Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration.
The pH-triggered change in diphtheria toxin conformation and the physical properties of the toxin above and below the transition pH have been examined. Exposure to low pH (less than or equal to 5 at 23 degrees C, less than or equal to 5.3 at 37 degrees C) triggers a rapid (t1/2 less than 30 s) change in toxin conformation; the transition occurs over a narrow pH range (0.2 unit). Below the transition pH, buried tryptophans become exposed, and the toxin becomes hydrophobic, binding very tightly to detergent. Aggregation is observed at low pH, probably due to this extreme hydrophobicity. Circular dichroism and fluorescence properties show that the low-pH conformation is not extensively unfolded. Therefore, the toxin "opens" at low pH without becoming a random coil. The conformation change is partly irreversible, and the degree of irreversibility parallels the degree of aggregation. Reduction of the disulfide bonds does not increase hydrophobicity at neutral pH. Furthermore, none of the structural variants of toxin (monomer or dimer, bound to ApUp or free, and nicked between subunits or intact) are hydrophobic at neutral pH or differ in transition pH markedly. Therefore, these factors do not mimic the effect of low pH. These observations are consistent with a functional role for the pH-triggered changes during penetration of the membranes of acidic organelles. The toxin may have adapted a conformational change similar to partial denaturation for a critical role in function. The possible nature of the pH-sensitive interactions and the effects of aggregation are discussed briefly.
D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D004167:Diphtheria Toxin / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D004167:Diphtheria Toxin; D046911:Macromolecular Substances; D014364:Tryptophan; D004229:Dithiothreitol