Recombinant Nautilus macromphalus Uncharacterized protein SMPP10

What is currently known about the structure and function of SMPP10?

SMPP10 is an uncharacterized protein isolated from Nautilus macromphalus (Bellybutton nautilus), a marine cephalopod. While the precise function remains unknown, it belongs to a class of proteins found in the Nautilus excretory system. The protein can be recombinantly expressed in various systems including E. coli, yeast, baculovirus, and mammalian cells, with purities reaching greater than 85% as determined by SDS-PAGE analysis . Given that Nautilus harbors unique bacterial symbionts in its pericardial appendage (excretory organ) that secretes ammonia-rich fluid, there may be potential relationships between SMPP10 and excretory or symbiotic functions, though this remains to be established through focused research .

What expression systems are most suitable for producing recombinant SMPP10?

The choice of expression system depends on your research goals. E. coli provides cost-effective expression with reasonable yields but lacks post-translational modifications. For structural studies requiring large quantities, E. coli is typically preferred. Yeast expression offers eukaryotic post-translational modifications with moderate yields. Baculovirus systems provide higher-order eukaryotic processing with excellent yields but require more complex methodology. Mammalian cell expression, while most expensive, provides the closest approximation to native protein processing .

When selecting an expression system, consider:

• Required protein yield

• Importance of post-translational modifications

• Budget constraints

• Downstream applications (structural studies vs. functional assays)

• Expression optimization parameters (temperature, induction conditions, etc.)

How should researchers design initial characterization experiments for SMPP10?

Initial characterization should follow a systematic approach:

• Sequence Analysis:

  • Conduct bioinformatic analysis using sequence alignment tools

  • Identify conserved domains, motifs, and potential functional sites

  • Perform phylogenetic comparisons with related proteins across species

• Expression Analysis:

  • Determine tissue distribution in Nautilus macromphalus

  • Quantify expression levels in different developmental stages

  • Assess subcellular localization using fractionation techniques

• Basic Biochemical Characterization:

  • Determine molecular weight, isoelectric point, and oligomerization state

  • Evaluate protein stability under various pH and temperature conditions

  • Assess post-translational modifications using mass spectrometry

• Preliminary Functional Assays:

  • Screen for enzymatic activities based on structural predictions

  • Perform protein-protein interaction studies using pull-down assays

  • Evaluate potential roles in symbiotic relationships with bacterial partners found in Nautilus

What approaches can be used to elucidate SMPP10's potential role in the unique dual symbiosis found in Nautilus?

Given the presence of a β-proteobacterium and coccoid spirochaete in the Nautilus pericardial appendage, investigating SMPP10's role in symbiosis requires multi-faceted approaches:

• Co-localization Studies:

  • Use fluorescence in situ hybridization (FISH) with specific probes for both the protein and symbiotic bacteria

  • Employ CARD-FISH (catalyzed reporter deposition-FISH) for enhanced sensitivity

  • Perform immunogold labeling with transmission electron microscopy (TEM) to visualize protein-bacteria interactions at ultrastructural level

• Functional Interaction Assays:

  • Develop bacterial culture systems mimicking the pericardial environment

  • Test recombinant SMPP10's effect on bacterial growth, metabolism, and gene expression

  • Perform co-immunoprecipitation to identify direct interactions between SMPP10 and bacterial proteins

• Metabolic Analysis:

  • Investigate SMPP10's potential role in nitrogen metabolism by examining interactions with ammonia processing

  • Test for interactions with genes involved in nitrogen cycling (amoA, nirS) that might be present in the symbiotic bacteria

  • Use isotope labeling to trace metabolic exchanges between host protein and symbionts

• Comparative Genomics/Proteomics:

  • Compare SMPP10 with proteins from other cephalopods lacking these specific symbionts

  • Perform differential proteomics on symbiont-rich versus symbiont-poor tissues

How should researchers approach structural analysis to predict SMPP10 function?

A comprehensive structural characterization would include:

• Primary Structure Analysis:

  • Perform multiple sequence alignments with related proteins

  • Identify conserved motifs and functional domains

  • Use disorder prediction algorithms to identify structured and unstructured regions

• Secondary Structure Determination:

  • Use circular dichroism (CD) spectroscopy to estimate α-helix and β-sheet content

  • Apply Fourier-transform infrared spectroscopy (FTIR) for complementary structure assessment

  • Implement predictive algorithms (PSIPRED, JPred) based on amino acid sequence

• Tertiary Structure Analysis:

  • X-ray crystallography for high-resolution structure (2.5Å or better)

  • NMR spectroscopy for solution structure and dynamics analysis

  • Cryo-electron microscopy for larger complexes or membrane-associated forms

• Computational Approaches:

  • Homology modeling using structurally characterized templates

  • Ab initio modeling for novel domains

  • Molecular dynamics simulations to predict functional movements and binding sites

• Functional Prediction from Structure:

  • Cavity and binding site prediction algorithms

  • Electrostatic surface analysis for interaction interfaces

  • Structure-based functional annotation using tools like ProFunc and COFACTOR

What considerations are important when designing knockdown/knockout experiments for SMPP10?

