Intramolecular disulfide bonds enhance the antimicrobial and lytic activities of protegrins at physiological sodium chloride concentrations.
Protegrins are 2-kDa antimicrobial peptides that contain 16-18 amino acid residues and two intramolecular disulfide bonds. We studied the contribution of these disulfide bonds to the bactericidal activity of protegrins in physiological concentrations of NaCl by comparing protegrin PG-1 with variants that lacked one or both cysteine disulfides. Whereas the bactericidal and liposome-lytic properties of protegrin PG-1 were enhanced by adding 100 mM NaCl to the phosphate-buffered medium, NaCl addition strongly inhibited the effects of its linearized, disulfide-free variant, [A6, A8, A13, A15]protegrin-1. Whereas protegrin PG-1 manifested beta-sheet structure by CD (circular dichroism) and ATR-FTIR (attenuated-total-reflectance-Fourier-transform-infrared) spectroscopy in buffer or membrane-mimetic environments, [A6, A8, A13, A15]protegrin-1 manifested disordered structure in phosphate buffer and alpha-helical characteristics in membrane-mimetic environments. Both single-disulfide protegrin variants, [A8, A13]protegrin-1 and [A6, A15]protegrin-1, assumed beta-sheet conformations with liposomes that simulated bacterial membranes, and both retained substantial bactericidal activity when 100 mM NaCl was present. These findings demonstrate that the intramolecular disulfide bonds of protegrins are required for their antiparallel beta-sheet conformation in membrane-mimetic environments and for their potent antimicrobial activity in media containing NaCl concentrations comparable to those found in serum and extracellular fluids.
Harwig
S S
SS
Department of Medicine, UCLA 90095-1690, USA.
Waring
A
A
Yang
H J
HJ
Cho
Y
Y
Tan
L
L
Lehrer
R I
RI
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D023181:Antimicrobial Cationic Peptides; D001419:Bacteria / Q000187:drug effects; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D017550:Spectroscopy, Fourier Transform Infrared
D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D005452:Fluoresceins; D008081:Liposomes; D011506:Proteins; C094880:protegrin-1; C007740:fluorexon