Recombinant Phyllomedusa sauvagei Dermaseptin-2

What is the structural composition of Dermaseptin-2 from Phyllomedusa sauvagei?

Dermaseptin-2 from Phyllomedusa sauvagei is a linear polycationic peptide with an amphipathic α-helical structure when in contact with hydrophobic environments that mimic biological membranes. The peptide belongs to the dermaseptin family, which generally consists of 28-34 amino acid residues . Like other dermaseptins, it contains a conserved tryptophan residue at position 3 and a positive net charge due to the presence of multiple lysine residues. When studied using circular dichroism (CD) spectroscopy, dermaseptins typically display random coil structures in aqueous solutions but transition to α-helical conformations in membrane-mimicking environments such as 50% trifluoroethanol (TFE) . This conformational flexibility is critical for their antimicrobial function, allowing them to interact with and disrupt microbial membranes while remaining relatively inactive in aqueous environments.

How does Dermaseptin-2 compare with other antimicrobial peptides from Phyllomedusa frogs?

Dermaseptin-2 shares common structural features with other members of the dermaseptin family but possesses unique characteristics that distinguish it from related peptides. All dermaseptins are derived from prepropeptides with remarkably similar signal peptide and acidic "spacer" regions, but they differ significantly in their mature peptide sequences . Compared to dermatoxin (SLGSFLKGVGTTLASVGKVVSDQFGKLLQAGQ) isolated from Phyllomedusa bicolor, which contains 32 residues , Dermaseptin-2 maintains the characteristic amphipathic structure but with a sequence optimized for its specific antimicrobial profile. When analyzing antimicrobial potency, various dermaseptins display different specificities; for example, dermaseptins from Phyllomedusa oreades and Phyllomedusa distincta (known as dermadistinctins K and L) exhibit potent activity against Trypanosoma cruzi without significant cytotoxicity to mammalian cells . The sequence variations among dermaseptins contribute to their diverse antimicrobial profiles while maintaining the fundamental membrane-disrupting mechanism.

What is the genetic organization of the Dermaseptin-2 precursor?

The Dermaseptin-2 precursor follows the canonical organization observed in other dermaseptin gene products. The precursor contains three distinct regions: (1) a highly conserved signal peptide directing secretion, (2) an acidic amino acid-rich "spacer" region, and (3) the mature peptide sequence following a typical Lys-Arg (-KR-) propeptide convertase processing site . This genetic organization is remarkably conserved across different Phyllomedusa species. For example, in Phyllomedusa coelestis, the novel dermaseptin DM-PC exhibits this same topological structure . The conservation of this genetic architecture suggests an evolutionary importance in the processing and production of these antimicrobial peptides. To study the genetic organization experimentally, researchers typically employ molecular cloning techniques using skin secretion-derived cDNA libraries, as demonstrated in the identification of DM-PC from Phyllomedusa coelestis . PCR analysis coupled with cDNA cloning and sequencing has revealed that dermaseptins (including dermatoxin) can be expressed not only in the skin but also in the intestine and brain of these frogs .

What is the primary mechanism of action for Dermaseptin-2?

Dermaseptin-2, like other members of the dermaseptin family, primarily acts by disrupting the integrity of microbial membranes. The mechanism involves several sequential steps: (1) initial electrostatic attraction between the positively charged peptide and the negatively charged microbial membrane, (2) conformational transition from random coil to α-helix upon membrane contact, (3) insertion of the amphipathic helix into the membrane, and (4) formation of ion-conducting channels or more extensive membrane disruption . Evidence for this mechanism comes from measurements of bacterial membrane potential, which reveal that the plasma membrane is the primary target of dermaseptins . Fluorescence microscopy studies of cells treated with dermaseptins show patterns consistent with membrane permeabilization rather than complete membrane solubilization . In the case of malarial parasites, dermaseptin derivatives have been shown to selectively lyse infected red blood cells while having minimal effects on healthy cells, with the ability to penetrate through multiple membrane barriers to reach the intracellular parasite . The antimicrobial potency of dermaseptins is influenced by their ability to form amphipathic helices, as demonstrated by circular dichroism spectroscopy studies in membrane-mimicking environments .

What are the optimal approaches for recombinant expression of Dermaseptin-2?

