Recombinant Litoria rubella Rubellidin-3.2

What is Rubellidin-3.2 and what is its origin?

Rubellidin-3.2 is a pentapeptide isolated from the skin secretions of the Australian desert tree frog (Litoria rubella) . It belongs to a diverse class of amphibian-derived bioactive peptides that have garnered significant scientific interest due to their potential biological activities. The peptide's name derives from the species name "rubella," reflecting its biological source. Understanding the evolutionary context of this peptide provides insight into its potential biological functions and mechanisms of action.

What is the primary structure and key physicochemical properties of Rubellidin-3.2?

Rubellidin-3.2 is characterized by the amino acid sequence VEFFT (Valine-Glutamic Acid-Phenylalanine-Phenylalanine-Threonine) . This relatively short peptide contains both hydrophobic residues (Val, Phe) and hydrophilic/charged residues (Glu, Thr), creating an interesting amphipathic profile. The presence of two consecutive phenylalanine residues is particularly notable, as this structural feature may contribute to specific biological activities through aromatic interactions or receptor binding capabilities.

How might the sequence of Rubellidin-3.2 relate to its potential functions?

The specific amino acid composition of Rubellidin-3.2 suggests several functional possibilities that warrant investigation. The N-terminal valine provides hydrophobicity that may facilitate membrane interactions, while the adjacent glutamic acid introduces negative charge and potential for ionic interactions. The central double-phenylalanine motif is particularly interesting as aromatic residues often participate in π-π stacking interactions and can contribute to antioxidant properties through their ability to donate electrons to free radicals . The C-terminal threonine offers hydrogen bonding potential and modest polarity. This unique sequence combination creates a peptide with balanced hydrophobic and hydrophilic regions that may enable specific molecular recognition or biological activities.

What expression systems are most suitable for recombinant Rubellidin-3.2 production?

For recombinant production of a short peptide like Rubellidin-3.2, researchers must consider several expression systems, each with distinct advantages and limitations. E. coli remains the most cost-effective and scalable system for initial research, though it requires careful design of expression constructs. The ideal approach involves fusion protein strategies to overcome the challenges associated with expressing small peptides. A methodological analysis should consider:

• Bacterial expression (E. coli):

  • Advantages: High yield, rapid growth, cost-effective

  • Challenges: Proteolytic degradation of short peptides, inefficient expression

  • Solution: Fusion with carrier proteins (MBP, GST, SUMO, thioredoxin)

• Yeast expression (P. pastoris, S. cerevisiae):

  • Advantages: Secretion capability, eukaryotic processing

  • Challenges: Longer production time, lower yield than bacteria

  • Best for: Cases where E. coli expression proves problematic

• Insect or mammalian cell systems:

  • Generally unnecessary for simple peptides like Rubellidin-3.2

  • Consider only if post-translational modifications are required

When developing a recombinant expression system, researchers should prioritize construct design with appropriate fusion partners and cleavage sites for subsequent purification of the native peptide sequence.

What are the critical factors for successful cloning and expression of Rubellidin-3.2?

Successful recombinant production of Rubellidin-3.2 requires careful optimization of the expression construct and conditions. The experimental design should address several key considerations:

• Gene design and optimization:

  • Codon optimization for the host organism

  • Incorporation of efficient ribosome binding sites

  • Addition of appropriate restriction sites for cloning flexibility

  • Inclusion of a precision protease cleavage site (TEV, Factor Xa, etc.)

• Expression vector selection:

  • Strong, inducible promoters (T7, tac, AOX1)

  • Selection markers compatible with the expression host

  • Fusion partner with high solubility (SUMO and thioredoxin often yield best results for small peptides)

• Expression condition optimization:

  • Temperature (often lower temperatures improve yield of fusion proteins)

  • Inducer concentration (IPTG, galactose, methanol depending on system)

  • Cell density at induction time

  • Duration of expression phase

• Harvest and initial processing:

  • Cell lysis methods that preserve peptide integrity

  • Initial clarification steps (centrifugation, filtration)

  • Immediate addition of protease inhibitors if needed

The most critical aspect is designing an expression construct that prevents proteolytic degradation while allowing efficient release of the target peptide after purification of the fusion protein.

How does recombinant synthesis compare with chemical synthesis for Rubellidin-3.2?

For a pentapeptide like Rubellidin-3.2, both recombinant and chemical synthesis approaches present viable options, each with distinct advantages that should be evaluated based on specific research requirements:

What analytical techniques are essential for confirming the identity and purity of Rubellidin-3.2?

