Abstract
The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at 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 natural membranes. We have studied the interaction of alpha-toxin with single bilayer vesicles prepared from asolectin using a hydrophobic photoactivable reagent, 2-[3H]diazofluorene ([3H]DAF) (Pradhan, D., and Lala, 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 models on the mode of action of alpha-toxin indicate that, on interaction with membranes, alpha-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound alpha-toxin oligomer. Cyanogen bromide fragmentation of [3H]DAF-labeled alpha-toxin gave several fragments, which were subjected to Edman degradation. We could thus sequence residues 1-19, 35-60, 114-139, 198-231, and 235-258. Radioactive analysis and 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 does not form the lipid-protein interface. The DAF labeling pattern indicated that the major structural motif in membrane-bound alpha-toxin was largely beta-sheet.
Highlights
The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level
A-Toxin is a soluble hemolytic protein toxin secreted by Staphylococcus aureus as a soluble 33-kDa monomer and a major factor contributing to the pathogenicity ofS. aureus
Hydrophobic photolabeling using apolar reagents can be used to establish the insertion of protein toxins in membranes and to identify protein segments that interact with the membrane hydrophobic core [24]
Summary
The identification of membrane-inserted segments of pore-forming soluble proteins is crucial to understanding the action of these proteins at the molecular level. Current models on the mode of action of a-toxin indicate that, on interaction with membranes, a-toxin forms an oligomer, which represents the active pore. In keeping with these models, we observe that [3H]DAF photolabels the membrane-bound a-toxin oligomer. More recent mutation studies suggest the involvement ofthe glycine-rich loop region in lining the membrane pore [19, 20]. While these studies have certainly helped in understanding the mode of action of a-toxin, we are still far from understanding it at the molecular level. This approach involves the use of hydrophobic or ampiphilic photoactivable reagents ethyllglycine; PVDF, polyvinylidene difluoride; PTH, phenylthiohydantoin
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