When designing genetic manipulation experiments for SMPP10 in Nautilus macromphalus, several factors must be considered:

• Model System Limitations:

  • Nautilus is challenging for genetic manipulation; consider developing surrogate models

  • Cell lines derived from Nautilus tissues may provide alternatives

  • Consider using CRISPR-Cas9 in cell culture before attempting whole-organism studies

• Targeting Strategy:

  • Design multiple sgRNAs targeting different regions of the SMPP10 gene

  • Create conditional knockdowns using inducible systems to avoid lethality

  • Consider knockdown approaches (RNAi, morpholinos) if complete knockout is problematic

• Validation Methods:

  • Confirm knockdown/knockout efficiency at both mRNA and protein levels

  • Use multiple validation techniques (qPCR, Western blot, immunofluorescence)

  • Include appropriate controls (scrambled sgRNAs, non-targeting controls)

• Phenotypic Analysis:

  • Examine effects on symbiotic bacteria population and localization

  • Assess impacts on excretory function and ammonia processing

  • Monitor organismal health and development throughout the experiment

• Rescue Experiments:

  • Design rescue constructs with codon optimization to avoid targeting

  • Consider structure-function analysis through domain-specific rescues

  • Include negative controls (rescue with mutated, non-functional protein)

What strategies can overcome challenges in purifying high-quality recombinant SMPP10?

Purification of uncharacterized proteins like SMPP10 presents unique challenges that require methodical approaches:

• Expression Optimization:

  • Test multiple expression systems (E. coli, yeast, baculovirus, mammalian) to identify optimal yield and solubility

  • Screen various fusion tags (His, GST, MBP, SUMO) for improved solubility and purification efficiency

  • Optimize induction conditions (temperature, inducer concentration, duration)

• Solubility Enhancement:

  • Co-express with molecular chaperones to improve folding

  • Test various buffer compositions (pH, salt concentration, additives)

  • Consider detergent screening if membrane association is suspected

• Purification Protocol Development:

  • Implement multi-step purification (affinity, ion exchange, size exclusion)

  • Optimize elution conditions to maintain protein stability

  • Monitor protein quality by dynamic light scattering and thermal shift assays

• Storage Optimization:

  • Test various storage buffers with stabilizing agents (glycerol, arginine, trehalose)

  • Determine optimal storage temperature (-20°C vs. -80°C for long-term)

  • Evaluate freeze-thaw stability and consider single-use aliquots

• Quality Control Measures:

  • Verify purity by SDS-PAGE (aim for >85% purity)

  • Confirm identity by mass spectrometry and N-terminal sequencing

  • Assess folding using circular dichroism or fluorescence spectroscopy

How can researchers develop reliable antibodies against SMPP10?

Developing specific antibodies against uncharacterized proteins requires careful planning:

• Antigen Design:

  • Use bioinformatic tools to identify immunogenic epitopes

  • Consider both full-length protein and peptide approaches

  • Select regions with high predicted surface exposure and low sequence conservation

• Production Strategy:

  • Compare polyclonal versus monoclonal approaches

  • For polyclonals, use multiple host species for cross-validation

  • For monoclonals, screen numerous clones for specificity and sensitivity

• Validation Protocol:

  • Test antibody specificity using Western blots against recombinant protein

  • Perform immunoprecipitation followed by mass spectrometry

  • Include knockout/knockdown controls to confirm specificity

  • Evaluate cross-reactivity against related proteins and host tissue samples

• Application Optimization:

  • Determine optimal conditions for each application (Western blot, immunofluorescence, ELISA)

  • Establish blocking conditions to minimize background

  • Document lot-to-lot variation and establish quality control procedures

What controls are essential when investigating protein-protein interactions involving SMPP10?

Robust protein-protein interaction studies require comprehensive controls:

• Negative Controls:

  • Non-relevant proteins of similar size and properties

  • Heat-denatured SMPP10 to detect non-specific binding

  • Host-cell extracts without recombinant protein expression

• Positive Controls:

  • Well-characterized protein interaction pairs

  • Tagged protein controls to validate detection methods

  • Spiked-in known interactors at varying concentrations

• Technique-Specific Controls:

  • For pull-down assays: beads-only controls, pre-clearing steps

  • For co-immunoprecipitation: isotype controls, reverse IP validation

  • For proximity ligation: single antibody controls, spatial resolution controls

• Validation Approaches:

  • Confirm interactions using multiple, orthogonal techniques

  • Perform domain mapping to identify specific interaction regions

  • Use competition assays with mutated versions to confirm specificity

  • Validate biological relevance through functional assays

How should researchers interpret phylogenetic analyses of SMPP10 in evolutionary context?