Recombinant expression of Dermaseptin-2 presents several challenges due to its cationic, amphipathic nature and potential toxicity to host cells. A multi-faceted approach is recommended for successful expression:

• Expression System Selection: E. coli-based systems using pET vectors with fusion partners such as thioredoxin or SUMO are preferred to neutralize peptide toxicity and enhance solubility. BL21(DE3) or C43(DE3) strains (designed for toxic protein expression) typically yield better results.

• Fusion Construct Design: The inclusion of an N-terminal fusion partner (thioredoxin/SUMO) followed by a precision protease cleavage site (TEV or Factor Xa) allows for efficient purification and subsequent release of the native peptide. Codon optimization for E. coli is critical for high-level expression.

• Culture Conditions: Expression at lower temperatures (16-20°C) following IPTG induction at OD600 ~0.6-0.8 can significantly improve soluble protein yield by reducing inclusion body formation. Supplementation with 0.5-1% glucose helps suppress basal expression and reduce toxicity.

• Purification Strategy: A two-step chromatography approach is recommended - initial IMAC purification via His-tag, followed by cleavage of the fusion partner and a second reverse-phase HPLC step to obtain pure peptide. Mass spectrometry verification is essential to confirm structural integrity .

For researchers specifically interested in studying structure-function relationships, site-directed mutagenesis can be incorporated into the recombinant expression workflow to generate Dermaseptin-2 variants with altered properties.

How can truncated derivatives of Dermaseptin-2 be designed to improve therapeutic index?

Designing truncated derivatives of Dermaseptin-2 with improved therapeutic indices involves systematic structure-activity relationship studies focusing on key structural determinants:

• N-terminal Truncation Strategy: Evidence from dermaseptin studies indicates that N-terminal truncated derivatives maintain significant antimicrobial activity while exhibiting reduced cytotoxicity . For Dermaseptin-2, initial truncation should preserve the critical amphipathic region (typically the first 19-20 residues) as demonstrated in the DMPC-19 derivative from Phyllomedusa coelestis, which retained potent antimicrobial activity .

• Charge Distribution Optimization: Increasing the net positive charge through strategic lysine substitutions at positions 4 and 20 (as in K4K20-S4) can enhance antimicrobial potency. This approach was shown to reduce the IC50 to as low as 0.2 μM against Plasmodium falciparum while maintaining selectivity .

• Hydrophobicity Balancing: Modifying the hydrophobic face of the amphipathic helix through substitutions like Ile→Leu or incorporating unnatural amino acids such as β-cyclohexyl-L-alanine (Cha) can fine-tune membrane interactions . This approach can enhance antimicrobial activity while minimizing hemolytic effects.

• Evaluation Protocol:

  • Antimicrobial activity assessment against Gram-positive and Gram-negative bacteria (MIC determination)

  • Hemolysis assays using mammalian erythrocytes (HC50 determination)

  • Calculation of therapeutic index (HC50/MIC ratio)

  • Membrane interaction studies using model liposomes

This systematic approach has been successful with other dermaseptins, where truncated derivatives maintained potent antimicrobial activity (MICs ~4μM) while exhibiting significantly reduced hemolytic activity (HC50 >100μM) .

What experimental methods are most effective for analyzing Dermaseptin-2's membrane disruption mechanism?

Multiple complementary techniques are required to comprehensively elucidate Dermaseptin-2's membrane disruption mechanism:

• Membrane Potential Measurements: Using fluorescent dyes like DiSC3(5) or membrane-impermeable potential-sensitive dyes to monitor changes in bacterial membrane potential following peptide treatment. This approach revealed that dermaseptins primarily target the plasma membrane of bacteria .

• Fluorescence Microscopy with DNA Staining: Applying DNA-staining fluorophores to observe bacterial cell morphology after peptide treatment. This method distinguishes between membrane permeabilization and complete solubilization mechanisms. Studies with dermatoxin showed patterns consistent with channel formation rather than detergent-like membrane disruption .

• Artificial Membrane Systems:

  • Lipid monolayer penetration assays to measure surface activity

  • Liposome leakage assays using calcein-entrapped vesicles to quantify membrane permeabilization

  • Planar lipid bilayer conductance measurements to characterize ion channel formation

• Structural Analysis in Membrane-Mimicking Environments:

  • Circular dichroism (CD) spectroscopy in increasing concentrations of TFE (0-50%) to track conformational transitions

  • Solid-state NMR to determine peptide orientation and dynamics within membranes

  • Molecular dynamics simulations to model peptide-membrane interactions at the atomic level

• Confocal Microscopy with Fluorescently Labeled Peptides: This approach demonstrated that dermaseptin derivatives could penetrate through multiple membrane barriers to directly interact with intracellular parasites in malaria-infected red blood cells .