Comprehensive characterization of Rubellidin-3.2 requires a multi-technique approach to establish identity, purity, and structural integrity. A methodical analytical workflow should include:

• Chromatographic analysis:

  • Reversed-phase HPLC using C18 columns with a gradient of 0.1% TFA/acetonitrile

  • Monitoring at dual wavelengths (216 nm for peptide bonds, 280 nm for aromatic residues)

  • Calculation of relative purity by peak area integration

  • Comparison with reference standards when available

• Mass spectrometric confirmation:

  • MALDI-TOF/TOF or ESI-MS for accurate molecular weight determination

  • MS/MS fragmentation for sequence verification

  • Expected molecular weight approximately 642 Da (depending on C-terminal modification)

• Amino acid analysis:

  • Edman degradation for unambiguous sequence confirmation

  • Amino acid composition analysis after acid hydrolysis

• Spectroscopic methods:

  • UV-Vis spectroscopy (characteristic absorption from aromatic residues)

  • Circular dichroism for any preferred conformational tendencies

Researchers should establish acceptance criteria for each analytical parameter, typically requiring chromatographic purity >95% and mass accuracy within 0.5 Da of theoretical values for confirmation of identity.

How can researchers determine the three-dimensional structure of Rubellidin-3.2?

Elucidating the three-dimensional structure of Rubellidin-3.2 presents unique challenges due to its small size but remains essential for understanding structure-function relationships. A comprehensive structural analysis requires combining experimental and computational approaches:

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Most suitable technique for small peptides in solution

  • Key experiments include:

    • 1H-1H TOCSY for spin system identification

    • NOESY/ROESY for through-space distance constraints

    • Temperature coefficients of amide protons to identify potential hydrogen bonds

  • 13C/15N labeling (via recombinant expression) for enhanced resolution

• Computational modeling:

  • Molecular dynamics simulations at elevated temperatures to sample conformational space

  • Initial structure generation followed by storage of multiple conformations (50-100) for optimization

  • Refinement using quantum mechanical approaches (DFT preferred for accuracy)

  • Validation against experimental NMR parameters

• Crystallography considerations:

  • Generally challenging for pentapeptides due to flexibility

  • Co-crystallization with binding partners or antibodies may be necessary

  • Valuable if achievable, providing atomic-level resolution

The most productive approach typically combines solution NMR data with computational refinement to generate an ensemble of structures representing the conformational preferences of Rubellidin-3.2 in physiologically relevant conditions.

What methods are available for quantifying Rubellidin-3.2 in experimental samples?

Accurate quantification of Rubellidin-3.2 is essential for experimental reproducibility and reliable determination of structure-activity relationships. Researchers should consider multiple approaches based on their specific experimental requirements:

• Spectrophotometric methods:

  • UV absorption at 280 nm (utilizing phenylalanine residues)

  • Calculated extinction coefficient based on amino acid composition

  • Linear range typically 0.1-1.0 mg/ml with appropriate dilution

• Chromatographic quantification:

  • HPLC with external calibration curves using standards

  • Area under curve (AUC) analysis with validation of linearity, accuracy, and precision

  • Limit of detection typically in low ng range with UV detection

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) for highest sensitivity

  • Stable isotope dilution with labeled internal standards

  • Absolute quantification possible in complex matrices

• Amino acid analysis:

  • Gold standard for absolute quantification

  • Requires acid hydrolysis followed by derivatization

  • Limited by sample requirements and throughput

For most laboratory applications, HPLC-UV quantification offers the best balance of accessibility, accuracy, and throughput when performed with appropriate validation and system suitability tests.

What are the most relevant bioassays for characterizing Rubellidin-3.2 activity?

Determining the biological activities of Rubellidin-3.2 requires a systematic screening approach spanning multiple potential mechanisms of action. Based on structural features and knowledge of similar amphibian-derived peptides, researchers should prioritize these key bioassays:

• Antioxidant capacity:

  • Chemical-based assays: ABTS and DPPH radical scavenging

  • Cellular ROS/RNS detection using fluorescent probes (DCFH-DA, DAF-FM)

  • Flow cytometry analysis for quantification of cellular responses

  • Comparison with reference antioxidants (Trolox, glutathione)

• Neuroprotective potential:

  • Microglial cell models (BV-2, HMC3)

  • Assessment of inflammatory mediator production

  • Protection against oxidative stress-induced neuronal damage

  • Evaluation in neuroblast cell lines (SK-N-BE(2))

• Antimicrobial activity:

  • Broth microdilution against reference bacterial strains

  • Determination of minimum inhibitory concentrations

  • Time-kill kinetics to establish bactericidal vs. bacteriostatic effects

• Safety profiling:

  • Hemolysis assays using human red blood cells

  • Cell viability assays with relevant human cell lines

  • Cytotoxicity concentration curves and therapeutic index calculation

Each assay should be performed with appropriate positive and negative controls, and results should be analyzed using suitable statistical methods to establish significance and reproducibility.

