Recombinant Aplysia californica Temptin

What is Aplysia californica temptin and what is its biological function?

Temptin is one of several water-borne protein pheromones secreted by the albumen gland of Aplysia californica during egg-laying. It acts in concert with other pheromones, particularly attractin, enticin, and seductin, to form a chemical complex that attracts potential mates to egg-laying sites . Structurally, temptin shows sequence homology to the epidermal growth factor (EGF)-like domains found in higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation . Behavioral studies demonstrate that while temptin alone is not attractive to Aplysia, the combination of attractin and temptin significantly increases attraction, doubling the number of animals attracted compared to control conditions .

How is the temptin gene organized and expressed in Aplysia californica?

The temptin gene in Aplysia californica was identified through differential library screening of an albumen gland cDNA library and is designated as Alb-172 . The gene encodes a precursor protein that includes a signal peptide sequence for secretion . Northern blot analysis confirms that temptin is highly expressed in the albumen gland of sexually mature Aplysia . Immunolocalization studies and secretion assays demonstrate that temptin is actively secreted during egg-laying events, with immunoblot analysis confirming its presence in egg cordon eluates . Comparative genomics analysis reveals that the Aplysia brasiliana temptin protein shares 91% sequence identity with its Aplysia californica homolog, suggesting evolutionary conservation of this pheromone across related species .

What are the structural characteristics of temptin protein?

Temptin's structural analysis reveals a predominantly β-sheet structure as confirmed by CD spectral analysis . The protein contains two experimentally determined disulfide bonds: one internal disulfide bond between Cys57 and Cys77 (predicted from alignments with class I EGF-like domains) and a second between Cys18 and Cys103 . This second disulfide bond likely protects temptin against proteolysis in seawater and stabilizes its interacting surface . Three-dimensional modeling based on the Ca²⁺-binding EGF-like domain of fibrillin indicates that two temptin residues, Trp52 and Trp79, align with cysteine residues conserved in fibrillins . These residues potentially stabilize the disulfide bonds and a proposed metal-binding loop . This structural arrangement suggests temptin may function analogously to fibrillin in controlling transforming growth factor-β concentration, but in temptin's case, by modulating pheromone signaling through direct binding to attractin .

What expression systems have been successfully used to produce recombinant temptin?

Recombinant full-length temptin has been efficiently expressed in Escherichia coli using a cold-shock promoter system . This "cold-shock" method for protein expression has proven particularly effective for producing temptin in a soluble form, which is critical for subsequent structural and functional studies . The method likely utilizes a cold-shock promoter (such as the cspA promoter) that becomes active when cultures are shifted to lower temperatures, slowing protein synthesis and improving proper folding. After expression, the recombinant temptin is purified using reverse-phase high-performance liquid chromatography (RP-HPLC) , yielding protein suitable for structural characterization including CD spectroscopy and disulfide bond determination.

What purification strategies are most effective for recombinant temptin?

For recombinant temptin purification, RP-HPLC has proven to be an effective method following expression in E. coli . This approach allows for the isolation of functional temptin protein that maintains its native structure, including proper disulfide bond formation . While specific details of the RP-HPLC protocol aren't fully described in the available search results, typical approaches would involve using C4 or C18 columns with acetonitrile gradients containing trifluoroacetic acid or heptafluorobutyric acid (HFBA) as ion-pairing agents. For native temptin (from Aplysia tissue), purification has been achieved using C18 Sep-Pak Vac cartridges, followed by lyophilization and fractionation using SDS-polyacrylamide gel electrophoresis . Immunoblot analysis using temptin-specific antisera can then be used to confirm the identity of the purified protein .

How can researchers optimize disulfide bond formation in recombinant temptin?

Since temptin contains two critical disulfide bonds (Cys57-Cys77 and Cys18-Cys103) that are essential for its structural integrity , ensuring proper disulfide bond formation is crucial for producing functional recombinant protein. While the search results don't provide explicit protocols for optimizing disulfide bond formation, the successful use of the cold-shock expression system suggests this approach facilitates proper folding and disulfide bridge formation .