These complementary approaches provide mechanistic insights while differentiating between various models of membrane disruption (carpet model, barrel-stave pores, toroidal pores, or detergent-like solubilization).

How do D-isomers of Dermaseptin-2 compare to L-isomers in antimicrobial activity and mechanism?

The comparison between D- and L-isomers of Dermaseptin-2 provides valuable insights into its mechanism of action and potential for therapeutic development:

• Antimicrobial Activity Comparison: D-isomers of dermaseptin derivatives (such as K4K20-S4 or K4-S4(1-13)a) demonstrate antimicrobial activity equivalent to their L-counterparts against various pathogens, including Plasmodium falciparum . This functional equivalence strongly suggests that antimicrobial activity is not mediated through stereospecific interactions with chiral cellular targets like protein receptors.

• Membrane Disruption Mechanism: The equal potency of D-isomers confirms that dermaseptins primarily act through physical disruption of microbial membranes rather than through specific receptor binding. Experimental evidence from transmembrane potential dissipation studies with both isomers supports this mechanism .

• Resistance to Proteolytic Degradation: D-isomers offer significant advantages in terms of stability, as they resist degradation by host and microbial proteases. This characteristic can be quantified by comparing peptide half-lives in serum or in the presence of specific proteases.

• Experimental Design for Isomer Comparison:

  • Parallel antimicrobial susceptibility testing (MIC determination)

  • Comparative hemolysis assays to assess selectivity

  • Time-course experiments measuring killing kinetics

  • Fluorescence spectroscopy to assess membrane binding and permeabilization

  • Protease resistance assays to quantify stability enhancement

The equivalent activity of D- and L-isomers not only elucidates the non-stereospecific nature of dermaseptin action but also suggests that D-isomers could serve as more stable therapeutic candidates with extended half-lives in biological environments, particularly important for systemic applications .

What analytical techniques are most appropriate for studying structure-activity relationships of Dermaseptin-2 derivatives?

A comprehensive analytical workflow for structure-activity relationship studies of Dermaseptin-2 derivatives requires multiple complementary techniques:

• Structural Characterization:

  • Mass Spectrometry: MALDI-TOF MS for molecular weight confirmation and purity assessment

  • Circular Dichroism (CD) Spectroscopy: For secondary structure analysis in different environments (buffer vs. membrane-mimicking conditions)

  • NMR Spectroscopy: For high-resolution 3D structure determination in membrane-mimicking environments

• Physicochemical Property Analysis:

  • Reversed-Phase HPLC: For retention time analysis correlating with hydrophobicity

  • Zeta Potential Measurements: For surface charge characterization

  • Helical Wheel Projections: For visualization of amphipathicity and charge distribution

• Functional Assays:

  • Antimicrobial Activity: Standard MIC determinations against diverse pathogens

  • Hemolysis Assays: HC50 determination against mammalian erythrocytes

  • Time-Kill Kinetics: To differentiate between bacteriostatic and bactericidal effects

  • Membrane Permeabilization: Using fluorescent dyes like SYTOX Green or propidium iodide

• Data Correlation Methods:

  • Multiple Linear Regression Analysis: To correlate structural parameters with activity

  • Principal Component Analysis: To identify key determinants of activity

  • Quantitative Structure-Activity Relationship (QSAR) Modeling: For prediction of activity based on structure

• Application-Specific Analyses:

  • For antimalarial applications: Stage-specific activity assessment against ring and trophozoite stages

  • For anti-trypanosomal applications: Activity against different developmental forms (trypomastigotes, epimastigotes)

This multi-technique approach enables researchers to systematically identify structure-activity relationships and rationally design improved derivatives with enhanced therapeutic indices.

What are the optimal conditions for solid-phase synthesis of Dermaseptin-2 and its derivatives?

