Recombinant Styela clava Clavanin-D

What is Clavanin D and how does it differ from other members of the clavanin family?

Clavanin D is an α-helical antimicrobial peptide derived from the hemocytes of the marine tunicate Styela clava. It belongs to the clavanin family, which comprises a group of antimicrobial peptides known for their potent activity against Gram-negative bacteria. The clavanin family consists of six peptides with varying degrees of sequence homology - five members share approximately 80% sequence similarity, while the sixth member (clavaspirin) possesses only about 30% similarity to Clavanin A, which is considered the archetypal member of this family .

Clavanin D differs from other family members through its specific amino acid sequence and unique structural characteristics. It contains multiple histidine residues (His10, His11, and His21) that play crucial roles in metal ion coordination and antimicrobial activity, distinguishing its functional properties from other clavanins.

What is the amino acid sequence of Clavanin D and how does it contribute to its function?

The amino acid sequence of Clavanin D is AFKLL GRIIH HVGNF VY'GFS HVF-NH2. This specific sequence is significant due to several structural and functional features:

• The presence of multiple histidine residues (particularly His10, His11, and His21) enables metal ion coordination with zinc and copper.

• At physiological pH (7.4), these histidine residues coordinate with metal ions through their imidazole rings and the amide nitrogen of His11.

• This metal ion coordination enhances antimicrobial efficacy by stabilizing the peptide structure and facilitating interactions with microbial membranes.

• The amino acid composition contributes to its ability to bind to negatively charged microbial membranes, facilitating membrane disruption.

These sequence-specific characteristics directly influence Clavanin D's antimicrobial mechanisms and spectrum of activity.

What antimicrobial activity spectrum does Clavanin D exhibit?

Clavanin D demonstrates significant antimicrobial activity against various pathogens, with particularly notable efficacy against multidrug-resistant Gram-negative bacteria. Key aspects of its activity spectrum include:

• Potent activity against multidrug-resistant Enterobacter cloacae species with a minimal inhibitory concentration (MIC) of 8 μM.

• Activity against both Gram-positive and Gram-negative bacteria, similar to other members of the clavanin family .

• Some antifungal properties, though these are generally less pronounced than its antibacterial effects .

The broad-spectrum activity makes Clavanin D particularly interesting for research into novel antimicrobial agents capable of addressing multidrug resistance challenges.

How does Clavanin D interact synergistically with other antimicrobial peptides?

Recent research has identified significant synergistic interactions between Clavanin D and other antimicrobial peptides, particularly against multidrug-resistant pathogens:

This synergistic capability has significant implications for developing combination therapies that may overcome resistance mechanisms while potentially allowing for lower therapeutic dosages of each individual peptide.

How does metal ion coordination affect Clavanin D's antimicrobial efficacy?

Clavanin D exhibits notable chemical interactions, particularly in its ability to form complexes with metal ions:

• At physiological pH (7.4), Clavanin D coordinates with metal ions such as zinc and copper through its histidine residues (His10, His11, and His21) and the amide nitrogen of His11.

• This coordination is significant for its biological activity, enhancing antimicrobial efficacy by stabilizing the peptide structure and facilitating interactions with microbial membranes.

• The metal ion complexation likely contributes to the peptide's dual mechanism of action: disrupting bacterial membranes and interfering with intracellular processes such as DNA synthesis.

These metal-peptide interactions represent a potential area for structure-based optimization of Clavanin D derivatives with enhanced antimicrobial properties.

What molecular dynamics characterize Clavanin D's interaction with membrane lipids?

Based on studies of related clavanins, particularly Clavanin A, we can infer important aspects of Clavanin D's interaction with membrane lipids:

• Molecular docking experiments with lipids like DOPC and DPPC provide models for peptide-lipid interactions that likely apply to Clavanin D as well .

• For these interactions, docking experiments typically consider both the shape and electrostatics of each molecule .

• Molecular dynamics simulations can be performed using software like GROMACS to analyze the stability and nature of these interactions over time (typically 50 ns simulations) .

• Analysis methods include root mean square deviation (RMSD) and standardized secondary structure assignment (DSSP) to characterize conformational changes during membrane interaction .

These molecular dynamics approaches offer valuable insights into the biophysical basis of Clavanin D's antimicrobial activity at the membrane level.

What expression systems are optimal for producing recombinant Clavanin D?

Recombinant Clavanin D can be produced using various expression systems, each with specific considerations:

• E. coli expression system: Commonly used for its simplicity and cost-effectiveness. Typically yields protein with >85% purity as determined by SDS-PAGE.