For optimizing disulfide bond formation in recombinant temptin, researchers should consider:

• Using an E. coli strain engineered for disulfide bond formation, such as Origami or SHuffle

• Expressing the protein in the oxidizing environment of the periplasmic space

• Adding oxidized/reduced glutathione pairs to the folding buffer during purification

• Including protein disulfide isomerase in refolding buffers to catalyze disulfide bond formation

Verification of correct disulfide bond formation can be performed using limited proteolysis followed by mass spectrometry, as demonstrated in the structural characterization of temptin .

How does temptin's structure relate to its function in pheromone signaling?

Temptin's structure bears significant homology to the Ca²⁺-binding, EGF-like domains found in the extracellular matrix protein fibrillin . This structural relationship provides insights into temptin's potential functional mechanisms. Three-dimensional modeling using the MPACK suite, based on the fibrillin EGF-like domain, reveals that temptin likely possesses a stabilized structure maintained by two disulfide bonds .

The presence of Trp52 and Trp79 residues, which align with conserved cysteine residues in fibrillins, suggests these residues stabilize temptin's disulfide bonds and a potential metal-binding loop . This arrangement facilitates temptin's role in modulating pheromone signaling. Specifically, docking studies with the NMR structure of attractin indicate that one face of temptin interacts directly with the attractin pheromone . This interaction may regulate attractin's accessibility to cellular receptors, similar to how fibrillin controls transforming growth factor-β concentration .

Gel shift experiments confirmed that temptin forms complexes with wild-type attractin, providing experimental validation of the predicted interactions . This molecular "glue" function could be crucial for the formation and stability of the pheromone complex in seawater environments, explaining why individual pheromones alone are less effective than combinations in behavioral assays .

What methods are used to determine temptin's disulfide bond arrangements?

The disulfide bond arrangements in temptin were determined through a combination of targeted proteolysis and mass spectrometry techniques . The methodology involved:

• Limited proteolysis of purified recombinant temptin

• Analysis of the resulting peptide fragments by mass spectrometry

• Identification of peptides containing cysteine residues

• Determination of which cysteine residues were linked by disulfide bonds

How can researchers develop accurate 3D models of temptin for interaction studies?

Developing accurate 3D models of temptin for interaction studies involves several complementary approaches:

• Homology modeling based on related proteins with known structures, particularly using the Ca²⁺-binding EGF-like domain of fibrillin as a template

• Incorporating experimental constraints from disulfide mapping studies

• Validating models with CD spectroscopy data that confirms temptin's predominantly β-sheet structure

• Refining models using computational docking with known binding partners, such as attractin

Researchers have successfully employed the MPACK suite to develop temptin models based on structural similarities to fibrillin . These models helped identify key structural features, including the roles of Trp52 and Trp79 in stabilizing disulfide bonds and a potential metal-binding loop .

For interaction studies, researchers can use these models for in silico docking simulations with other pheromone structures, such as the NMR structure of attractin . The predicted interactions can then be experimentally validated using techniques like gel shift assays, which have confirmed temptin-attractin complex formation . This integrated approach combining computational modeling with experimental validation provides powerful insights into the molecular mechanisms of pheromone complex formation and function.

How is temptin's role in mate attraction experimentally assessed?

Temptin's role in mate attraction is primarily assessed using T-maze behavioral assays with live Aplysia specimens. These assays evaluate the animals' attraction to various stimuli, including individual pheromones and pheromone combinations. The methodology includes:

• Setting up a T-maze apparatus containing 6 liters of artificial seawater

• Placing cages with or without non-laying Aplysia in each upper arm of the maze

• Adding test pheromones to the seawater adjacent to one cage

• Introducing a test animal at the base of the maze and observing its behavior for up to 20 minutes

A positive response is recorded if the animal travels to and remains in contact with the stimulus cage for 5 minutes. A negative response occurs if the animal chooses the opposite arm, and "no choice" is recorded if the animal does neither .