The solid-phase peptide synthesis (SPPS) of Dermaseptin-2 and its derivatives requires careful optimization at multiple steps to ensure high purity and yield:

• Resin Selection and Loading:

  • For C-terminal amidated peptides: Rink Amide MBHA resin with 0.3-0.5 mmol/g substitution level is optimal

  • For C-terminal free acids: 2-chlorotrityl chloride resin offers gentle cleavage conditions

  • Lower initial loading (0.1-0.2 mmol/g) improves synthesis of difficult sequences

• Protection Strategy and Coupling Reagents:

  • Fmoc/tBu protection chemistry is preferred over Boc/Bzl due to milder conditions

  • HBTU/HOBt with DIEA in DMF provides efficient coupling for most residues

  • For difficult couplings (particularly subsequent to Lys, Arg, or β-branched amino acids), COMU or HATU coupling reagents demonstrate superior performance

• Microwave-Assisted Synthesis Protocol:

  • Deprotection: 20% piperidine in DMF, 1 min at 40°C followed by 5 min at 75°C

  • Coupling: 5 equivalents amino acid, 4.9 eq HBTU, 5 eq HOBt, 10 eq DIEA, 5 min at 75°C

  • Double coupling for difficult residues and residues beyond position 15

• Cleavage and Post-Synthetic Processing:

  • Cleavage mixture: 95% TFA, 2.5% TIS, 2.5% water for 3 hours at room temperature

  • Cold ether precipitation followed by RP-HPLC purification using a C18 column

  • Gradient elution with acetonitrile/water containing 0.1% TFA

• Quality Control:

  • MALDI-TOF MS for molecular weight confirmation

  • Analytical RP-HPLC for purity assessment (>95% purity standard)

  • Amino acid analysis for quantification

This protocol has been successfully applied to synthesize dermaseptin derivatives with purities exceeding 95%, suitable for both structural and functional studies .

How can researchers effectively evaluate the potential synergistic effects between Dermaseptin-2 and conventional antibiotics?

Evaluating synergistic interactions between Dermaseptin-2 and conventional antibiotics requires systematic approaches:

• Checkerboard Assay Protocol:

  • Create a matrix of 2-fold serial dilutions of both Dermaseptin-2 (0.25× to 4× MIC) and antibiotic (0.25× to 4× MIC)

  • Calculate Fractional Inhibitory Concentration Index (FICI) using the formula:
    FICI = (MIC of Dermaseptin-2 in combination/MIC of Dermaseptin-2 alone) + (MIC of antibiotic in combination/MIC of antibiotic alone)

  • Interpretation: FICI ≤ 0.5 indicates synergy; 0.5 < FICI ≤ 1.0 indicates additivity; 1.0 < FICI < 4.0 indicates indifference; FICI ≥ 4.0 indicates antagonism

• Time-Kill Kinetics Analysis:

  • Monitor bacterial growth over 24 hours in the presence of:

    • Dermaseptin-2 alone at 0.5× MIC

    • Antibiotic alone at 0.5× MIC

    • Combination at these concentrations

  • Synergy definition: ≥ 2 log10 decrease in CFU/mL with the combination compared to the most active agent alone

• Mechanistic Investigation:

  • Membrane permeabilization assays using fluorescent dyes (propidium iodide, SYTOX Green)

  • Intracellular antibiotic accumulation measurements using fluorescent antibiotics or LC-MS/MS quantification

  • Gene expression analysis of resistance mechanisms using RT-qPCR

• Advanced Models for Combination Testing:

  • Biofilm susceptibility testing using Calgary Biofilm Device or flow cell systems

  • Ex vivo infection models using primary human cells

  • In vivo efficacy in appropriate animal infection models

• Data Analysis:

  • Isobologram construction for visual representation of interactions

  • Response surface modeling for complex combinations

  • Statistical analysis using two-way ANOVA with Bonferroni post-tests

This systematic approach enables quantitative assessment of synergistic potential, which is particularly valuable for dermaseptins given their membrane-disruptive properties that may enhance antibiotic penetration into bacterial cells.

What techniques can be used to investigate the immunomodulatory potential of Dermaseptin-2?

