Recombinant Viola hederacea Cycloviolacin-H2

What is Cycloviolacin-H2 and how does it compare structurally to other cyclotides?

Cycloviolacin-H2 is a macrocyclic peptide belonging to the cyclotide family, characterized by a unique knotted arrangement of three disulfide bonds forming a cyclic cystine knot (CCK) motif. Cyclotides are plant-derived peptides typically containing 28-37 amino acid residues with the entire primary amino acid chain covalently cyclized via peptide bonds .

The structural characteristics of Cycloviolacin-H2 include:

• A circular backbone providing resistance to exopeptidases

• Three conserved disulfide bonds forming the cystine knot

• Six conserved cysteine residues

• Hydrophobic surface patches that contribute to its bioactivity

Cyclotides are classified into two main subfamilies based on their structures:

• Möbius subfamily (e.g., kalata B1) - contains a conceptual twist in the backbone

• Bracelet subfamily (e.g., cycloviolacins) - lack the twist and typically have a larger number of residues

Cycloviolacin-H2 belongs to the bracelet subfamily and shares sequence homology with other cycloviolacins, particularly in the conserved cysteine framework that is critical for structural stability.

What expression systems are most efficient for producing recombinant Cycloviolacin-H2?

Multiple expression systems can be utilized for the recombinant production of Cycloviolacin-H2, each with specific advantages depending on research objectives:

For laboratory-scale research applications, E. coli systems are often preferred due to cost efficiency, while for structural studies requiring precise folding, yeast or mammalian systems might yield better results. The choice should be guided by the specific research questions being addressed and the downstream applications of the recombinant cyclotide.

How can researchers verify the correct cyclization and disulfide bond formation in recombinant Cycloviolacin-H2?

Verifying correct cyclization and disulfide bond formation in recombinant Cycloviolacin-H2 requires a multi-analytical approach:

• Mass Spectrometry Analysis:

  • Intact mass analysis to confirm the expected molecular weight

  • Reduction of disulfide bonds followed by alkylation should result in a mass shift corresponding to the number of cysteine residues (six for cyclotides)

• Enzymatic Digestion Pattern:

  • Properly folded cyclotides display characteristic fragmentation patterns when digested with enzymes like trypsin and endoGluC

  • Compare digestion fragments with those of native Cycloviolacin-H2 using tandem MS

• NMR Spectroscopy:

  • Chemical shift comparisons with known cyclotide structures

  • 2D NMR experiments (TOCSY, NOESY) to confirm the three-dimensional structure

• Bioactivity Assays:

  • Functional tests (e.g., anti-HIV activity, hemolytic assays) as properly folded cyclotides exhibit characteristic bioactivities

The quality control process should include comparison with reference standards, as misfolded cyclotides typically show altered retention times in RP-HPLC and different bioactivity profiles.

What are the optimized methods for extracting and purifying Cycloviolacin-H2 from recombinant sources?

The purification of recombinant Cycloviolacin-H2 involves a systematic workflow:

• Initial Extraction:

  • For E. coli systems: Cell lysis using sonication or homogenization in appropriate buffer systems

  • For secretion-based systems (yeast/mammalian): Collection of culture supernatant and concentration

• Primary Purification:

  • Immobilized metal affinity chromatography (IMAC) if an affinity tag is incorporated

  • Strong cation exchange chromatography leveraging the typically positive net charge of cyclotides at physiological pH

• Tag Removal and Cyclization (if applicable):

  • Enzymatic cleavage of fusion tags

  • Oxidative folding in glutathione redox buffers to promote correct disulfide formation

• Final Purification:

  • Reversed-phase HPLC using C18 columns with acetonitrile/water gradient systems

  • Typical retention time for Cycloviolacin-H2 is relatively late in the gradient due to its hydrophobic nature

• Quality Assessment:

  • SDS-PAGE (with expected purity >85%)

  • Mass spectrometry confirmation

  • Circular dichroism to confirm secondary structure elements

The entire purification protocol should be performed under conditions that minimize oxidation and maintain the integrity of the disulfide bonds, which are crucial for cyclotide activity.

How can researchers effectively assess the anti-HIV activity of Cycloviolacin-H2 in laboratory settings?

