Translocation of the catalytic domain of diphtheria toxin across the endosomal membrane to the cytosolic compartment depends on low-pH-triggered insertion of the toxin's T (transmembrane) domain into the membrane. The T domain, consisting of nine α-helices arranged in three layers, was cloned and expressed as a discrete protein in Escherichia coli, and mutant forms were prepared and characterized. To investigate the relative movements of the three layers under various conditions, we generated two mutant forms of the domain, each containing an artificial intramolecular disulfide bridge linking the buried apolar hairpin (TH8–TH9) to one of the other two layers. Both disulfides inhibited exposure of the domain's apolar regions in solution at low pH, as determined by 2-p-toluidinylnaphthalene-6-sulfonate binding, and blocked its ability to form channels in artificial bilayers. Reduction of the bridges abolished these effects. Reduced forms of the mutant proteins were reacted with pyrenylmaleimide, a fluorescent probe, to monitor separation of the layers. Strong excimer bands seen in both mutants at neutral pH were undiminished at pH 5, indicating the retention of gross conformation in solution under acidic conditions. The addition of phospholipid vesicles at pH 5, but not at pH 7.5, quenched excimer fluorescence, reflecting the physical separation of the TH8-—TH9 hairpin from the other layers upon the T domain's interaction with the bilayer. The results indicate that (i) the conformation of the isolated T domain closely resembles that seen in the whole toxin, (ii) the TH8—TH9 hairpin separates from both of the other layers of the domain as an essential step of membrane insertion, and (iii) this separation is triggered by contact of the domain with the membrane under acidic conditions.
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Dynamic Transitions of the Transmembrane Domain of Diphtheria Toxin : Disulfide Trapping and Fluorescence Proximity Studies. / Zhan, Hangjun; Collier, R. John; Choe, Senyon; Eisenberg, David; Huynh, Paul D.; Finkelstein, Alan.In: Biochemistry, Vol. 33, No. 37, 01.09.1994, p. 11254-11263.
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