The pH-dependent conformational change of diphtheria toxin.
Labeling by a hydrophobic photoactivatable reagent and limited proteolysis have been used to study conformational changes of diphtheria toxin related to its pH-dependent membrane insertion and translocation. TID (3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine) labels diphtheria toxin at pH 5 much more efficiently than at pH 7, both in the presence and absence of lipid vesicles. In the absence of membranes, the extent of labeling is greater and the pH dependence is stronger. As analyzed on sodium dodecyl sulfate-polyacrylamide gels and by high pressure liquid chromatography, both the A- and B-subunits and most of the cyanogen bromide fragments of the toxin are labeled by TID at acid pH. The products of trypsin cleavage of diphtheria toxin at pH 5 are different from those seen at neutral pH. Trypsin-susceptible sites were identified by gel electrophoresis of the trypsin fragments, combined with electrophoresis and high pressure liquid chromatography of CNBr digests of trypsin-treated toxin. At neutral pH, the main sites of digestion are at the junction between the A- and B-fragments and near the NH2 terminus of the A-fragment. At pH 5.2, these sites are less efficiently cut, and new sites appear near the NH2 terminus of the B-fragment, in an amphipathic portion of the sequence. Thus, even in the absence of membranes, acid pH induces a significant conformational change in diphtheria toxin. This change involves burial of some previously accessible sites, exposure of previously inaccessible sites, and the formation of hydrophobic regions over an extensive portion of the polypeptide chain.
Dumont
M E
ME
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06511.
Richards
F M
FM
D000345:Affinity Labels / Q000378:metabolism; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004167:Diphtheria Toxin; D006863:Hydrogen-Ion Concentration; D010446:Peptide Fragments; D010782:Photolysis; D011487:Protein Conformation; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D004167:Diphtheria Toxin; D010446:Peptide Fragments; D014357:Trypsin; D003488:Cyanogen Bromide