These behavioral studies have yielded several important findings:

• Temptin alone is not significantly attractive to Aplysia

• A binary blend of attractin and temptin (1 nmol each) significantly increases attraction, with 45% of animals responding positively

• This attraction level is comparable to that observed with egg cordons alone, suggesting the binary blend effectively mimics natural attraction cues

Statistical significance is assessed using the G test, providing quantitative measures of pheromone effectiveness . These behavioral assays are crucial for establishing the functional significance of temptin in mate attraction and for understanding how different pheromone components interact to elicit behavioral responses.

What evidence supports temptin's interaction with other pheromones in the Aplysia attraction complex?

Multiple lines of evidence support temptin's interaction with other pheromones in the Aplysia attraction complex:

• Behavioral studies: Binary blends of attractin and temptin double the number of animals attracted compared to controls, indicating synergistic effects that suggest molecular interaction .

• Biochemical evidence: Gel shift assays demonstrate that temptin forms complexes with wild-type attractin, providing direct evidence of molecular interaction .

• Structural modeling: Docking results using temptin's 3D model and the NMR structure of attractin suggest that one face of temptin interacts with attractin, potentially controlling its access to cellular receptors .

• Co-secretion patterns: Immunoblot analyses of egg cordon eluates show that temptin is secreted alongside attractin, enticin, and seductin during egg-laying events, indicating they function together in nature .

• Comparative potency: The combination of attractin, enticin, temptin, and seductin diffusing from freshly laid egg cordons comprises a "bouquet of scents" that is more attractive than individual components, suggesting they form a functional complex .

This multi-faceted evidence strongly supports the model that temptin serves as a "glue" in the water-borne attractin pheromone complex, modulating the activity of attractin through direct binding interactions . This mechanism may be analogous to how fibrillin controls transforming growth factor-β concentration in higher organisms .

How does temptin's stability in seawater affect its pheromonal activity?

The structural features of temptin contribute significantly to its stability in seawater, which is crucial for its function as a water-borne pheromone. The disulfide bond between Cys18 and Cys103 appears particularly important, as it likely protects temptin against proteolysis in seawater and stabilizes its interacting surface . This protection mechanism extends the functional lifespan of temptin in marine environments, allowing it to maintain its activity as a pheromonal signal over meaningful ecological timeframes.

The predominantly β-sheet structure of temptin, confirmed by CD spectral analysis, likely contributes additional stability in the marine environment . Beta-sheet structures are generally more resistant to denaturation in high-salt conditions than alpha-helical structures, providing temptin with structural resilience in seawater.

Additionally, temptin's ability to form complexes with other pheromones like attractin may provide mutual stabilization . These complexes could protect vulnerable regions of each pheromone from environmental degradation, explaining why pheromone combinations are more effective at eliciting behavioral responses than individual components .

While the search results don't provide direct measurements of temptin's half-life in seawater, its structural features strongly suggest adaptations for stability in marine environments. This stability is likely crucial for temptin's role in facilitating the formation and maintenance of egg-laying and mating aggregations in natural Aplysia populations .

How conserved is temptin across different Aplysia species?

Analysis of temptin across Aplysia species reveals remarkable conservation, suggesting strong evolutionary pressure to maintain the protein's structure and function. Specifically, the Aplysia brasiliana temptin protein shares 91% sequence identity with its Aplysia californica homolog . This high degree of conservation across species indicates the fundamental importance of temptin in Aplysia reproductive biology and pheromone communication systems.

The conservation extends beyond just primary sequence to functional aspects as well. Behavioral studies using Aplysia brasiliana in T-maze attraction assays demonstrate that the pheromone systems function similarly across species . The combination of attractin and temptin effectively attracts Aplysia brasiliana individuals, mirroring the responses seen in Aplysia californica .

