Recombinant Litoria ewingi Caeridin-7.1

What is Caeridin-7.1 and where was it originally discovered?

Caeridin-7.1 is a peptide first discovered in 1997 by Steinborner et al. from the dorsal glandular skin extract of the brown tree frog, Litoria ewingi (also spelled ewingii) . It belongs to the Caeridin family of peptides that were originally studied in Australian tree frogs. Caeridins are small peptides (approximately 1000-1500 Da) comprising 12-15 amino acid residues with specific sequence characteristics, typically sharing the sequence -Gly-Leu-Leu/Phe- at the N-terminal ends and -Leu/Ile (NH₂) at the C-terminal ends .

These peptides can form α-helical structures with clearly delineated hydrophilic and hydrophobic zones, which may facilitate their binding to biological membranes . The discovery of Caeridin-7.1 came after the initial identification of Caeridins 1-6 from Litoria caerulea and other Australian frog species in 1993 by Waugh et al .

What are the structural characteristics of Caeridin-7.1?

Based on the information available, Caeridin-7.1 has similar structural properties to other members of the Caeridin family but with some distinctive characteristics:

As shown in the data, Caeridin-7.1 possesses the highest hydrophobic moment (0.615) among all characterized Caeridins . This property indicates a more pronounced amphipathic structure, with a stronger separation between hydrophobic and hydrophilic faces when the peptide adopts an α-helical conformation.

Like other Caeridins, it likely adopts an α-helical secondary structure in membrane-mimicking environments, as demonstrated by circular dichroism (CD) spectroscopy for related Caeridins which show positive bands at 190 nm and double-negative bands at 208 nm and 222 nm in solutions containing 50% TFE (trifluoroethanol) .

How does Caeridin-7.1 compare to other peptides isolated from Litoria species?

Caeridin-7.1 belongs to a distinct family of peptides from Litoria species that differs from other peptide families like Caerins and Caeruleins. The comparative analysis reveals:

• Size and structure: Caeridins (including Caeridin-7.1) are smaller (12-15 amino acids, 1000-1500 Da) compared to Caerins, which are typically larger antimicrobial peptides .

• Physicochemical properties: When comparing Caeridin-7.1 to other Caeridins (see Table 3 below), it has:

  • Mid-range hydrophobicity (0.757) compared to others (ranging from 0.725 to 0.878)

  • The highest hydrophobic moment (0.615) among all characterized Caeridins

  • Net charge of -1, similar to most other Caeridins except Caeridin-5 and Caeridin-6

Table 3: Comparative Physicochemical Properties of Caeridins

• Bioactivity profile: While some Litoria-derived peptides like Caeruleins show hypotensive bioactivity and Caerins display antimicrobial activity, the bioactivity profile of Caeridin-7.1 specifically hasn't been fully characterized in the available literature .

What are the potential bioactivities of recombinant Caeridin-7.1 based on studies of related Caeridins?

While specific bioactivity data for Caeridin-7.1 is limited, its potential bioactivities can be extrapolated from studies of related Caeridins and its unique physicochemical properties:

• Antimicrobial activity: Given its high hydrophobic moment (0.615), Caeridin-7.1 may exhibit antimicrobial properties similar to or potentially stronger than Caeridin-a1, which demonstrates potent activity against Gram-positive bacteria (S. aureus, MRSA, E. faecalis), Gram-negative bacteria (E. coli), and yeast (C. albicans) . The enhanced amphipathicity indicated by this high hydrophobic moment suggests potentially stronger membrane interactions.

• Smooth muscle modulatory effects: Related Caeridins show tissue-specific effects on smooth muscle function. For instance:

  • Caeridin-1 produces contraction of rat bladder smooth muscle at nanomolar concentrations

  • S5-Caeridin-1 induces relaxation of rat ileum smooth muscle, also at nanomolar concentrations

• Membrane interactions: The amphipathic nature of Caeridin-7.1, as indicated by its exceptionally high hydrophobic moment, suggests it may interact with biological membranes differently than other Caeridins. This interaction could result in:

  • Different mechanisms of membrane permeabilization

  • Potential cell-penetrating properties

  • Altered selectivity between microbial and mammalian cell membranes

Experimental validation through comparative bioactivity assays would be necessary to confirm these hypothesized activities for recombinant Caeridin-7.1.

How might the high hydrophobic moment of Caeridin-7.1 influence its membrane interaction mechanisms?

