Chilukuri Subbalakshmi

Requirements for antibacterial and hemolytic activities in the bovine neutrophil derived 13-residue peptide indolicidin

The antimicrobial and hemolytic activities of the 13‐residue peptide indolicidin (ILPWKWPWWPWRR‐NH2), present in bovine neutrophils, and its analogs have been determined with a view to gaining insight into the structural roles of tryptophan and proline. Peptides where proline was replaced by alanine and tryptophan by phenylalanine showed antibacterial activities comparable to that of indolicidin. The peptides do not exhibit a strong propensity to occur in either helical or β‐sheet conformation. The peptides also do not appear to exert their activity by permeabilizing the bacterial plasma membrane unlike other endogenous antibacterial peptides. The presence of tryptophan appears to be essential for hemolytic activity as the phenylalanine analog does not exhibit any hemolytic activity.

Biological activities of C-terminal 15-residue synthetic fragment of melittin: design of an analog with improved antibacterial activity

Melittin, the 26‐residue predominant toxic peptide from bee venom, exhibits potent antibacterial activity in addition to its hemolytic activity. The synthetic peptide of 15 residues corresponding to its C‐terminal end (MCF), which encompasses its most amphiphilic segment, is now being shown to possess antibacterial activity about 5–7 times less compared to that of melittin. MCF, however, is 300 times less hemolytic. An analog of MCF, MCFA, in which two cationic residues have been trans‐positioned to the N‐terminal region from the C‐terminal region, exhibits antibacterial activity comparable to that of melittin, but is only marginally more hemolytic than MCF. The biophysical properties of the peptides, like folding and aggregation, correlate well with their biological properties.

Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes

Indolicidin is a 13-residue broad-spectrum antibacterial peptide isolated from bovine neutrophils. The primary structure of the peptide ILPWKWPWWPWRR-amide (IL) reveals an unusually high percentage of tryptophan residues. IL and its analogues where proline residues have been replaced by alanine (ILA) and trp replaced by phe (ILF) show comparable antibacterial activitieso While IL and ILA are haemolytic, ILF does not have this property. Since aromatic residues would strongly favour partitioning of the peptide into the lipid bilayer interface, the biological activities of IL and its analogues could conceivably arise due perturbation of the lipid bilayer of membranes. We have therefore investigated the interaction of IL and its analogues with lipid vesicles. Peptides IL and ILA bind to lipid vesicles composed of phosphatidylcholine and phosphatidylethanol amine: phosphatidyl glycerol: cardiolipin. The position of λmax and I- quenching experiments suggest that the trp residues are localized at the membrane interface and not associated with the hydrophobic core of the lipid bilayer in both the peptides. Hence, membrane permeabilization is likely to occur due to deformation of the membrane surface rather than formation of transmembrane channels by indolicidin and its analogues. Peptides ILA, IL and ILF cause the release of entrapped carboxyfluorescein from phosphatidyl choline vesicles. The peptide-lipid ratios indicate that ILF is less effective than IL and ILA in permeabilizing lipid vesicles, correlating with their haemolytic activities.

Self-assembly of short peptides composed of only aliphatic amino acids and a combination of aromatic and aliphatic amino acids

The morphology of structures formed by the self‐assembly of short N‐terminal t‐butyloxycarbonyl (Boc) and C‐terminal methyl ester (OMe) protected and Boc‐deprotected hydrophobic peptide esters was investigated. We have observed that Boc‐protected peptide esters composed of either only aliphatic hydrophobic amino acids or aliphatic hydrophobic amino acids in combination with aromatic amino acids, formed highly organized structures, when dried from methanol solutions. Transmission and scanning electron microscopic images of the peptides Boc‐Ile‐Ile‐OMe, Boc‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe and Boc‐Trp‐Ile‐Ile‐OMe showed nanotubular structures. Removal of the Boc group resulted in disruption of the ability to form tubular structures though spherical aggregates were formed. Both Boc‐Leu‐Ile‐Ile‐OMe and H‐Leu‐Ile‐Ile‐OMe formed only spherical nanostructures. Dynamic light scattering studies showed that aggregates of varying dimensions were present in solution suggesting that self‐assembly into ordered structures is facilitated by aggregation in solution. Fourier transform infrared spectroscopy and circular dichroism spectroscopy data show that although all four of the protected peptides adopt well‐defined tertiary structures, upon removal of the Boc group, only H‐Phe‐Phe‐Phe‐Ile‐Ile‐OMe had the ability to adopt β‐structure. Our results indicate that hydrophobic interaction is a very important determinant for self‐assembly and presence of charged and aromatic amino acids in a peptide is not necessary for self‐assembly. Copyright

Identification of the region that plays an important role in determining antibacterial activity of bovine seminalplasmin

Seminalplasmin (SPLN) is a 47‐residue protein isolated from bovine seminal plasma having potent antimicrobial activity against a broad spectrum of microorganisms. SPLN, also known as caltrin, acts as a calcium transport regulator in bovine sperms. Analysis of the sequence of SPLN reveals a 27‐residue stretch with the sequence SLSRYAKLANRLANPKLLETFLSKWIG more hydrophobic than the rest of the protein. It is demonstrated that a synthetic peptide corresponding to this 27‐residue segment has antimicrobial activity comparable to that of SPLN. It does not exhibit hemolytic activity at concentrations where antibacterial activity is observed. Since P27 can be conveniently obtained in large amounts by chemical synthesis, it could serve not only as a starting compound to obtain peptides with improved antibacterial activity but also to understand the role of SPLN in reproductive physiology.

Identification of a second membrane-active 13-residue peptide segment in the antimicrobial protein, bovine seminalplasmin

Seminalplasmin (SPLN) is a 47‐residue protein from bovine seminalplasma having broad‐spectrum antibacterial activity. The protein has no hemolytic activity. SPLN interacts with lipid vesicles and its antibacterial activity appears to stem from its ability to permeabilize the bacterial plasma membrane. Analysis of SPLNs primary structure, with respect to its relative hydrophobicity and hydrophilicity, revealed a segment, PKLLETFLSKWIG, more hydrophobic than the rest of the protein. A synthetic peptide corresponding to this region had not only antibacterial activity but also hemolytic properties. Analysis of the SPLN sequence based on hydrophobic moment plots has revealed a second segment, SLSRYAKLANRLA, which could be membrane active. A synthetic peptide corresponding to this region shows only antibacterial activity with no hemolytic activity.