Design of Non-Cysteine-Containing Antimicrobial β-Hairpins:  Structure−Activity Relationship Studies with Linear Protegrin-1 Analogues

• Published in: Biochemistry (Easton) Vol. 41; no. 42; pp. 12835 - 12842
• Main Authors: Lai, Jonathan R, Huck, Bayard R, Weisblum, Bernard, Gellman, Samuel H
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 22.10.2002
• Subjects: Amino Acid Sequence; Animals; Anti-Bacterial Agents - chemical synthesis; Anti-Bacterial Agents - isolation & purification; Antimicrobial Cationic Peptides - chemical synthesis; Antimicrobial Cationic Peptides - isolation & purification; Bacillus subtilis - drug effects; Bacillus subtilis - growth & development; Circular Dichroism; Cysteine - chemistry; Disulfides - chemistry; Enterococcus faecium - drug effects; Enterococcus faecium - growth & development; Escherichia coli - drug effects; Escherichia coli - growth & development; Molecular Sequence Data; Protein Conformation; Protein Isoforms - chemical synthesis; Protein Isoforms - isolation & purification; Protein Structure, Secondary; Proteins - chemical synthesis; Proteins - isolation & purification; Staphylococcus aureus - drug effects; Staphylococcus aureus - growth & development; Structure-Activity Relationship; Swine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi026127d

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.