TOPOLOGY OF DIPHTHERIA-TOXIN B FRAGMENT INSERTED IN LIPID VESICLES

Diphtheria toxin (DT) is a bacterial protein that crosses the membrane of endosomes of target cells in response to the low endosomal pH. In this paper, we have inserted diphtheria toxin in asolectin vesicles at pH 5.0 and treated the reconstituted system with pronase. The peptide s that were protected from digestion were separated by gel electrophor esis, transferred to a membrane and their N-terminal sequences were de termined. All peptides belong to the B fragment of DT and cover residu es 194-223, 265-375 and 429-528. The secondary structures of the pepti des inserted in the membrane, determined by Fourier-transformed infrar ed spectroscopy, were shown to be mostly alpha-helices and beta-sheets (44% and 53%, respectively). On the basis of these data and the recen tly published X-ray structure of DT, we are proposing a topology for t he DTB fragment in the membrane.