Dynamics and orientation of amphipathic peptides in solution and bound to membranes: a steady-state and time-resolved fluorescence study of staphylococcal delta-toxin and its synthetic analogues

The environment of both the hydrophilic and hydrophobic sides of alpha -hel ical delta -toxin are probed by tryptophanyl (Trp) fluorescence, when self- association occurs in solution and on binding to membranes. The fluorescenc e parameters of staphylococcal delta -toxin (Trp15 on the polar side of the amphipathic helix) and synthetic analogues with single Trp at position 5 o r 16 (on the apolar side) were studied. The time-resolved fluorescence deca ys of the peptides in solution show that the local environment of their sin gle Trp is always heterogeneous. Although the self-association degree incre ases with concentration, as shown by fluorescence anisotropy decays, the li fetimes (and their statistical weight) of Trp16 do not change, contrary to what is observed for Trp15. The first step of self-association is then driv en by hydrophobic interactions between apolar sides of alpha -helices, whil st further oligomerization involves their polar side (Trp15) via electrosta tic interactions. This is supported by dissociation induced by salt. For al l self-associated peptides, the polarity of the Trp microenvironment was no t significantly modified upon binding to phospholipid vesicles, as indicate d by the small shifts of the fluorescence emission spectra and lifetime val ues. However, the relative populations of the lifetime classes vary with bo und-peptide density similar to the rates of their global motions in bilayer s or smaller particles. Quenching experiments by water or lipid-soluble com pounds show changes of the orientation of membrane-inserted peptides, from probably dimers lying flat at the interface at low peptide density, to olig omers spanning the membrane and inducing membrane fragmentation at high pep tide density.