Investigating the immunomodulatory properties of Dermaseptin-2 requires a comprehensive approach spanning from molecular to in vivo analyses:

• In Vitro Immune Cell Assays:

  • Cytokine profiling: Measure pro- and anti-inflammatory cytokine production (IL-1β, TNF-α, IL-6, IL-10) from peptide-treated macrophages or dendritic cells using ELISA or multiplex assays

  • Chemotaxis assays: Evaluate neutrophil and monocyte migration using Boyden chamber or transwell systems

  • Phagocytosis assays: Quantify the effect on macrophage phagocytic activity using fluorescent particles or labeled bacteria

• Signaling Pathway Analysis:

  • NF-κB activation assessment using reporter cell lines or phospho-specific Western blotting

  • MAPK pathway analysis (phosphorylation of p38, ERK, JNK)

  • Inflammasome activation markers (caspase-1 activation, IL-1β processing)

• Transcriptomic and Proteomic Approaches:

  • RNA-seq analysis of immune cells treated with Dermaseptin-2 to identify global gene expression changes

  • Proteomics analysis to identify post-translational modifications and protein expression changes

  • Pathway enrichment analysis to identify immunomodulatory networks

• Ex Vivo Tissue Models:

  • Precision-cut lung slices or skin explants to evaluate tissue-specific immune responses

  • Whole blood stimulation assays to assess systemic immune effects

• In Vivo Models:

  • Neutrophil recruitment in sterile inflammation models

  • Infection models with immunological readouts (cytokine profiling, immune cell infiltration)

  • Delayed-type hypersensitivity reactions to assess T-cell mediated immunity

This multi-level approach provides comprehensive insights into both innate and adaptive immune modulation by Dermaseptin-2, which complements its direct antimicrobial activities and may contribute to its therapeutic potential.

How should researchers address contradictory findings in Dermaseptin-2 structure-activity relationship studies?

Reconciling contradictory findings in structure-activity relationship studies of Dermaseptin-2 requires a systematic analytical approach:

• Methodological Standardization and Comparison:

  • Create a standardized table comparing experimental conditions across studies: peptide concentration ranges, buffer compositions, incubation times, temperature, and target organism strains

  • Reconstruct dose-response curves using raw data when available to identify threshold effects or biphasic responses

  • Replicate key experiments using standardized protocols to directly compare results

• Context-Dependent Activity Analysis:

  • Systematically investigate environmental factors influencing activity:

    • Ionic strength effects (physiological vs. low salt conditions)

    • pH-dependent activity profiles (pH 5.5-7.4)

    • Presence of serum proteins or divalent cations

  • These factors can significantly alter peptide conformation and membrane interactions

• Target Membrane Composition Consideration:

  • Compare lipid compositions used in different studies (PE/PG ratio for bacteria, cholesterol content for mammalian cells)

  • Perform parallel testing against identical bacterial strains from multiple sources (lab strains vs. clinical isolates)

  • Membrane fluidity and surface charge density can dramatically influence antimicrobial peptide activity

• Statistical Approaches for Data Integration:

  • Meta-analysis techniques to quantitatively combine results from multiple studies

  • Bayesian hierarchical modeling to account for inter-study variability

  • Sensitivity analysis to identify key variables driving discrepancies

• Computational Structure-Function Reconciliation:

  • Molecular dynamics simulations under different conditions to explain context-dependent effects

  • Quantitative structure-activity relationship (QSAR) models incorporating conditional variables

This systematic approach helps identify whether contradictions arise from methodological differences, context-dependent mechanisms, or fundamental differences in peptide-membrane interactions across experimental systems.

What statistical approaches are most appropriate for analyzing the selectivity index of Dermaseptin-2 derivatives?

Robust statistical analysis of selectivity indices for Dermaseptin-2 derivatives requires specialized approaches:

• Selectivity Index Calculation and Uncertainty Estimation:

  • Primary selectivity index (SI) = HC50/MIC (hemolytic concentration / minimum inhibitory concentration)

  • Calculate 95% confidence intervals using propagation of error methods

  • For each derivative, present SI as: SI = median (lower CI - upper CI)

• Comparative Statistical Analysis:

  • Non-parametric tests for comparing SIs across multiple derivatives:

    • Kruskal-Wallis test followed by Dunn's multiple comparison

    • Mann-Whitney U test for paired comparisons

  • Calculate effect sizes (Cohen's d) to quantify meaningful differences

• Multivariate Analysis of Structure-Selectivity Relationships:

  • Principal Component Analysis (PCA) to identify key physicochemical determinants of selectivity

  • Cluster analysis to group derivatives with similar selectivity profiles

  • Partial Least Squares (PLS) regression to model relationships between structural features and selectivity

• Data Visualization Techniques:

  • Log-scale scatter plots of HC50 vs. MIC with isolines representing equal selectivity indices

  • Heatmaps clustering derivatives by both antimicrobial potency and hemolytic activity

  • Radar plots displaying multiple selectivity indices against different pathogens

• Probability-Based Decision Models:

  • Bayesian approaches for incorporating prior knowledge about structurally similar peptides

  • Monte Carlo simulations to model propagation of experimental uncertainties

  • Calculation of probability that SI exceeds clinically relevant thresholds

These statistical approaches provide a rigorous framework for comparing selectivity across derivatives and identifying structural determinants that enhance therapeutic potential while minimizing potential toxicity.

