PHOTOLABELING OF A PORE-FORMING TOXIN WITH THE HYDROPHOBIC PROBE 2-[H-3]DIAZOFLUORENE - IDENTIFICATION OF MEMBRANE-INSERTED SEGMENTS OF STAPHYLOCOCCUS-AUREUS ALPHA-TOXIN

The identification of membrane-inserted segments of pore-forming solub le proteins is crucial to understanding the action of these proteins a t the molecular level. A distinct member of this class of proteins is alpha-toxin, a 293-amino acid long 33-kDa hemolytic toxin secreted by Staphylococcus aureus that can form pores in both artificial and natur al membranes, We have studied the interaction of alpha-toxin with sing le bilayer vesicles prepared from asolectin using a hydrophobic photoa ctivable reagent, 2-[H-3]diazofluorene ([H-3]DAF) (Pradhan, D., and La la, A. K. (1987) J, Biol, Chem, 262, 8242-8251), This reagent readily partitions into the membrane hydrophobic core and on photolysis labels the lipid and protein segments that penetrate the membrane. Current m odels on the mode of action of alpha-toxin indicate that, on interacti on with membranes, alpha-toxin forms an oligomer, which represents the active pore. Ln keeping with these models, we observe that [H-3]DAF p hotolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide f ragmentation of [H-3]DAF-labeled alpha-toxin gave several fragments, w hich were subjected to Edman degradation, We could thus sequence resid ues 1-19, 35-60, 114-139, 198-231, and 235-258, Radioactive analysis a nd phenylthiohydantoin-derivative analysis during sequencing permitted analysis of DAF insertion sites, The results obtained indicated that the N and C termini (residues 235-258) have been extensively labeled. The putative pore-forming glycine-rich central hinge region was poorly labeled, indicating that the apposing side of the lumen of the pore d oes not form the lipid-protein interface, The DAF labeling pattern ind icated that the major structural motif in membrane-bound alpha-toxin w as largely beta-sheet.