Assessment of anti-HIV activity of Cycloviolacin-H2 should follow established protocols:

• XTT-Based Viability Assays:

  • Incubate HIV-infected cells (typically MT-4 or TZM-bl cell lines) with different concentrations of the cyclotide

  • Measure cell viability using XTT tetrazolium salt reduction to determine EC₅₀ values

  • Include appropriate controls (untreated infected cells, AZT as positive control)

• Viral Replication Assays:

  • Quantify p24 antigen levels in the supernatant of infected cells using ELISA

  • Monitor viral RNA levels using RT-PCR

• Mechanism Studies:

  • Time-of-addition experiments to determine which stage of the viral life cycle is inhibited

  • Test against a panel of HIV-1 strains with different tropisms and drug resistance profiles

• Selectivity Index Calculation:

  • Determine cytotoxicity (CC₅₀) in uninfected cells

  • Calculate selectivity index (SI = CC₅₀/EC₅₀) to assess therapeutic potential

Based on comparative studies, highly hydrophobic cyclotides like Cycloviolacin Y5 have demonstrated potent anti-HIV activity with EC₅₀ values around 40 nM . As a member of the cycloviolacin family, Cycloviolacin-H2 would be expected to show similar activity profiles, with efficacy correlating with its relative hydrophobicity and membrane-disrupting potential.

What structural modifications can enhance the therapeutic potential of Cycloviolacin-H2 while preserving stability?

Strategic structural modifications can enhance Cycloviolacin-H2's therapeutic potential:

• Surface Residue Substitutions:

  • Modifying hydrophobic/hydrophilic balance by substituting exposed residues can fine-tune membrane interactions

  • Introducing positively charged residues may enhance antimicrobial activity while reducing hemolytic potential

• Loop Grafting:

  • The cyclotide scaffold allows grafting of bioactive peptide sequences into specific loops

  • Target-specific sequences (e.g., receptor-binding epitopes) can be incorporated while maintaining the core CCK motif

• Chemical Conjugation:

  • Site-specific PEGylation to reduce immunogenicity and extend half-life

  • Conjugation with targeting moieties for tissue-specific delivery

• Disulfide Engineering:

  • Substitution of native disulfides with diselenide bonds can increase stability

  • Selective reduction/alkylation of specific disulfides can alter conformational flexibility

When designing modifications, researchers should consider:

• The correlation between hydrophobicity and activity (as observed for cycloviolacins)

• The impact on three-dimensional structure and disulfide bonding

• Potential effects on recombinant production efficiency

Structural modification studies should employ molecular modeling prior to experimental validation, and modified variants should be systematically compared against the native cyclotide using standardized bioactivity assays.

How does the membrane-disrupting mechanism of Cycloviolacin-H2 compare to other bioactive cyclotides?

The membrane-disrupting mechanism of Cycloviolacin-H2 and related cyclotides involves:

• Initial Binding:

  • Electrostatic interactions between positively charged residues and negatively charged membrane components

  • Hydrophobic interactions between exposed non-polar residues and membrane lipids

• Insertion and Pore Formation:

  • Partial insertion into the lipid bilayer

  • Potential oligomerization leading to pore formation or membrane thinning

• Membrane Disruption Consequences:

  • Altered membrane permeability leading to ion leakage

  • Potential disruption of membrane-dependent processes in target cells

Based on structure-activity relationships of related cyclotides, the following factors likely influence Cycloviolacin-H2's membrane activity:

Research with related cycloviolacins has demonstrated a strong correlation between hydrophobicity and both anti-HIV activity and hemolytic potential . This suggests that Cycloviolacin-H2's activity profile is likely determined by its specific hydrophobic surface presentation rather than simply its amino acid composition.

What are the key challenges in scaling up recombinant Cycloviolacin-H2 production for research applications?