How should researchers approach antioxidant activity testing for Rubellidin-3.2?

Comprehensive assessment of Rubellidin-3.2's antioxidant properties requires a multi-faceted approach that progresses from chemical assays to cellular models. A methodical workflow should include:

• Primary chemical-based screening:

  • ABTS radical scavenging: Measures ability to quench ABTS radical cation

    • Procedure: Incubate Rubellidin-3.2 (0.031-0.25 mg/ml) with ABTS radical

    • Analysis: Calculate IC50 values compared to reference antioxidants

  • DPPH assay: Assesses hydrogen-donating capacity

    • Procedure: Monitor absorbance decrease at 517 nm

    • Analysis: Determine concentration-dependent free radical neutralization

• Cellular oxidative stress models:

  • Preparation: Culture appropriate cell lines (BV-2 microglial cells, neuronal models)

  • Treatments:

    • Controls: Culture medium only, PMA (100 nM) as oxidative stress inducer

    • Test conditions: PMA + Rubellidin-3.2 (50-100 μM), Rubellidin-3.2 alone

  • Detection methods:

    • ROS quantification: DCFH-DA probe followed by flow cytometry

    • RNS assessment: DAF-FM probe for nitric oxide detection

    • Data collection: 10,000 events per sample, FL-1 channel (515-545 nm)

• Mechanistic investigations:

  • Antioxidant enzyme activity measurements

  • Evaluation of gene expression changes in oxidative stress response pathways

  • Direct comparison with other antioxidant peptides of similar size

This systematic approach allows researchers to establish both the direct radical scavenging capacity of Rubellidin-3.2 and its ability to modulate cellular redox processes in physiologically relevant models.

What factors should be considered when evaluating Rubellidin-3.2's potential neuroprotective effects?

Investigation of Rubellidin-3.2's neuroprotective properties requires careful experimental design considering both direct and indirect mechanisms. Researchers should address these key methodological considerations:

• Selection of appropriate cellular models:

  • Microglial cells: BV-2 (murine) or HMC3 (human) for neuroinflammation studies

  • Neuronal cells: SK-N-BE(2) or primary neurons for direct neuroprotection assessment

  • Mixed cultures: To evaluate neuron-glia interactions

• Oxidative/nitrosative stress induction:

  • PMA (100 nM) for respiratory burst activation in microglia

  • H2O2, glutamate, or Aβ peptides as neurotoxic challenges

  • Oxygen-glucose deprivation for ischemia modeling

• Treatment paradigms:

  • Pre-treatment: To assess preventive effects

  • Co-treatment: For direct neutralization capacity

  • Post-treatment: To evaluate recovery-promoting properties

  • Concentration range: Typically 10-100 μM based on preliminary cytotoxicity data

• Outcome measurements:

  • Cell viability: MTT, resazurin reduction, or LDH release assays

  • Oxidative stress markers: ROS/RNS quantification by flow cytometry

  • Inflammatory mediators: Cytokine production, NO release

  • Morphological assessment: Cell shape, neurite outgrowth, phagocytic activity

• Controls and comparators:

  • Vehicle controls (accounting for any DMSO effects)

  • Positive controls: Established antioxidants (Trolox, N-acetylcysteine)

  • Related peptides for structure-activity insights

By systematically investigating these aspects, researchers can establish whether Rubellidin-3.2 exhibits neuroprotective effects and determine the underlying mechanisms, which may involve direct antioxidant activity, modulation of microglial activation, or enhancement of neuronal resilience.

How can researchers design structure-activity relationship studies for Rubellidin-3.2?

Structure-activity relationship (SAR) studies provide critical insights into the molecular determinants of Rubellidin-3.2's biological functions. A comprehensive SAR investigation should follow this methodological framework:

• Systematic analog design:

  • Alanine scanning: Replace each residue with alanine to identify essential positions

  • Conservative substitutions: Maintain side chain properties while altering specific features

  • Terminal modifications: N-acetylation, C-amidation to enhance stability

  • D-amino acid substitutions: Alter backbone conformation while maintaining side chains

  • Sequence truncation: N- and C-terminal deletions to identify minimal active sequence

• Synthesis strategies:

  • Solid-phase peptide synthesis for rapid generation of multiple analogs

  • Parallel synthesis approaches for efficiency

  • Purification to >95% homogeneity by RP-HPLC

  • Comprehensive characterization by MS and analytical HPLC

• Primary activity screening:

  • Select key bioassays based on established Rubellidin-3.2 activities

  • Standardized testing protocols for comparative analysis

  • Concentration-response curves for each analog

  • Determination of relative potency and efficacy

• Structure-function correlation:

  • Computational modeling of all analogs

  • Pharmacophore development

  • Identification of critical functional groups

  • Quantitative structure-activity relationship (QSAR) modeling

This systematic approach will reveal which structural elements of Rubellidin-3.2 are essential for activity, providing direction for the development of optimized derivatives with enhanced potency, selectivity, or stability.