The exceptionally high hydrophobic moment (0.615) of Caeridin-7.1 compared to other Caeridins suggests significant implications for its membrane interaction mechanisms:

• Enhanced amphipathicity: The high hydrophobic moment indicates a more pronounced separation between hydrophobic and hydrophilic faces in the α-helical structure, which can intensify interactions with membrane interfaces where polar head groups meet hydrophobic tails .

• Potential mechanisms of action based on amphipathicity:

  • Initial membrane binding may be stronger due to more defined electrostatic interactions between the negatively charged ASP residue and positively charged membrane components

  • The highly amphipathic structure might favor mechanisms like toroidal pore formation or carpet-model membrane disruption

  • Potentially lower concentrations needed for membrane insertion compared to Caeridins with lower hydrophobic moments

• Selectivity implications: The balance between hydrophobicity (0.757) and amphipathicity (0.615) may influence Caeridin-7.1's selectivity between:

  • Bacterial membranes (rich in negatively charged phospholipids)

  • Mammalian membranes (more neutral, with cholesterol)

  • Fungal membranes (containing ergosterol)

• Experimental approaches to investigate these mechanisms would include:

  • Lipid vesicle leakage assays with varying lipid compositions

  • Membrane potential measurements

  • Atomic force microscopy to visualize membrane perturbations

  • Surface plasmon resonance to quantify binding kinetics

What methodological challenges might arise when analyzing structure-function relationships of recombinant Caeridin-7.1?

Investigating structure-function relationships of recombinant Caeridin-7.1 presents several methodological challenges that researchers should anticipate:

• Expression and folding challenges:

  • Small, amphipathic peptides often exhibit cytotoxicity to expression hosts

  • Achieving the correct secondary structure (α-helical) may require specific folding conditions

  • Potential aggregation during expression or purification due to hydrophobic interactions

• Structural analysis limitations:

  • Short peptides may not adopt stable structures in aqueous solutions, requiring membrane-mimicking environments for accurate structural determination

  • Different membrane-mimetic environments (micelles, bicelles, liposomes) may induce different conformations

  • Reconciling solution-phase structures with functional membrane-bound conformations

• Bioactivity assay considerations:

  • Standardization of antimicrobial testing conditions (media composition, growth phase, inoculum size) to enable reliable comparisons with other Caeridins

  • Appropriate control peptides (both positive and negative) must be included, as demonstrated in studies with other Caeridins using melittin and bradykinin

  • Salt sensitivity and serum stability may significantly impact activity measurements

• Mechanistic studies challenges:

  • Distinguishing between direct membrane permeabilization and receptor-mediated effects

  • Time-dependent changes in peptide-membrane interactions

  • Concentration-dependent mechanism switches (from membrane binding to disruption)

• Sequence-activity correlation challenges:

  • Limited natural sequence diversity within the Caeridin family may restrict natural SAR analysis

  • Need for synthetic variants with systematic modifications

  • Isolating the contribution of individual physicochemical parameters (charge, hydrophobicity, amphipathicity)

These challenges necessitate a multi-technique approach combining recombinant expression, chemical synthesis, structural biology, and functional assays to establish robust structure-function relationships.

What expression systems are optimal for recombinant production of Caeridin-7.1?

Selecting the optimal expression system for recombinant Caeridin-7.1 requires careful consideration of several factors to maximize yield, ensure proper folding, and maintain bioactivity:

• Bacterial expression systems:

  • E. coli remains the most cost-effective and well-established system for small peptides

  • Recommended strategies to overcome challenges:

    • Fusion partners: Thioredoxin, SUMO, or MBP to enhance solubility and prevent proteolytic degradation

    • Codon optimization for E. coli to improve translation efficiency

    • Lower temperature expression (16-20°C) to improve folding

    • Periplasmic targeting to facilitate disulfide bond formation if present

  • Limitations: Potential endotoxin contamination, limited post-translational modifications

• Yeast expression systems:

  • Pichia pastoris offers advantages for secreted peptide production

  • Benefits include:

    • Eukaryotic protein processing machinery

    • High-density fermentation possible

    • Secretion into media simplifies purification

    • Lower endotoxin concerns compared to E. coli

  • Consideration: Expression optimization may require screening multiple clones

• Cell-free protein synthesis:

  • Particularly valuable for potentially toxic peptides

  • Advantages:

    • Rapid production (hours instead of days)

    • Direct incorporation of non-natural amino acids if desired

    • Avoids host cell toxicity issues

  • Limitation: Higher cost and typically lower yields than cellular systems

• Purification strategy considerations:

  • Initial capture using affinity chromatography (His-tag, GST, etc.)

