Protegrins are short, cationic peptides that display potent, broad-spectrum antimicrobial activity. PG-1, the first of the five natural analogues discovered, forms a rigid antiparallel two-stranded β-sheet that is stabilized by two disulfide bonds. The two strands of the sheet are linked by a short two-residue loop segment. Removal of the disulfide bridges (e.g., in Cys → Ala analogues) is known to cause marked loss of antimicrobial activity. We have used basic principles of β-hairpin design to develop linear analogues of PG-1 that lack cysteine but nevertheless display PG-1-like activity. Our most potent reengineered molecules contain three essential design features: (i) the four cysteine residues of PG-1 are replaced by residues that have high propensity for β-strand conformation, (ii) D-proline is placed at the i + 1 position of the reverse turn to promote a type II′ β-turn, and (iii) amino functionality is incorporated at the γ-carbon of the D-proline residue to mimic the charge distribution of the natural β-hairpin. Structural studies revealed that the antimicrobial potency of the non-disulfide-bonded peptides can be correlated to the stability of the β-hairpin conformations they adopt in aqueous solution. The presence of 150 mM NaCl was found to have little effect on the antimicrobial activity of PG-1, but one of our linear analogues loses some potency under these high salt conditions. Despite this discrepancy in salt sensitivity, NMR and CD data indicate that neither PG-1 nor our linear analogue experiences a significant decrease in β-hairpin conformational stability in the presence of 150 mM NaCl. Thus, salt inactivation is not due to destabilization of the β-hairpin conformation. Furthermore, our results show that β-sheet design principles can be used to replace conformation-stabilizing disulfide bridges with noncovalent conformation-stabilizing features.
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