• Yeast expression system: Offers post-translational modifications that may better mimic the native peptide structure.

• Baculovirus expression system: Suitable for larger-scale production with eukaryotic processing.

• Mammalian cell expression system: Provides the most native-like post-translational modifications but at higher cost.

Selection of the appropriate expression system should be based on research requirements, including scale needed, budget constraints, and whether specific post-translational modifications are critical for the intended applications.

What are the recommended storage and handling protocols for recombinant Clavanin D?

Proper storage and handling of recombinant Clavanin D are essential for maintaining its structural integrity and antimicrobial activity:

• Storage form: Typically supplied as a lyophilized powder which offers greater stability during shipping and long-term storage.

• Reconstitution protocol: Briefly centrifuge the vial before opening. Reconstitute the protein in sterile deionized water to a concentration of 0.1-1.0 mg/mL.

• Long-term storage: Add glycerol to a final concentration of 5-50% (standard is 50%), aliquot, and store at -20°C/-80°C for long-term stability.

• Working storage: Store working aliquots at 4°C for up to one week to minimize degradation.

• Critical precaution: Avoid repeated freezing and thawing cycles as these can significantly diminish peptide activity.

Following these protocols helps ensure experimental reproducibility and maintains the structural and functional integrity of the peptide.

What analytical methods can be employed to characterize the structure of Clavanin D?

Several analytical approaches can be used to characterize the structural properties of Clavanin D, based on techniques applied to related clavanins:

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • 1H-1H Total Correlation Spectroscopy (TOCSY)

  • 1H-1H Nuclear Overhauser Effect Spectroscopy (NOESY)

  • 1H-15N So-fast Heteronuclear Multiple Quantum Coherence (sf-HMQC)

  • 1H-13C Heteronuclear Single Quantum Coherence (HSQC)

• Structural refinement and validation:

  • Ramachandran plot analysis to validate dihedral angles

  • QUEEN analyses to assess agreement between model structures and experimental data

  • Root mean square deviation (RMSD) calculations to evaluate structural consistency

• Computational methods:

  • Molecular dynamics simulations using packages like GROMACS

  • Analysis of secondary structure through DSSP (Define Secondary Structure of Proteins)

These techniques provide complementary data for comprehensive structural characterization of Clavanin D.

How can researchers assess the synergistic antimicrobial activity of Clavanin D?

To evaluate potential synergistic interactions between Clavanin D and other antimicrobial peptides or conventional antibiotics, researchers should employ multiple complementary methods:

• Checkerboard assay: This method allows systematic testing of combinations of two antimicrobial agents at different concentrations to identify synergistic, additive, or antagonistic interactions .

• Time-kill kinetics assay: This approach measures the rate of bacterial killing over time when exposed to individual peptides versus combination treatments, providing dynamic information about antimicrobial activity .

• Fractional Inhibitory Concentration (FIC) index calculation: Used to quantify the degree of synergy between antimicrobial agents, with values <0.5 typically indicating synergy .

• Testing against clinically relevant resistant strains: Particularly important is evaluation against multidrug-resistant isolates such as E. cloacae 0136, which has demonstrated susceptibility to Clavanin D combinations .

Using multiple methods provides more robust evidence of synergy, as demonstrated by the finding that Clavanin D and clavaspirin show synergy in both checkerboard and time-kill kinetics assays .

What techniques are appropriate for studying Clavanin D's membrane interactions?

Understanding Clavanin D's interactions with bacterial membranes requires specialized biophysical and computational techniques:

• Molecular docking:

  • Software such as Hex 6.1 can be used to construct complexes between Clavanin D and membrane lipids

  • Both shape and electrostatics should be considered in docking experiments

  • Resulting complexes should be clustered using appropriate RMS cut-offs (e.g., 3 Å)

• Molecular dynamics simulations:

  • Parameterization of lipid structures using tools like PRODRG server

  • Simulation in cubic water boxes with appropriate ions for charge neutralization

  • Energy minimization followed by pressure and temperature normalization

  • Extended simulations (e.g., 50 ns) at normalized pressure and temperature

• Biophysical characterization:

  • Lipid binding assays to assess direct interactions

  • Membrane leakage assays to quantify membrane disruption

  • Fluorescence microscopy with labeled peptides to visualize membrane localization

These approaches provide complementary insights into how Clavanin D interacts with and disrupts bacterial membranes as part of its antimicrobial mechanism.