The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.

Biophysical techniques such as size-exclusion chromatography, sedimentation equilibrium analytical ultracentrifugation, and non-denaturing gel electrophoresis are the classical methods for determining the selfassociation of molecules into dimers, trimers, or other higher order species. However, these techniques usually require high (mg/ml) loading concentrations to detect self-association and also possess a lower size limit that is dependent on the ability of the technique to resolve monomeric from higher order species. Here we describe a novel, sensitive method with no upper or lower molecular size limits that indicates self-association of molecules driven together by the hydrophobic effect under aqueous conditions. "Temperature profiling in reversed-phase chromatography" analyzes the retention behavior of a sample over the temperature range of 5-80°C during gradient elution reversed-phase highperformance liquid chromatography. Because this technique greatly increases the effective concentration of analyte upon adsorption to the column, it is extremely sensitive, requiring very small sample quantities (microgram or less). In contrast, the classical techniques mentioned above decrease the effective analyte concentration during analysis, decreasing sensitivity by requiring larger amounts of analyte to detect molecular self-association. We demonstrate the utility of this technique with 14-residue cyclic and linear cationic peptides (<2000 Da) based on the sequence of the de novo-designed cytolytic peptide, GS14. The only requirements for the analyte molecule when using this technique are its ability to be retained on the reversed-phase column and to be subsequently removed from the column during gradient elution.The detection of molecular self-association and aggregation is an important concern for molecules intended for biological applications, such as proteins, peptides, and small organic drug molecules. A plethora of biophysical techniques already exist for the detection of molecular self-association in aqueous solution, including spectroscopic (NMR, CD, Fourier-transform infrared spectroscopy (FTIR), fluorescence, light scattering), chromatographic (affinity, size-exclusion (SEC) 1 /gel filtration), and other techniques (matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), non-denaturing PAGE, sedimentation equilibrium analytical ultracentrifugation). However, some of the classical techniques commonly used for measuring self-association (sedimentation equilibrium analytical ultracentrifugation, non-denaturing PAGE, and size-exclusion chromatography) possess a lower limit to the molecular size that can be clearly resolved, such that fewer methods exist for detecting self-association in smaller molecules. With a view to solving this problem, we describe here a novel method for measuring self-association, referred to as "temperature profiling in reversed-phase chromatography," based on observation of the conformation-dependent response of peptides to RP-HPLC under changing temperature.Much of...

In the present study we have utilized the structural framework of the analog GS14K4 (cyclo(VKLd-KVd-YPL KVKLd-YP, where d denotes a d-amino acid)), to examine the role of hydrophobicity in microbial activity and specificity. The hydrophobicity of GS14K4 was systematically altered by residue replacements in the hydrophobic sites of the molecule to produce a series of analogs that were either less or more hydrophobic than the parent compound. Circular dichroism spectroscopy and reversed-phase high performance liquid chromatography analysis showed that the molecules were structurally similar and only differed in overall hydrophobicity. The hydrophobicity of GS14K4 was found to be the midpoint for hemolytic activity, with more hydrophobic analogs exhibiting increased hemolytic activity and less hydrophobic analogs showing decreased hemolytic activity. For antimicrobial activity there were differences between the hydrophobicity requirements against Gram-positive and Gram-negative microorganisms. The hydrophobicity of GS14K4 was sufficient for maximum activity against Gram-negative microorganisms and yeast, with no further increases in activity occurring with increasing hydrophobicity. With Gram-positive microorganisms significant increases in activity with increasing hydrophobicity were seen in three of the six microorganisms tested. A therapeutic index (calculated as a measure of specificity of the peptides for the microorganisms over human erythrocytes) served to define the boundaries of a therapeutic window within which lay the optimum peptide hydrophobicity for each microorganism. The therapeutic window was found to be at a lower hydrophobicity level for Gram-negative microorganisms than for Gram-positive microorganisms, although the limits were more variable for the latter. Our results show that the balance between activity and specificity in the present cyclic peptides can be optimized for each microorganism by systematic modulation of hydrophobicity.