Conformational and membrane interaction studies of the antimicrobial peptide alyteserin-1c and its analogue [E4K]alyteserin-1c.

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH(2)), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria. The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu(2) and Val(21). Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys(3) and Val(21). Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala(19) do not interact with the micelles.

In vitro activities of synthetic host defense propeptides processed by neutrophil elastase against cystic fibrosis pathogens.

The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

Metabolic and structural properties of human obestatin {1-23} and two fragment peptides.

Obestatin is a peptide produced in the oxyntic mucosa of the stomach and co-localizes with ghrelin on the periphery of pancreatic islets. Several studies demonstrate that obestatin reduces food and water intake, decreases body weight gain, inhibits gastrointestinal motility, and modulates glucose-induced insulin secretion. In this study we evaluated the acute metabolic effects of human obestatin {1-23} and fragment peptides {1-10} or {11-23} in high-fat fed mice, and then investigated their solution structure by NMR spectroscopy and molecular modelling. Obestatins {1-23} and {11-23} significantly reduced food intake (86% and 90% respectively) and lowered glucose responses to feeding, whilst leaving insulin responses unchanged. No metabolic changes could be detected following the administration of obestatin {1-10}. In aqueous solution none of the obestatin peptides possessed secondary structural features. However, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure of obestatin {1-23} was characterized by an alpha-helix followed by a single turn helix conformation between residues Pro(4) and Gln(15) and His(19) and Ala(22) respectively. Obestatin {1-10} showed no structural components whereas {11-23} contained an alpha-helix between residues Val(14) and Ser(20) in a mixed solvent. These studies are the first to elucidate the structure of human obestatin and provide clear evidence that the observed alpha-helical structures are critical for in vivo activity. Future structure/function studies may facilitate the design of novel therapeutic agents based on the obestatin peptide structure.

Development of potent anti-infective agents from Silurana tropicalis: conformational analysis of the amphipathic, alpha-helical antimicrobial peptide XT-7 and its non-haemolytic analogue [G4K]XT-7.

Peptide XT-7 (GLLGP(5)LLKIA(10)AKVGS(15)NLL.NH(2)) is a cationic, leucine-rich peptide, first isolated from skin secretions of the frog, Silurana tropicalis (Pipidae). The peptide shows potent, broad-spectrum antimicrobial activity but its therapeutic potential is limited by haemolytic activity (LC(50)=140 microM). The analogue [G4K]XT-7, however, retains potent antimicrobial activity but is non-haemolytic (LC(50)>500 microM). In order to elucidate the molecular basis for this difference in properties, the three dimensional structures of XT-7 and the analogue have been investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, both peptides lack secondary structure. In a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O mixed solvent system, XT-7 is characterised by a right handed alpha-helical conformation between residues Leu(3) and Leu(17) whereas [G4K]XT-7 adopts a more restricted alpha-helical conformation between residues Leu(6) and Leu(17). A similar conformation for XT-7 in 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellular media was observed with a helical segment between Leu(3) and Leu(17). However, differences in side chain orientations restricting the hydrophilic residues to a smaller patch resulted in an increased hydrophobic surface relative to the conformation in TFE-H(2)O. Molecular modelling of the structures obtained in our study demonstrates the amphipathic character of the helical segments. It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly(4)-->Lys in XT-7 arises from a decrease in both helicity and hydrophobicity. These studies may facilitate the development of potent but non-toxic anti-infective agents based upon the structure of XT-7.

Conformational analysis of the broad-spectrum antibacterial peptide, ranatuerin-2CSa: identification of a full length helix-turn-helix motif.

Design of clinically valuable antibacterial agents based upon naturally occurring peptides requires the use of spectroscopic methods, particularly NMR, to determine the three-dimensional structure of the native peptide so that analogues with improved therapeutic properties can be made. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAG KLLETLKCKITGC), first isolated from skin secretions of the Cascades frog, Rana cascadae, represents a promising candidate for drug development. The peptide shows potent growth inhibitory activity against Escherichia coli (MIC=5 microM) and Staphylococcus aureus (MIC=10 microM) but displays haemolytic activity against human erythrocytes (LC(50)=160 microM). The solution structure of ranatuerin-2CSa was investigated by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide lacks secondary structure but, in a 2,2,2-trifluoroethanol (TFE-d(3))-H(2)O solvent mixture, the structure is characterised by a full length helix-turn-helix conformation between residues I(2)-L(21), L(22)-L(25) and K(26)-T(30) respectively. This structural information will facilitate the design of novel therapeutic agents based upon the ranatuerin-2CSa structure with improved antimicrobial potencies but decreased cytolytic activities against mammalian cells.