  • Tag removal with specific proteases (TEV, thrombin, Factor Xa)

  • Final purification via RP-HPLC, similar to methods used for natural Caeridins

  • Quality control by mass spectrometry to confirm identity and purity

• Analytical quality control:

  • Secondary structure verification by CD spectroscopy in membrane-mimicking environments (50% TFE), as performed for other Caeridins

  • Activity assays compared to chemically synthesized standards

The optimal approach would likely involve testing multiple expression systems in parallel, with particular attention to maintaining the amphipathic structural characteristics that are likely crucial for Caeridin-7.1's bioactivity.

What are the most effective methods for assessing the antimicrobial activity of recombinant Caeridin-7.1?

Based on methodologies used for related antimicrobial peptides, a comprehensive assessment of recombinant Caeridin-7.1's antimicrobial activity should include:

• Standardized susceptibility testing:

  • Minimum Inhibitory Concentration (MIC) determination using broth microdilution

  • Minimum Bactericidal/Fungicidal Concentration (MBC/MFC) assessment

  • Testing against reference strains similar to those used for Caeridin-a1:

    • Gram-positive: S. aureus (NCTC10788), MRSA (NCTC12493), E. faecalis (NCTC12697)

    • Gram-negative: E. coli (NCTC10418)

    • Fungi: C. albicans (NCYC1467)

  • Inclusion of appropriate control peptides (melittin as positive control, bradykinin as negative control)

• Time-kill kinetics:

  • Assessment of killing rate at different concentrations (0.5× MIC to 4× MIC)

  • Sampling at multiple timepoints (0, 1, 2, 4, 8, 24h)

  • Comparison with conventional antibiotics

• Membrane permeabilization studies:

  • SYTOX Green fluorescence assay to detect membrane compromise, as used for Caeridin-a1

  • Measurement of concentration-dependent and time-dependent effects

  • Comparison with membrane-active controls (melittin) and non-membrane-active controls (bradykinin)

Table 4: Example Antimicrobial Activity Data Format (based on Caeridin-a1 results)

• Mechanism of action studies:

  • Electron microscopy to visualize membrane effects

  • Leakage assays with artificial liposomes of varying composition

  • Gene expression analysis to identify stress responses in target organisms

• Resistance development assessment:

  • Serial passage in sub-inhibitory concentrations

  • Assessment of resistance development frequency

  • Cross-resistance with other antimicrobial peptides and conventional antibiotics

This comprehensive approach would provide detailed characterization of Caeridin-7.1's antimicrobial properties and potential applications.

What analytical techniques are most appropriate for confirming the structure and purity of recombinant Caeridin-7.1?

A multi-technique analytical approach is essential for comprehensive characterization of recombinant Caeridin-7.1's structure and purity:

• Mass spectrometry techniques:

  • ESI-MS for molecular weight confirmation (expected around 1000-1500 Da based on typical Caeridin size)

  • MS/MS fragmentation for sequence verification, following methodologies used for other Caeridins

  • MALDI-TOF for high-resolution mass determination

  • Top-down proteomics approaches for complete sequence coverage

• Chromatographic methods:

  • Reversed-phase HPLC for purity assessment, using similar conditions to those applied for natural Caeridin isolation

  • Size-exclusion chromatography to detect potential aggregation

  • Hydrophilic interaction chromatography (HILIC) as a complementary separation technique

  • Analytical ultracentrifugation for higher-order structure assessment

• Structural characterization:

  • Circular Dichroism (CD) spectroscopy in membrane-mimicking environments (50% TFE) to confirm α-helical structure, as performed for other Caeridins

  • Key CD spectral features to verify:

    • Positive band at 190 nm

    • Double-negative bands at 208 nm and 222 nm

  • NMR spectroscopy for high-resolution structural analysis

  • FTIR for additional secondary structure confirmation

• Electrophoretic techniques:

  • Tricine-SDS-PAGE optimized for small peptides

  • Isoelectric focusing to confirm charge characteristics

  • Capillary electrophoresis for high-resolution purity assessment

• Functional fingerprinting:

  • Bioactivity assays as structural confirmation

  • Membrane interaction studies using model membranes

  • Comparison with synthetic reference standard

The combined data from these complementary techniques provides a comprehensive characterization package that confirms identity, purity, correct folding, and functional activity of the recombinant Caeridin-7.1 preparation.

How should researchers interpret variations in antimicrobial activity when comparing recombinant versus synthetic Caeridin-7.1?

