling analysis to identify functionally important residues

  • Motif scanning for calcium-binding or mineral interaction sites

• Structure prediction methods:

  • AlphaFold2 or RoseTTAFold for protein structure prediction

  • Molecular dynamics simulations to evaluate conformational dynamics

  • Protein-mineral docking simulations

  • Modeling of post-translational modifications

• Systems biology approaches:

  • Protein-protein interaction network analysis

  • Pathway enrichment analysis

  • Co-expression network construction from transcriptomic data

  • Evolutionary analysis across molluscan lineages

The most effective approach combines multiple computational methods with experimental validation of key predictions, particularly focusing on regions predicted to interact with calcium carbonate or other shell components.

What techniques are most effective for analyzing IMPP17 interactions with calcium carbonate?

Several complementary techniques provide insights into shell protein-mineral interactions:

• In vitro crystallization assays:

  • Ammonium carbonate diffusion method to form calcium carbonate crystals

  • Analysis of resulting crystals using SEM, TEM, and XRD

  • Time-lapse microscopy to observe crystal growth kinetics

• Advanced microscopy:

  • Atomic force microscopy (AFM) for direct observation at nanoscale resolution

  • Force spectroscopy to measure protein-crystal binding strengths

  • Cryo-electron microscopy of protein-mineral interfaces

• Spectroscopic methods:

  • Solid-state NMR spectroscopy to analyze protein structure when bound to minerals

  • FTIR spectroscopy to detect changes in protein secondary structure upon mineral binding

  • Raman spectroscopy for polymorph identification and protein detection

• Computational approaches:

  • Molecular dynamics simulations of protein-mineral interactions

  • Density functional theory calculations of binding energetics

  • Machine learning for pattern recognition in experimental data

These techniques provide complementary information about how IMPP17 might influence calcium carbonate formation and crystal properties in the nautilus shell.

What are the challenges and solutions in expressing cephalopod shell proteins in heterologous systems?

Expression of cephalopod shell proteins in heterologous systems presents several challenges:

Successful strategies often involve:

• Testing multiple expression constructs with different tags and truncations

• Screening various expression conditions (temperature, induction time, media composition)

• Exploring multiple host systems from bacterial to mammalian cells

• Developing specialized refolding protocols when necessary

How can researchers effectively study post-translational modifications of IMPP17?

Post-translational modifications (PTMs) are crucial for shell protein function and require specialized approaches:

• Identification of native PTMs:

  • High-resolution mass spectrometry of native shell proteins

  • Targeted enrichment methods for specific modifications (phosphopeptide enrichment, glycopeptide enrichment)

  • Comparison between native and recombinant protein mass profiles

• Expression system selection:

  • Mammalian or insect cell systems for complex PTMs

  • Engineered yeast strains with humanized glycosylation

  • Cell-free systems supplemented with modifying enzymes

• PTM analysis methods:

  • Tandem mass spectrometry with electron transfer dissociation (ETD)

  • Site-directed mutagenesis of modification sites

  • PTM-specific detection methods (ProQ Diamond for phosphorylation, periodic acid-Schiff for glycosylation)

• Functional impact assessment:

  • Comparative crystallization assays between modified and unmodified proteins

  • Structure determination incorporating PTMs

  • Binding studies with shell components and minerals

Common PTMs in shell proteins include phosphorylation of serine/threonine residues, glycosylation, and sulfation, each potentially critical for mineral interactions and proper shell formation.

How can interdisciplinary approaches enhance our understanding of IMPP17 function?

Understanding shell proteins like IMPP17 benefits significantly from interdisciplinary collaboration:

• Molecular biology and biochemistry:

  • Recombinant protein expression and purification

  • Functional characterization of protein activities

• Structural biology:

  • Protein structure determination

  • Structural analysis of protein-mineral interfaces

• Materials science:

  • Characterization of mechanical properties

  • Analysis of crystal structure and organization

• Computational science:

  • Molecular dynamics simulations

  • Machine learning for pattern recognition in complex datasets

• Evolutionary biology:

  • Comparative genomics across molluscan lineages

  • Reconstruction of ancestral protein sequences

Effective collaboration frameworks include:

• Establishing shared terminology and standard protocols

• Implementing data management systems for multi-omics datasets

• Developing integrated analytical pipelines that combine multiple data types

• Creating collaborative networks that span academic disciplines

This interdisciplinary approach can resolve contradictions that might arise when viewing the protein from a single disciplinary perspective.

What experimental designs best elucidate the role of IMPP17 in the hierarchical structure of the nautilus shell?

To understand how IMPP17 contributes to hierarchical shell structure, multi-scale experimental approaches are required:

• Molecular-scale analysis:

  • In vitro crystallization assays with purified IMPP17

  • Characterization of protein-mineral binding sites

  • Analysis of protein self-assembly properties

• Microscale studies:

  • Immunolocalization of IMPP17 in developing shells

  • Correlative microscopy combining protein detection with mineral characterization

  • In vitro reconstitution of shell matrix assembly

• Macroscale investigations:

  • Mechanical testing of shell samples with varying protein content

  • Analysis of shell morphology in relation to protein expression patterns

  • Computational modeling of shell mechanical properties based on protein distribution

Experimental design should incorporate:

• Time-course studies during shell formation and repair

• Comparison between different regions of the nautilus shell

• Control experiments with related proteins from other mollusks

• Quantitative analysis methods to correlate protein parameters with structural outcomes

This multi-scale approach can reveal how molecular interactions translate into the remarkable hierarchical structure of the nautilus shell.