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 peptides that were protected from digestion were separated by gel electrophoresis, transferred to a membrane and their N-terminal sequences were determined. All peptides belong to the B fragment of DT and cover residues 194-223, 265-375 and 429-528. The secondary structures of the peptides inserted in the membrane, determined by Fourier-transformed infrared spectroscopy, were shown to be mostly alpha-helices and beta-sheets (44% and 53%, respectively). On the basis of these data and the recently published X-ray structure of DT, we are proposing a topology for the DTB fragment in the membrane.
Cabiaux
V
V
Laboratoire des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Quertenmont
P
P
Conrath
K
K
Brasseur
R
R
Capiau
C
C
Ruysschaert
J M
JM
D000595:Amino Acid Sequence; D003353:Corynebacterium diphtheriae / Q000737:chemistry*; D004167:Diphtheria Toxin / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010713:Phosphatidylcholines; D010743:Phospholipids / Q000737:chemistry; D011402:Pronase; D017433:Protein Structure, Secondary; D017421:Sequence Analysis; D017550:Spectroscopy, Fourier Transform Infrared
D004167:Diphtheria Toxin; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; C039171:diphtheria toxin fragment B; C024990:asolectin; D011402:Pronase