This cross-species conservation makes temptin a valuable model for studying the evolution of reproductive pheromones in marine gastropods and potentially in other marine invertebrates. The fact that temptin shares structural features with EGF-like domains found in many organisms suggests it may have evolved from ancient signaling molecules with roles in cell-cell communication .

What related proteins share structural similarities with temptin?

Temptin shares significant structural similarities with several important protein families:

• EGF-like domains: Sequence homology analysis indicates temptin is related to the epidermal growth factor (EGF)-like domains of higher organisms, particularly those that mediate protein-cell surface contact during fertilization and blood coagulation .

• Fibrillin: Three-dimensional modeling suggests temptin's structure is similar to the Ca²⁺-binding, EGF-like domain of the extracellular matrix protein fibrillin . This similarity extends to functional aspects, as temptin may modulate pheromone signaling in a manner analogous to how fibrillin controls transforming growth factor-β concentration .

• Mammalian fertilins: Structure prediction results indicate that enticin (another Aplysia pheromone that works with temptin) may be related to the Ca²⁺ binding, EGF-like domains of mammalian fertilins and other proteins that mediate intercellular surface contacts . This suggests a potential evolutionary relationship between invertebrate pheromone systems and vertebrate fertilization proteins.

These structural relationships highlight the potential evolutionary conservation of protein domains involved in intercellular signaling and reproductive processes across diverse animal phyla. The EGF-like domain structure appears to be a versatile protein module that has been adapted for various functions, including chemical communication in marine invertebrates.

How do temptin expression patterns compare to other Aplysia pheromones?

Temptin expression exhibits both similarities and differences compared to other Aplysia pheromones:

Similarities in expression patterns:

• Like attractin, enticin, and seductin, temptin is highly expressed in the albumen gland of sexually mature Aplysia californica .

• All four pheromones are secreted during egg-laying events, as confirmed by immunoblot analysis of egg cordon eluates .

• The genes encoding temptin (Alb-172), attractin, enticin (Alb-24), and seductin are all significantly upregulated in the albumen gland, suggesting coordinated expression .

Differences in expression patterns:

• Unlike some other albumen gland proteins (Alb-23 and Alb-69), which are membrane-associated, temptin (like enticin) is highly abundant in the soluble fraction of albumen gland extracts .

• Expression levels may vary among the pheromones, with immunoblot analysis showing potentially different concentrations in egg cordon eluates .

This coordinated expression pattern supports the model that these pheromones function together as a complex to attract potential mates during egg-laying. The co-secretion of these pheromones creates a "bouquet of scents" that diffuses from freshly laid egg cordons, serving as a chemical signal to attract conspecifics . The similarities in expression patterns across multiple pheromones suggest evolutionary pressure to maintain this coordinated system for reproductive success.

How can researchers effectively design mutants to study temptin structure-function relationships?

Designing effective temptin mutants to study structure-function relationships should focus on key structural elements identified through previous research:

• Disulfide bond mutations: Create single and double cysteine-to-alanine mutations at positions 18, 57, 77, and 103 to disrupt the two characterized disulfide bonds (Cys57-Cys77 and Cys18-Cys103) . These mutations would help determine the contribution of each disulfide bond to temptin's stability and function.

• Tryptophan substitutions: Generate mutations at Trp52 and Trp79, which align with conserved cysteine residues in fibrillins and likely stabilize the disulfide bonds and metal-binding loop . Substituting these residues with alanine or phenylalanine would test their role in maintaining temptin's structure.

• Putative attractin-binding interface: Based on docking results that suggest one face of temptin interacts with attractin , create single and combination mutations in residues on this predicted interface. This would help map the exact binding surface between the two pheromones.

• Potential metal-binding loop mutations: If temptin indeed contains a metal-binding loop similar to fibrillin , mutating residues in this region would test the importance of metal binding for temptin function.

