Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186.
Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully and rapidly reactive with several sulfhydryl-specific reagents, indicating superficial location. Coupling of SH-groups with fluorescein-maleimide or biotin-maleimide was tolerated without loss of hemolytic activity at position 130, and the formed hexamers were visible on target cells by fluorescence microscopy and could be detected on electroblots by reaction with streptavidin-peroxidase. At the two other positions, modification caused significant loss of activity. However, the labeled proteins still bound to red cells, as shown by fluorescence microscopy and electroblotting. Intrinsically labeled alpha-toxin represents a novel tool to study the interaction of this pore-former with target membranes.
Institute of Medical Microbiology, University of Mainz, Germany.
M
M
Palmer
R
R
Jursch
U
U
Weller
A
A
Valeva
K
K
Hilgert
M
M
Kehoe
S
S
Bhakdi
D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D011994:Recombinant Proteins; C021091:staphylococcal alpha-toxin; D004228:Dithionitrobenzoic Acid; D003545:Cysteine