Scaling up recombinant Cycloviolacin-H2 production presents several significant challenges:

• Proper Folding and Cyclization:

  • The complex disulfide-rich structure requires carefully optimized oxidative folding conditions

  • Incorrect isomers can form, reducing yield of correctly folded product

• Expression System Limitations:

  • Potential toxicity to host cells due to membrane-disrupting activity

  • Need for specialized secretion strategies to achieve proper processing

• Purification Challenges:

  • Separation of correctly folded cyclotide from misfolded variants

  • Removal of host cell proteins with similar physicochemical properties

• Analytical Hurdles:

  • Validation of correct structure across batches

  • Development of robust quality control methods for consistency assessment

• Bioactivity Standardization:

  • Establishing reproducible bioactivity assays for batch validation

  • Correlation of structural variations with functional outcomes

Potential solutions include:

• Development of specialized fusion protein systems with self-cleaving capabilities

• Optimization of in vitro folding protocols using redox buffer systems

• Implementation of orthogonal purification strategies combining charge-based and hydrophobicity-based separations

• Utilization of MS/MS fingerprinting for quality control

How can researchers differentiate between the direct anti-HIV activity of Cycloviolacin-H2 and its cytotoxic effects?

Differentiating between anti-HIV activity and cytotoxicity requires methodical experimental design:

• Selectivity Index Determination:

  • Calculate the ratio between cytotoxic concentration (CC₅₀) and effective antiviral concentration (EC₅₀)

  • Higher selectivity indices (>10) indicate better therapeutic potential

• Time-Course Studies:

  • Compare the kinetics of antiviral effects versus cytotoxic effects

  • True antiviral activity may occur at earlier time points or lower concentrations than cytotoxicity

• Mechanistic Investigations:

  • Employ specific assays targeting different stages of the HIV life cycle:

    • Virus attachment assays

    • Fusion inhibition assays

    • Reverse transcriptase inhibition assays

    • Integration inhibition assays

• Structure-Activity Relationship Studies:

  • Create a panel of variants with systematic modifications

  • Map regions responsible for antiviral activity versus those causing cytotoxicity

• Membrane Selectivity Assessment:

  • Compare effects on model membranes mimicking viral envelopes versus host cell membranes

  • Utilize fluorescent lipid mixing assays and membrane leakage assays

What advanced MS/MS sequencing strategies are most effective for characterizing recombinant Cycloviolacin-H2?

Advanced MS/MS sequencing of recombinant Cycloviolacin-H2 requires specialized approaches due to its cyclic nature and disulfide bonds:

• Sample Preparation Strategies:

  • Reduction of disulfide bonds using DTT or TCEP

  • Alkylation of free cysteines (iodoacetamide or iodoacetic acid)

  • Controlled enzymatic digestion with multiple proteases:

    • Trypsin (cleaves at Lys/Arg)

    • EndoGluC (cleaves at Glu)

    • Chymotrypsin (cleaves at aromatic residues)

• MS/MS Fragmentation Techniques:

  • Electron Transfer Dissociation (ETD) for improved fragmentation of disulfide-rich peptides

  • Collision-Induced Dissociation (CID) for linear fragments after reduction

  • Higher-energy Collisional Dissociation (HCD) for improved fragment ion series

• Data Analysis Approaches:

  • De novo sequencing algorithms optimized for cyclic peptides

  • Database searches with custom parameters for cyclotides

  • Manual interpretation of spectra for critical regions

• Confirmation Strategies:

  • Amino acid analysis for composition verification

  • Comparison with synthetic fragments or standards

  • Orthogonal sequencing approaches like Edman degradation for linearized fragments

For example, a typical workflow for Cycloviolacin-H2 characterization would include:

• Initial intact mass determination

• Reduction and alkylation

• Parallel digestions with multiple enzymes

• LC-MS/MS analysis of all digests

• Fragment mapping and sequence assembly

• Verification of the complete sequence through overlapping fragments

This comprehensive approach has been successfully applied to related cyclotides like those from Viola yedoensis, allowing complete sequence determination and structure prediction .

How does the anti-infective activity of Cycloviolacin-H2 compare with other medicinal plant-derived cyclotides?

Comparative analysis of Cycloviolacin-H2 with other medicinal plant cyclotides reveals important patterns:

The anti-infective activity profile of Cycloviolacin-H2 would be expected to correlate with its:

• Hydrophobic surface area

• Surface charge distribution

• Specific amino acid composition in exposed loops

Research with related cyclotides has demonstrated that these peptides typically exhibit a spectrum of activities beyond anti-HIV effects, including insecticidal, anthelmintic, and antibacterial properties . The multi-functional nature of cyclotides makes them valuable scaffolds for developing versatile anti-infective agents.

