Determination of disulphide bridges in PG-2, an antimicrobial peptide from porcine leukocytes.
We determined the cysteine connectivity of protegrin PG-2, a leukocyte-derived antimicrobial peptide, by performing sequential enzyme digestions with chymotrypsin and thermolysin, and monitoring each digest by direct liquid chromatography-electrospray mass spectrometric analysis. This approach resolved the disulphide pairing pattern unambiguously with only picomolar amounts of PG-2. The inferred cysteine connectivity was confirmed by traditional amino acid composition analyses using nanomolar amounts of the protegrin. The results suggest that protegrins will assume a tachyplesin-like, disulphide-stabilized anti-parallel beta-sheet configuration in solution.
Harwig
S S
SS
Department of Medicine, University of California, Los Angeles 90095, USA.
Swiderek
K M
KM
Lee
T D
TD
Lehrer
R I
RI
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000890:Anti-Infective Agents / Q000737:chemistry* / Q000302:isolation & purification; D023181:Antimicrobial Cationic Peptides; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D007962:Leukocytes / Q000166:cytology; D013058:Mass Spectrometry; D015394:Molecular Structure; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012996:Solutions; D013552:Swine; D013820:Thermolysin
D000596:Amino Acids; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D004220:Disulfides; D011506:Proteins; D012996:Solutions; C084516:protegrin-2; D002918:Chymotrypsin; D013820:Thermolysin; D003545:Cysteine