What emerging technologies could enhance the therapeutic potential of recombinant Dermaseptin-2?

Several cutting-edge technologies show promise for enhancing the therapeutic potential of recombinant Dermaseptin-2:

• Peptide Delivery Systems:

  • Lipid nanoparticle encapsulation to protect from proteolytic degradation

  • Cell-penetrating peptide conjugation for enhanced cellular uptake

  • Stimuli-responsive polymeric carriers for targeted release at infection sites

  • These approaches could overcome stability limitations while reducing potential hemolytic effects

• Genetic Code Expansion for Unnatural Amino Acid Incorporation:

  • Site-specific incorporation of unnatural amino acids with enhanced stability properties

  • Incorporation of click-chemistry compatible residues for post-expression modification

  • Fluorinated amino acids to enhance helicity and membrane interactions

  • This technology enables precise tuning of physicochemical properties beyond natural amino acid constraints

• Computational Design and Artificial Intelligence:

  • Machine learning algorithms trained on antimicrobial peptide databases to predict optimal sequence modifications

  • Molecular dynamics simulations to refine membrane interactions

  • De novo design of hybrid peptides combining Dermaseptin-2 motifs with complementary antimicrobial peptides

  • These computational approaches can drastically accelerate the optimization process

• Antimicrobial Peptide Immobilization Technologies:

  • Covalent attachment to medical device surfaces via flexible linkers

  • Layer-by-layer deposition with controlled release properties

  • Biofilm-responsive tethering systems for on-demand release

  • Surface immobilization creates long-lasting antimicrobial surfaces while minimizing systemic exposure

• CRISPR-Cas-Based Delivery Systems:

  • Engineered probiotic bacteria expressing recombinant Dermaseptin-2

  • CRISPR-Cas delivery of Dermaseptin-2 genes to pathogen-adjacent cells

  • Phage-based delivery systems targeting specific bacterial pathogens

These emerging technologies address the key limitations of antimicrobial peptides (stability, delivery, selectivity) while leveraging the fundamental membrane-disruptive mechanism that makes Dermaseptin-2 less prone to conventional resistance mechanisms.

How might genome mining approaches be used to discover novel Dermaseptin-2 variants with enhanced properties?

Genome mining represents a powerful approach for discovering novel Dermaseptin-2 variants with potentially enhanced therapeutic properties:

• Comparative Genomics Across Phyllomedusa Species:

  • Whole-genome sequencing of diverse Phyllomedusa species, with particular focus on those inhabiting extreme environments

  • Identification of dermaseptin gene clusters and regulatory elements

  • Phylogenetic analysis to trace evolutionary relationships and diversification patterns

  • This approach leverages natural evolution as a source of functional diversity

• Advanced Bioinformatic Search Strategies:

  • Profile Hidden Markov Models constructed from known dermaseptins to identify distant homologs

  • Synteny analysis to identify conserved genomic contexts around dermaseptin genes

  • Codon usage and selective pressure analysis to identify functionally important residues

• Transcriptome Analysis Under Diverse Conditions:

  • RNA-seq analysis of Phyllomedusa skin under various challenges (bacterial exposure, pH stress, temperature variation)

  • Identification of differentially expressed dermaseptin variants

  • Alternative splicing analysis to detect novel isoforms

  • This approach identifies context-dependent expression patterns that may indicate specialized functions

• Functional Screening Platforms:

  • High-throughput expression of candidate sequences in bacterial or yeast display systems

  • Automated screening against diverse pathogen panels

  • Selection under varying conditions (pH, salt, temperature) to identify robust variants

  • Droplet microfluidics for single-cell analysis of antimicrobial activity

• Database Integration and Analysis:

  • Creation of comprehensive dermaseptin database with structure-function annotations

  • Machine learning models to predict activity based on sequence features

  • Network analysis to identify co-evolving residues important for function