Secondary structure of diphtheria toxin and its fragments interacting with acidic liposomes studied by polarized infrared spectroscopy.
We used infrared attenuated total reflection spectroscopy to study the structure of diphtheria toxin (DT) and its fragments A, B, CB1, and CB4 as a function of the pH in the absence and in the presence of phospholipid vesicles. Binding of DT to asolectin or DL-alpha-dipalmitoylphosphatidylcholine-DL-alpha-dipalmitoylphosphatidic acid liposomes at pH 7.3 results in a 10% increase in its alpha-helix content. At pH 4, in the presence of liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds which did not exist before pH lowering. DT fragment B displays little conformational change upon pH lowering in the presence of liposomes. However, the alpha-helix content of CB1 increases by 10%, and polarization measurements indicate that the alpha-helices of CB1 at pH 4 are oriented parallel to the lipid acyl chains. On the other hand, the alpha-helix content of CB4 decreases dramatically while the low frequency beta-sheet content increases. Dichroism measurements demonstrate that this sheet lies close to a parallel to the bilayer surface. The fragment A of DT experiences a large conformational change upon pH lowering and binds to the liposome membrane even in the absence of DT fragment B. The conformational modification of DT fragment A is fully reversed when pH is brought back to 7.3.
Cabiaux
V
V
Laboratoire de Chimie Physique des Macromolécules aux Interfaces, Université Libre de Bruxelles, Belgium.
Brasseur
R
R
Wattiez
R
R
Falmagne
P
P
Ruysschaert
J M
JM
Goormaghtigh
E
E
D015060:1,2-Dipalmitoylphosphatidylcholine; D000577:Amides; D004167:Diphtheria Toxin; D005583:Fourier Analysis; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D010446:Peptide Fragments; D010712:Phosphatidic Acids; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared
D000577:Amides; D004167:Diphtheria Toxin; D008081:Liposomes; D010446:Peptide Fragments; D010712:Phosphatidic Acids; D015060:1,2-Dipalmitoylphosphatidylcholine