title: Design of non-cysteine-containing antimicrobial beta-hairpins: structure-activity relationship studies with linear protegrin-1 analogues.
abstract: 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 beta-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 beta-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 beta-strand conformation, (ii) D-proline is placed at the i + 1 position of the reverse turn to promote a type II' beta-turn, and (iii) amino functionality is incorporated at the gamma-carbon of the D-proline residue to mimic the charge distribution of the natural beta-hairpin. Structural studies revealed that the antimicrobial potency of the non-disulfide-bonded peptides can be correlated to the stability of the beta-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 beta-hairpin conformational stability in the presence of 150 mM NaCl. Thus, salt inactivation is not due to destabilization of the beta-hairpin conformation. Furthermore, our results show that beta-sheet design principles can be used to replace conformation-stabilizing disulfide bridges with noncovalent conformation-stabilizing features.
authors.affiliation: Graduate Program in Biophysics and Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Lai
authors.forename: Bayard R
authors.initials: BR
authors.lastname: Huck
authors.forename: Bernard
authors.initials: B
authors.lastname: Weisblum
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Gellman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000138:chemical synthesis* / Q000302:isolation & purification; D023181:Antimicrobial Cationic Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification; D001412:Bacillus subtilis / Q000187:drug effects / Q000254:growth & development; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D016984:Enterococcus faecium / Q000187:drug effects / Q000254:growth & development; D004926:Escherichia coli / Q000187:drug effects / Q000254:growth & development; D008969:Molecular Sequence Data; D011487:Protein Conformation; D020033:Protein Isoforms / Q000138:chemical synthesis / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011506:Proteins / Q000138:chemical synthesis* / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000187:drug effects / Q000254:growth & development; D013329:Structure-Activity Relationship; D013552:Swine
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D020033:Protein Isoforms; D011506:Proteins; C094880:protegrin-1; D003545:Cysteine