When comparing antimicrobial activity between recombinant and synthetic Caeridin-7.1 preparations, researchers should consider several factors that might explain observed variations:

• Structural considerations:

  • Secondary structure differences: Recombinant and synthetic peptides may adopt slightly different conformational distributions, affecting activity

  • Post-purification modifications: Oxidation of sensitive residues or chemical modifications during purification

  • C-terminal amidation: Ensure both preparations have identical C-terminal status (amidated vs. free acid), as C-terminal amidation is common in natural Caeridins and affects activity

• Purity factors:

  • Contaminant effects: Low-level contaminants from expression systems might synergize with or antagonize antimicrobial activity

  • Counter-ion differences: Variation in TFA or acetate content between preparations can affect activity measurements

  • Endotoxin contamination in recombinant preparations may interfere with certain assays

• Methodological analysis:

  • Perform parallel testing under identical conditions

  • Calculate potency ratios rather than comparing absolute MIC values

  • Use multiple bacterial strains to establish a pattern of differences

  • Test across a concentration range to generate complete dose-response curves

• Statistical approach:

  • Apply appropriate statistical tests (paired t-tests or ANOVA) to determine if differences are significant

  • Calculate 95% confidence intervals for MIC/MBC values

  • Perform multiple independent preparations to assess batch-to-batch variability

• Reconciliation strategies:

  • Detailed characterization of both preparations (MS, CD, HPLC)

  • Bioassay-guided fractionation if activity differences persist

  • Consider testing synthetic peptide with deliberate modifications to match recombinant product

Understanding the source of variations is crucial for determining whether differences reflect true structural/functional relationships or are artifacts of production methods.

What statistical approaches are most appropriate for analyzing dose-response data from Caeridin-7.1 biological assays?

Robust statistical analysis of dose-response data from Caeridin-7.1 biological assays requires appropriate methodologies tailored to the experimental design:

• Nonlinear regression modeling:

  • Fit dose-response data to sigmoidal curves (four-parameter logistic model)

  • Calculate EC50/IC50 values with 95% confidence intervals

  • Compare Hill slopes to understand cooperativity or mechanistic differences

  • Test for constraints in maximum or minimum responses

• Data transformation considerations:

  • Log-transform concentration data to normalize the distribution

  • Consider Box-Cox transformations for heteroscedastic data

  • Use arcsin transformation for proportional data (e.g., % inhibition)

• Comparison between conditions:

  • Extra sum-of-squares F-test to compare EC50 values between different conditions

  • Two-way ANOVA to assess interaction between Caeridin-7.1 and experimental variables

  • Mixed-effects models for repeated measures designs

  • Multiple comparison corrections (Bonferroni, Dunnett's, Tukey's) when appropriate

• Handling variability and experimental design:

  • Include sufficient replicates (minimum n=3, ideally n=6)

  • Use both biological and technical replicates

  • Calculate coefficient of variation to assess assay reproducibility

  • Apply weighted regression for heteroscedastic data

• Specialized applications for antimicrobial testing:

  • Calculate fractional inhibitory concentration indices for synergy studies

  • Time-kill curve modeling with area under the curve analysis

  • Survival analysis for time-dependent effects

  • Bootstrap resampling for non-parametric confidence intervals

• Reporting standards:

  • Report exact p-values rather than significance thresholds

  • Include effect sizes and confidence intervals

  • Present raw data alongside fitted curves

  • Verify model assumptions and report goodness-of-fit statistics

Applying these statistical approaches ensures robust, reproducible, and meaningful interpretation of Caeridin-7.1's biological activity data.

How can researchers establish structure-activity relationships between Caeridin-7.1 and other Caeridins?

Establishing comprehensive structure-activity relationships (SAR) between Caeridin-7.1 and other Caeridins requires a multifaceted approach combining bioinformatic, structural, and functional analyses:

• Sequence alignment and analysis:

  • Multiple sequence alignment of all known Caeridins

  • Identification of conserved and variable regions

  • Calculation of sequence similarity/identity matrices

  • Evolutionary analysis to understand relationships between Caeridin variants

• Physicochemical property correlation:

  • Analyze the relationship between measured activities and parameters from Table 3 :

    • Hydrophobicity (ranging from 0.725 to 0.878)

    • Hydrophobic moment (ranging from 0.369 to 0.615)

    • Net charge (-1 to 0)

    • Charged residue distribution

  • Generate quantitative structure-activity relationship (QSAR) models using these parameters

• Structural comparison techniques:

  • Circular dichroism (CD) spectroscopy in membrane-mimicking environments

  • Nuclear magnetic resonance (NMR) spectroscopy if feasible

  • In silico molecular modeling and dynamics simulations

  • Helical wheel projections to visualize amphipathicity differences

• Systematic functional comparison:

