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Fluorescence resonance energy transfer studies on anthrax lethal toxin
Author(s) -
Croney John C.,
Cunningham Kristina M.,
Collier R.John,
Jameson David M.
Publication year - 2003
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)00870-6
Subject(s) - förster resonance energy transfer , anthrax toxin , cysteine , alexa fluor , chemistry , residue (chemistry) , mutagenesis , toxin , fluorescence , covalent bond , biophysics , maleimide , biochemistry , stereochemistry , fusion protein , biology , mutation , enzyme , polymer chemistry , recombinant dna , organic chemistry , physics , quantum mechanics , gene
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.