Fluorescence resonance energy transfer studies on anthrax lethal toxin.
Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA(63) heptamer and for one residue (E126) on LF(N), the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF(N) was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF(N), whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF(N) binds near the top of the ring-shaped (PA(63))(7) heptamer.
Croney
John C
JC
Department of Cell and Molecular Biology, Room A209, John A. Burns School of Medicine, University of Hawaii at Manoa, 1960 East-West Rd., Honolulu, HI 96822, USA.
Cunningham
Kristina M
KM
Collier
R John
RJ
Jameson
David M
DM
D000818:Animals; D000942:Antigens, Bacterial; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D006224:Cricetinae; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006834:Hydrazines / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009282:Naphthalenesulfonates / Q000737:chemistry; D011487:Protein Conformation
C417561:Alexa 488 hydrazide; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D005456:Fluorescent Dyes; D006834:Hydrazines; D009282:Naphthalenesulfonates; C030325:anthrax toxin