Recombinant Rana temporaria Ranacyclin-T (RNCT)

What is Ranacyclin-T and how was it discovered?

Ranacyclin-T is a cyclic 17-residue antimicrobial peptide discovered through screening a cDNA library from the European common frog Rana temporaria. It belongs to a distinct family of peptides called ranacyclins, which have unique structural and functional properties that differentiate them from most other antimicrobial peptides. The initial discovery was reported alongside Ranacyclin-E, which was isolated directly from Rana esculenta skin secretions. These peptides were identified as having antimicrobial and antifungal activity despite being structurally related to pLR, an 18-mer peptide from Rana pipiens that previously had no reported antimicrobial activity .

What is the primary structure and key characteristics of Ranacyclin-T?

Ranacyclin-T has the primary sequence GALRGCWTKSYPPKPCK. Like other ranacyclins, it features a highly conserved region of 13 amino acids that form a cyclic undecapeptide through a disulfide bridge connecting Cys5 and Cys15. This structural characteristic is considered a hallmark of the ranacyclin family. The peptide adopts approximately 29.41% extended strand and 70.59% random coil secondary structure in solution, which is notable because most antimicrobial peptides adopt predominantly alpha-helical or beta-sheet conformations .

How does the mechanism of action of Ranacyclin-T differ from conventional antimicrobial peptides?

Unlike many antimicrobial peptides that primarily interact with negatively charged bacterial membrane components, Ranacyclin-T exhibits several distinctive properties:

• It maintains a significant portion of random coil structure (70.59%) when interacting with membranes, as demonstrated by ATR-FTIR and CD spectroscopy .

• It binds similarly to both zwitterionic and negatively charged membranes, as shown by tryptophan fluorescence and surface plasmon resonance studies .

• It inserts into the hydrophobic core of the membrane and forms transmembrane pores without damaging the bacterial cell wall, as confirmed by SPR, ATR-FTIR, and transmission electron microscopy .

• It interacts primarily with the hydrophobic core of cell membranes rather than with negatively charged lipid head groups, allowing it to intercalate into various membrane types .

These properties suggest a membrane permeabilization mechanism that is less dependent on electrostatic interactions than is typical for most antimicrobial peptides.

What is the spectrum of antimicrobial activity for Ranacyclin-T?

Ranacyclin-T demonstrates variable effectiveness against different bacterial strains, with the following minimum inhibitory concentrations (MICs):

These values indicate that while Ranacyclin-T has broad-spectrum activity, it is particularly effective against B. megaterium and Y. pseudotuberculosis, while showing moderate activity against other tested strains .

How does the antimicrobial activity of Ranacyclin-T compare to other ranacyclins?

Despite high structural similarity, ranacyclins demonstrate different antimicrobial activity spectra. For example, Ranacyclin-E shows no activity against E. coli D21 (compared to Ranacyclin-T's MIC of 30 μM) but is more effective against S. lentus with an MIC of 7 μM (compared to Ranacyclin-T's 10 μM). Additionally, Ranacyclin-E demonstrates antifungal activity against several Candida species and Phytophthora nicotianae spores .

In contrast, newer members of the family such as Ranacyclin-NF from Pelophylax nigromaculatus show negligible direct antimicrobial activity (MIC >512 μM against all tested bacteria including E. coli, P. aeruginosa, K. pneumoniae, S. aureus, MRSA, and E. faecalis), but demonstrate significant trypsin inhibitory activity and can enhance the effectiveness of conventional antibiotics against resistant bacteria .

What methods are recommended for evaluating the membrane interactions of Ranacyclin-T?

For researching Ranacyclin-T's membrane interactions, several complementary techniques should be employed:

• ATR-FTIR and CD spectroscopy to determine secondary structure changes upon membrane binding. These techniques revealed that Ranacyclin-T maintains approximately 50% random coil structure in membrane environments .

• Tryptophan fluorescence to monitor peptide-membrane binding kinetics and determine binding affinity. The tryptophan residue in Ranacyclin-T (W7) serves as an intrinsic fluorescent probe .

• Surface Plasmon Resonance (SPR) using a BIAcore biosensor to quantify binding parameters to different membrane compositions and measure insertion dynamics .

• Transmission Electron Microscopy (TEM) to visualize membrane morphological changes and potential pore formation without bacterial wall damage .

This multi-technique approach provides comprehensive insights into Ranacyclin-T's interactions with different membrane types.

How can researchers effectively produce recombinant Ranacyclin-T for experimental studies?

Based on approaches used with similar antimicrobial peptides, researchers should consider the following methodological steps for recombinant Ranacyclin-T production:

• Gene synthesis and vector design: Optimize the coding sequence for expression in the chosen host system (typically E. coli).

• Expression system selection: Use a fusion protein approach with partners like thioredoxin or SUMO to enhance solubility and prevent toxicity to the host.

