title: Photolabeling of a pore-forming toxin with the hydrophobic probe 2-[3H]diazofluorene. Identification of membrane-inserted segments of Staphylococcus aureus alpha-toxin.
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.
authors.affiliation: Department of Chemistry, Indian Institute of Technology Bombay, Powai.
authors.forename: A K
authors.initials: AK
authors.lastname: Lala
authors.forename: S M
authors.initials: SM
authors.lastname: Raja
mesh_terms: D000345:Affinity Labels; D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D003979:Diazonium Compounds; D010450:Endopeptidases; D005449:Fluorenes; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000378:metabolism; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010713:Phosphatidylcholines; D010743:Phospholipids; D010782:Photolysis; D013211:Staphylococcus aureus; D014316:Tritium
chemical_list: D000345:Affinity Labels; D001427:Bacterial Toxins; D003979:Diazonium Compounds; D005449:Fluorenes; D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; C021091:staphylococcal alpha-toxin; D014316:Tritium; C024990:asolectin; C052770:2-diazofluorene; D010450:Endopeptidases; D003488:Cyanogen Bromide