What computational tools can predict molecular targets and mechanisms of Rubellidin-3.2?

Computational approaches offer valuable insights into potential molecular interactions and mechanisms of Rubellidin-3.2, guiding experimental design. A comprehensive in silico investigation should incorporate:

• Structural conformational analysis:

  • Molecular dynamics simulations at elevated temperatures (e.g., 1000K)

  • Storage of multiple conformations (50-100) for analysis

  • Classification of predominant structural motifs

  • Identification of key intramolecular interactions

• Reactivity prediction:

  • Condensed-to-atoms Fukui indices (CAFI) calculation

  • Local softness determination to identify reactive sites

  • Donor-acceptor mapping (DAM) for electron transfer potential

  • Comparison with established antioxidant compounds (glutathione, Trolox)

• Protein-peptide interaction modeling:

  • Reverse virtual screening against protein databases

  • Molecular docking to candidate targets

  • Binding energy calculation and pose analysis

  • Molecular dynamics simulations of promising complexes

• Membrane interaction simulation:

  • Lipid bilayer models of varying composition

  • Assessment of insertion depth, orientation, and stability

  • Calculation of free energy of membrane association

  • Evaluation of potential membrane-disrupting activity

These computational approaches generate testable hypotheses about Rubellidin-3.2's molecular targets and mechanisms, prioritizing experimental directions and reducing the need for extensive screening studies.

How does TFA from synthesis and purification affect biological assays with Rubellidin-3.2?

Trifluoroacetic acid (TFA) is commonly used in peptide synthesis and HPLC purification but can significantly impact biological assays, requiring careful consideration in experimental design:

• TFA retention in peptide preparations:

  • Synthetic peptides like Rubellidin-3.2 typically contain 10-45% TFA as counterions

  • TFA associates particularly with basic residues, though less problematic for Rubellidin-3.2 which contains acidic glutamic acid

  • Can persist through lyophilization and reconstitution

• Potential interference with biological systems:

  • TFA in nanomolar concentrations can affect cellular proliferation

  • Can act as an unintended allosteric modulator of certain receptors

  • May alter local pH in assay systems, affecting enzyme kinetics or receptor binding

  • Can contribute to experimental variability and irreproducibility

• Recommended mitigation strategies:

  • TFA removal via repeated lyophilization with HCl or acetate buffer

  • Use of counter-ion exchange techniques prior to biological testing

  • Inclusion of appropriate vehicle controls matching TFA content

  • Validation of key findings with TFA-removed peptide preparations

• Assays of particular concern:

  • Cell-based functional assays are most susceptible to TFA interference

  • Electrophysiological studies can be affected by TFA's ion channel effects

  • pH-sensitive enzymatic assays may show artificial inhibition or activation

Researchers should document TFA content in Rubellidin-3.2 preparations and implement appropriate controls or removal procedures to ensure biological findings are not artifacts of TFA contamination.

How can researchers differentiate between specific and non-specific effects in Rubellidin-3.2 biological assays?

Distinguishing specific molecular interactions from non-specific effects is critical for accurately characterizing Rubellidin-3.2's biological activities. A methodical approach to this challenge should include:

• Concentration-response relationship analysis:

  • Test broad concentration range (typically nanomolar to high micromolar)

  • Establish complete concentration-response curves

  • Differentiate between sigmoidal (specific) and linear (often non-specific) responses

  • Calculate EC50/IC50 values for comparison with known ligands

• Structural specificity controls:

  • Scrambled sequence peptide with identical composition

  • D-amino acid version (retains composition but alters 3D structure)

  • Single residue substitutions at key positions

  • N- and C-terminally modified versions

• Competitive binding studies:

  • Displacement assays with known ligands for suspected targets

  • Radioligand or fluorescent ligand binding competition

  • Kinetic analysis (association/dissociation rates)

  • Selective antagonists to block specific pathways

• Mechanistic validation:

  • Genetic approaches (siRNA knockdown of candidate targets)

  • Pharmacological inhibitors of downstream signaling

  • Receptor subtype-selective compounds

  • Cross-desensitization experiments

• Direct binding demonstration:

  • Surface plasmon resonance with purified targets

  • Isothermal titration calorimetry for thermodynamic parameters

  • Fluorescence polarization with labeled peptide

  • Pull-down assays followed by proteomics identification

This comprehensive approach enables researchers to confidently distinguish Rubellidin-3.2's specific molecular interactions from non-specific effects such as membrane disruption or aggregation, establishing a solid foundation for mechanistic understanding.