  • Standardized antimicrobial testing against identical microbial panels

  • Parallel smooth muscle assays under identical conditions

  • Membrane permeabilization assays using fluorescent dyes (SYTOX Green)

  • Host cell toxicity assessments

• Synthetic variant studies:

  • Design of chimeric peptides combining regions from different Caeridins

  • Alanine scanning to identify critical residues

  • Point mutations at positions that differ between Caeridins with different activities

  • N- and C-terminal truncation studies

• Data integration and visualization:

  • Heat maps correlating sequence features with activity measurements

  • Principal component analysis to identify key determinants of activity

  • Hierarchical clustering of Caeridins based on multiple parameters

  • Network analysis connecting structural features to functional outcomes

By systematically applying these approaches, researchers can establish a detailed understanding of how specific structural features of Caeridin-7.1 contribute to its unique properties compared to other members of this peptide family.

What are the most promising therapeutic applications for recombinant Caeridin-7.1 based on its properties?

Based on the physicochemical properties of Caeridin-7.1 and activities observed in related Caeridins, several therapeutic applications warrant investigation:

• Antimicrobial applications:

  • The high hydrophobic moment (0.615) of Caeridin-7.1 suggests potential antimicrobial activity, particularly against Gram-positive bacteria like S. aureus and MRSA, similar to Caeridin-a1

  • Potential applications include:

    • Topical antimicrobial formulations for wound infections

    • Antimicrobial coatings for medical devices

    • Combination therapy with conventional antibiotics to combat resistance

    • Narrow-spectrum antimicrobial targeting specific pathogens

• Smooth muscle modulatory applications:

  • If Caeridin-7.1 demonstrates smooth muscle effects similar to other Caeridins:

    • Potential treatments for bladder dysfunction (like Caeridin-1)

    • Gastrointestinal motility disorders (like S5-Caeridin-1)

    • Respiratory conditions involving bronchial smooth muscle

    • Vascular applications related to smooth muscle tone

• Peptide-based drug delivery:

  • The amphipathic nature suggested by its high hydrophobic moment could enable:

    • Cell-penetrating peptide applications for intracellular drug delivery

    • Enhancement of transdermal drug delivery

    • Development of peptide-drug conjugates with improved pharmacokinetics

• Diagnostic applications:

  • Development of peptide-based biosensors for detecting specific pathogens

  • Fluorescently labeled derivatives for membrane research

Each application would require specific optimization strategies, beginning with confirmation of activity, followed by structure-activity relationships to enhance desired properties while minimizing potential toxicity.

What novel experimental techniques might advance our understanding of Caeridin-7.1's mechanism of action?

Emerging experimental techniques could significantly advance our understanding of Caeridin-7.1's mechanism of action:

• Advanced imaging approaches:

  • Super-resolution microscopy to visualize peptide-membrane interactions at nanoscale resolution

  • Time-resolved fluorescence microscopy to track real-time membrane interactions

  • Atomic force microscopy to observe membrane topographical changes

  • Cryo-electron microscopy to visualize peptide-induced membrane structures

• Single-molecule techniques:

  • Patch-clamp fluorometry to simultaneously measure membrane conductance and peptide binding

  • Single-molecule FRET to analyze conformational changes upon membrane binding

  • Optical tweezers to measure forces involved in membrane penetration

  • Nanopore sensing to study peptide translocation across membranes

• Label-free biosensing:

  • Quartz crystal microbalance with dissipation monitoring (QCM-D) to quantify peptide-membrane binding kinetics

  • Surface plasmon resonance (SPR) for real-time binding analysis

  • Bio-layer interferometry to measure association/dissociation kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic binding parameters

• Advanced spectroscopic methods:

  • Solid-state NMR to determine peptide orientation in membranes

  • Neutron reflectometry to measure peptide insertion depth

  • Time-resolved CD spectroscopy to capture conformational transitions

  • Raman spectroscopy for label-free structural analysis

• Computational and systems biology approaches:

  • Molecular dynamics simulations at extended timescales

  • Machine learning to predict activity from sequence/structural features

  • Multi-scale modeling combining quantum mechanics and molecular mechanics

  • Transcriptomic/proteomic analysis of cellular responses to the peptide

• Microfluidic and high-throughput platforms:

  • Droplet-based microfluidics for high-throughput screening

  • Organ-on-chip technology to assess tissue-specific effects

  • Artificial membrane systems with controlled composition

  • Real-time antimicrobial resistance development monitoring

These advanced techniques would provide unprecedented insights into the molecular mechanisms underlying Caeridin-7.1's interactions with biological systems.