Phylogenetic analysis of SMPP10 requires careful interpretation:

• Sequence Selection:

  • Include diverse taxonomic groups focusing on cephalopods and mollusks

  • Consider both closely related and distantly related sequences

  • Include SMPP17 and other related uncharacterized proteins from Nautilus for comparison

• Alignment Quality Assessment:

  • Evaluate alignment quality using statistical measures

  • Manually inspect alignments for errors and artifacts

  • Consider structure-guided alignments where possible

• Tree Construction Methods:

  • Implement multiple algorithms (Maximum Likelihood, Bayesian, Neighbor-Joining)

  • Use appropriate evolutionary models based on likelihood ratio tests

  • Perform bootstrap analysis (>1000 replicates) to assess node confidence

• Evolutionary Interpretation:

  • Analyze rates of evolution across different lineages

  • Identify signatures of selection (dN/dS ratios)

  • Consider gene duplication events and functional divergence

  • Correlate evolutionary patterns with species having symbiotic relationships similar to Nautilus

• Functional Inference:

  • Map known functional data onto the phylogenetic tree

  • Identify co-evolution with symbiont presence across related species

  • Consider convergent evolution in species with similar ecological niches

What bioinformatic approaches are most useful for predicting SMPP10 function?

A comprehensive bioinformatic analysis should include:

• Sequence-Based Predictions:

  • PSI-BLAST and HHpred for distant homology detection

  • InterProScan for domain and motif identification

  • SignalP and TMHMM for cellular localization signals

  • NetPhos and other PTM prediction tools

• Structural Predictions:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • CASTp and SiteMap for binding pocket identification

  • Molecular docking with potential substrates/interactors

  • MD simulations to assess conformational flexibility

• Network-Based Approaches:

  • Co-expression analysis across tissues and conditions

  • Protein-protein interaction network prediction

  • Phylogenetic profiling to identify functionally related proteins

  • Metabolic pathway gap identification

• Integration Methods:

  • Machine learning approaches combining multiple features

  • Bayesian integration of diverse prediction methods

  • Literature mining for related uncharacterized proteins

  • Cross-species comparison focusing on organisms with similar symbiotic relationships

How can researchers resolve contradictory experimental results when studying SMPP10?

When faced with contradictory results:

• Systematic Troubleshooting:

  • Catalog all experimental variables between contradictory studies

  • Evaluate differences in reagents, expression systems, and methodologies

  • Assess protein quality metrics (purity, activity, folding state)

• Sequential Validation:

  • Replicate experiments using standardized protocols

  • Introduce controlled variables one at a time to identify sources of variation

  • Use multiple orthogonal techniques to test the same hypothesis

• Technical Considerations:

  • Evaluate antibody specificity and potential cross-reactivity

  • Consider post-translational modifications affecting function

  • Assess buffer composition effects on protein behavior

  • Examine potential contamination with bacterial components from expression systems

• Biological Complexity Assessment:

  • Consider tissue-specific or developmental differences

  • Evaluate potential isoforms or splice variants

  • Assess context-dependent protein function (e.g., symbiont presence/absence)

  • Examine potential species-specific differences in protein function

• Collaborative Resolution:

  • Implement multi-laboratory validation studies

  • Standardize protocols and materials across research groups

  • Establish minimal reporting standards for SMPP10 experiments

How might SMPP10 relate to the excretory function of Nautilus pericardial appendages?

Given the localization of symbiotic bacteria in the excretory organs of Nautilus, several research avenues could elucidate SMPP10's potential role:

• Functional Localization Studies:

  • Perform immunohistochemistry to map SMPP10 distribution within pericardial tissues

  • Determine if SMPP10 co-localizes with the β-proteobacterium and coccoid spirochaete in the baso-medial region of pericardial villi

  • Investigate presence in ultrafiltration and reabsorption regions

• Excretory Process Investigation:

  • Examine SMPP10 involvement in ammonia-rich fluid secretion

  • Test interactions with ion channels or transporters involved in excretion

  • Analyze potential roles in acid-base regulation within the excretory system

• Symbiont Relationship Studies:

  • Investigate whether SMPP10 influences symbiont distribution or activity

  • Test if SMPP10 mediates nutrient exchange between host and symbionts

  • Examine potential protective functions against symbiont overgrowth

• Comparative Physiology:

  • Compare SMPP10 expression and function with other cephalopods lacking the unique dual symbiosis

  • Analyze evolutionary adaptations in excretory proteins across nautiloid lineages

  • Investigate functional analogs in other marine organisms with similar excretory challenges

What high-throughput approaches would be most valuable for characterizing SMPP10?

Modern high-throughput technologies offer powerful approaches for uncharacterized proteins:

• Interactome Mapping:

  • Yeast two-hybrid or BioID proximity labeling to identify binding partners

  • IP-MS (immunoprecipitation-mass spectrometry) to identify protein complexes

  • Cross-linking mass spectrometry to capture transient interactions

• Functional Genomics:

  • CRISPR screening in cell culture models to identify genetic interactions

  • Transcriptomics before and after SMPP10 knockdown to identify affected pathways

  • Ribosome profiling to assess translational impacts

• Structural Genomics:

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural analysis

  • Cryo-EM for complex structural assemblies

  • Fragment-based screening to identify potential ligands

• Systems Biology Integration:

  • Multi-omics integration (proteomics, metabolomics, transcriptomics)

  • Network analysis to position SMPP10 within cellular pathways

  • Machine learning approaches to predict function from integrated datasets