Mechanism and kinetics of delta-lysin interaction with phospholipid vesicles.
Delta-lysin is a 26 amino acid, hemolytic peptide toxin secreted by Staphylococcus aureus. It has been reported to form an amphipathic helix upon binding to lipid bilayers and is often cited as a typical example of the barrel-stave model for pore formation in lipid bilayer membranes. However, the exact mechanism by which it lyses cells and the physical basis of its target specificity are still unknown. Moreover, the evidence for delta-lysin insertion and pore formation in the membrane stems largely from theoretical modeling of the toxin and lacks experimental confirmation. We investigated binding and insertion of delta-lysin into phospholipid bilayer vesicles. The kinetics of these processes were studied by stopped-flow fluorescence with two types of experiments: (a) carboxyfluorescein release from the vesicles upon peptide-vesicle interaction, with concomitant relief of dye self-quenching; (b) fluorescence energy transfer from the intrinsic tryptophan of the peptide to a membrane-bound lipid probe. We formulated a detailed kinetic mechanism with explicit molecular rate constants for peptide binding, association, and insertion, obtaining a quantitative description of the experimental results. delta-Lysin insertion is strongly dependent on the peptide-to-lipid ratio, suggesting that association of a critical number of monomers on the membrane is required for activity. However, we found no evidence for a stable membrane-inserted pore. Rather, the peptide appears to cross the membrane rapidly and reversibly and cause release of the lipid vesicle contents in this process.
Pokorny
Antje
A
Department of Chemistry, University of North Carolina at Wilmington, Wilmington, North Carolina 28403, USA.
Birkbeck
T Harry
TH
Almeida
Paulo F F
PF
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D003374:Coumarins / Q000378:metabolism; D004735:Energy Transfer; D005452:Fluoresceins / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013211:Staphylococcus aureus / Q000737:chemistry; D014364:Tryptophan / Q000378:metabolism; D014867:Water
C102149:4-(N,N-dimethyl-N-(n-tetradecyl)ammoniummethyl)-7-(N,N-dimethylamino)coumarin chloride; D001426:Bacterial Proteins; D003374:Coumarins; D005452:Fluoresceins; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010743:Phospholipids; D012996:Solutions; D014867:Water; C024098:6-carboxyfluorescein; C025994:delta hemolysin protein, Staphylococcus aureus; D014364:Tryptophan; C028694:1-palmitoyl-2-oleoylphosphatidylcholine