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Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study
Author(s) -
Quertenmont Pierre,
Wattiez Ruddy,
Falmagne Paul,
Ruysschaert JeanMarie,
Cabiaux Veronique
Publication year - 1996
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1996.851446.x
Subject(s) - biology , proteolysis , diphtheria toxin , vesicle , membrane , toxin , microbiology and biotechnology , topology (electrical circuits) , computational biology , biochemistry , enzyme , mathematics , combinatorics
Summary The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH 5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non‐specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier‐transform infrared spectroscopy (ATR‐FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu‐C), led us to propose a topological model of the N‐terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two a‐helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third o‐helix could adopt a transmembrane topology only in a small proportion of DT molecules.