After creating these mutants, researchers should assess:

• Structural integrity using CD spectroscopy to confirm maintenance of β-sheet structure

• Ability to form complexes with attractin using gel shift assays

• Functional activity in T-maze behavioral assays when combined with other pheromones

• Stability in seawater environments

This systematic mutational analysis would provide valuable insights into the structural determinants of temptin function and its interactions with other components of the Aplysia pheromone system.

What methodological challenges exist in studying temptin-receptor interactions?

Studying temptin-receptor interactions presents several methodological challenges that researchers must address:

• Receptor identification: The specific cellular receptors that interact with temptin or the temptin-attractin complex remain unidentified in the search results. Identifying these receptors would require approaches such as:

  • Cross-linking studies with labeled temptin

  • Affinity purification coupled with mass spectrometry

  • Expression cloning from Aplysia neural tissues

  • Candidate receptor approaches based on related signaling systems

• Membrane protein expression: If temptin receptors are membrane proteins (likely G-protein coupled receptors), expressing them in recombinant systems can be challenging due to:

  • Proper folding and trafficking issues

  • Requirements for specific lipid environments

  • Potential need for accessory proteins

• Complex pheromonal interactions: Since temptin functions as part of a pheromone complex rather than individually , studying its receptor interactions requires considering:

  • Whether temptin directly interacts with receptors or primarily modulates attractin's receptor interactions

  • If the temptin-attractin complex forms before or during receptor binding

  • The potential for multiple receptor types responding to different components of the pheromone blend

• Marine environment considerations: Any in vitro receptor binding assays would need to account for the ionic conditions of seawater, which differ significantly from standard buffer systems used in receptor studies.

• Temporal dynamics: The kinetics of temptin-receptor interactions in natural settings may be complex, potentially involving:

  • Concentration-dependent effects as pheromones diffuse in seawater

  • Sequential binding events among multiple pheromones and receptors

  • Changes in receptor sensitivity or expression during reproductive periods

Addressing these challenges requires integrating multiple experimental approaches, including molecular, biochemical, and behavioral methods, to fully characterize the temptin signaling system.

How might recombinant temptin be used to develop novel research tools?

Recombinant temptin offers several promising applications as a research tool:

• Biosensor development: Labeled recombinant temptin could be used to develop biosensors for studying pheromone-receptor interactions in real-time. Fluorescently tagged temptin combined with imaging techniques could visualize the distribution and dynamics of pheromone binding in Aplysia tissues.

• Affinity chromatography: Immobilized recombinant temptin could serve as an affinity reagent to:

  • Purify interacting proteins, including potential receptors

  • Isolate the complete pheromone complex from natural egg cordon eluates

  • Study the binding parameters of temptin-attractin interactions

• Structural biology platforms: The ability to produce recombinant temptin in E. coli provides a platform for:

  • X-ray crystallography studies of temptin alone or in complex with attractin

  • NMR studies to complement the existing structural models

  • Cryo-EM analysis of larger pheromone complexes

• Model system for EGF-domain interactions: Given temptin's structural similarity to EGF-like domains involved in protein-cell surface contacts during fertilization and blood coagulation , recombinant temptin could serve as a model system for studying these evolutionarily conserved interaction mechanisms.

• Chemical ecology research: Recombinant temptin, combined with other Aplysia pheromones, could be used in field studies to:

  • Manipulate Aplysia aggregation and reproductive behaviors

  • Study population dynamics in response to controlled pheromone releases

  • Investigate species-specificity of pheromone responses

• Synthetic biology applications: The understanding of temptin's structure-function relationships could inform the design of novel protein-based signaling systems with applications in:

  • Cell-based biosensors

  • Controlled cellular aggregation systems

  • Biomimetic adhesives based on temptin's "glue-like" properties

These applications highlight how recombinant temptin production not only advances our understanding of marine invertebrate chemical ecology but also provides valuable tools for broader research areas in protein biochemistry and cell biology.