What experimental approaches can determine the in vivo stability and pharmacokinetics of recombinant Cycloviolacin-H2?

Investigating the in vivo stability and pharmacokinetics of recombinant Cycloviolacin-H2 requires multiple experimental approaches:

• Serum Stability Assays:

  • Incubation in serum at physiological temperature

  • Time-course sampling and LC-MS analysis to monitor degradation

  • Comparison with linear peptide controls to demonstrate cyclotide stability advantage

• Radiolabeling or Fluorescent Labeling Studies:

  • Site-specific labeling with minimal impact on structure

  • Tracking tissue distribution following administration

  • Determination of half-life and clearance routes

• Ex Vivo Tissue Distribution:

  • Organ harvesting at different time points after administration

  • Extraction and quantification of intact cyclotide

  • Immunohistochemistry with specific antibodies if available

• Metabolism Studies:

  • Identification of potential metabolites using LC-MS/MS

  • In vitro studies with liver microsomes or hepatocytes

  • Determination of major metabolic pathways

• Bioavailability Assessment:

  • Comparison of different administration routes

  • Quantification of active cyclotide reaching systemic circulation

  • Correlation with observed pharmacodynamic effects

What are the most promising applications of Cycloviolacin-H2 in addressing antimicrobial resistance challenges?

Cycloviolacin-H2 offers several promising applications for addressing antimicrobial resistance:

• Novel Mechanism Against Resistant Pathogens:

  • Membrane-disrupting mechanism differs from conventional antibiotics

  • Lower propensity for resistance development due to physical rather than enzymatic targeting

  • Potential activity against multi-drug resistant bacterial strains

• Anti-HIV Applications:

  • Activity against HIV strains resistant to current antiretrovirals

  • Potential combination therapy with conventional antiretrovirals

  • Topical microbicide development for prevention

• Agricultural Applications:

  • Control of crop pathogens with reduced environmental impact

  • Management of insect pests resistant to conventional pesticides

  • Potential for transgenic expression in crops for enhanced resistance

• Scaffold for Antimicrobial Peptide Design:

  • Template for designing stable antimicrobial peptides

  • Structure-guided modifications to enhance specificity

  • Platform for multivalent peptide display

• Biofilm Disruption:

  • Potential activity against biofilm-forming pathogens

  • Combination with conventional antibiotics to enhance penetration

The exceptional stability of cyclotides like Cycloviolacin-H2 makes them particularly valuable in harsh environments where conventional antimicrobials might be degraded. Their membrane-active mechanism also suggests potential synergy with existing antimicrobials, potentially revitalizing the activity of drugs to which resistance has developed.

How can computational approaches enhance the design of Cycloviolacin-H2 variants with improved therapeutic indices?

Computational approaches offer powerful tools for rational design of improved Cycloviolacin-H2 variants:

• Molecular Dynamics Simulations:

  • Analysis of cyclotide-membrane interactions

  • Identification of key residues involved in membrane binding

  • Assessment of structural flexibility and its relationship to activity

• Quantitative Structure-Activity Relationship (QSAR) Modeling:

  • Correlation of physicochemical properties with bioactivity data

  • Development of predictive models for both therapeutic and toxic effects

  • Optimization of hydrophobic/hydrophilic balance

• Docking Studies:

  • Investigation of potential specific protein targets beyond membrane disruption

  • Modeling interactions with viral proteins or bacterial cell wall components

  • Design of targeted binding interfaces

• De Novo Design Approaches:

  • Computational scaffold design maintaining the cyclotide framework

  • Optimization of sequence while preserving critical structural elements

  • In silico validation before experimental testing

• Machine Learning Integration:

  • Training on existing cyclotide bioactivity datasets

  • Prediction of properties for novel variants

  • Design of cyclotide libraries with optimized properties

Research has demonstrated that hydrophobicity correlates strongly with both anti-HIV activity and hemolytic properties of cyclotides . Computational approaches can help identify the optimal balance, designing variants that maintain anti-infective activity while reducing cytotoxicity. For example, models could predict variants where hydrophobic patches are optimally sized and positioned for interaction with viral membranes while minimizing interactions with host cell membranes.