• Purification strategy: Implement affinity chromatography followed by enzymatic cleavage of the fusion tag, with special attention to preserving the disulfide bridge formation.

• Proper folding verification: Confirm the correct cyclization through the Cys5-Cys15 disulfide bond using mass spectrometry and circular dichroism to ensure the peptide has achieved its native conformation.

• Activity validation: Compare the antimicrobial activity of the recombinant peptide against known MIC values to confirm functional properties.

This approach will help ensure production of correctly folded, biologically active Ranacyclin-T for experimental studies.

What structural factors influence Ranacyclin-T's antimicrobial effectiveness?

Research indicates several structural elements critical to Ranacyclin-T's function:

• Secondary structure composition: Despite having only 29.41% extended strand structure and maintaining 70.59% random coil, Ranacyclin-T exhibits significant antimicrobial activity. This challenges the conventional wisdom that a high proportion of defined secondary structure (α-helix or β-sheet) is necessary for antimicrobial effectiveness .

• Disulfide bridge: The cyclic structure formed by the Cys5-Cys15 disulfide bond creates a constrained loop region essential for activity and stability.

• Membrane penetration efficiency: The specific amino acid composition affects Ranacyclin-T's ability to penetrate different bacterial cell walls before reaching the cytoplasmic membrane. Structure-activity relationship studies with related peptides suggest that increasing the fraction of secondary structure and reducing peptide assembly in the membrane facilitates diffusion through bacterial cell walls .

• Residues outside the conserved loop: Studies with related ranacyclins suggest that amino acids outside the trypsin inhibitory loop may influence antimicrobial efficacy, as demonstrated by the different activity profiles of structurally similar ranacyclins .

How might researchers investigate potential synergistic effects between Ranacyclin-T and conventional antibiotics?

Based on findings with Ranacyclin-NF, which showed synergy with Gentamicin against MRSA despite lacking direct antimicrobial activity , researchers should consider the following approach:

• Checkerboard assay methodology: Systematically test combinations of Ranacyclin-T with various antibiotic classes at different concentration ratios against target bacteria.

• Time-kill kinetics: Measure bacterial killing rates for Ranacyclin-T alone, antibiotics alone, and combinations to identify potential synergistic or antagonistic interactions.

• Membrane permeabilization assays: Determine if Ranacyclin-T enhances antibiotic uptake by increasing membrane permeability using fluorescent dyes or radiolabeled antibiotics.

• Resistance development monitoring: Evaluate whether combination therapy delays or prevents the emergence of resistance compared to single-agent treatment.

• Mechanism investigations: Explore whether synergy occurs through enhanced antibiotic penetration, inhibition of efflux pumps, or other mechanisms.

This systematic approach will help establish potential therapeutic combinations that leverage Ranacyclin-T's unique membrane-interacting properties.

How can researchers reconcile contradictory findings regarding Ranacyclin-T's effectiveness?

When facing contradictory results about Ranacyclin-T's efficacy, consider these methodological approaches:

• Standardize testing conditions: Ensure consistent peptide preparation, bacterial growth phase, and media composition, as these factors significantly impact antimicrobial activity measurement.

• Account for bacterial strain differences: Even within the same species, different strains may show variable susceptibility to Ranacyclin-T, as seen with the peptide's differential activity against various bacterial types .

• Evaluate membrane composition effects: Since Ranacyclin-T interacts with membrane hydrophobic cores, variations in lipid composition between experimental systems could explain contradictory findings.

• Consider peptide aggregation state: Ranacyclin-T's random coil content and potential for self-assembly may affect its availability for antimicrobial action in different experimental settings.

• Implement multiple activity measurement techniques: Use both growth inhibition and direct bacterial killing assays to comprehensively assess antimicrobial activity.

This structured approach helps identify sources of variability and resolve apparently contradictory findings about Ranacyclin-T's effectiveness.

What techniques are most appropriate for studying Ranacyclin-T's structural dynamics during membrane interaction?

To effectively study Ranacyclin-T's structural transitions when interacting with membranes, researchers should employ:

• Time-resolved CD spectroscopy: Monitor secondary structure changes during membrane binding, especially given Ranacyclin-T's unusual maintenance of significant random coil structure (70.59%) in membrane environments .

• Solid-state NMR spectroscopy: Determine precise conformational changes and orientation within the membrane bilayer.

• Molecular dynamics simulations: Model Ranacyclin-T's interaction with different membrane compositions based on experimentally determined parameters.

• Site-directed fluorescence labeling: Track specific amino acid position movements during membrane penetration using environment-sensitive fluorophores.

• Neutron reflectometry: Analyze the depth of peptide penetration into model membranes with different lipid compositions.

These complementary techniques provide a comprehensive view of the structural dynamics underlying Ranacyclin-T's unique membrane interaction mechanism.