Effect of low pH on the conformation of Pseudomonas exotoxin A.
Previously we examined factors involved in the entry mechanism of Pseudomonas exotoxin A (PTx) at the level of lipid-protein interactions (Farahbakhsh, Z. T., Baldwin, R. L., and Wisnieski, B. J. (1986) J. Biol. Chem. 261, 11404-11408). Exposure to a low pH environment appears to be an obligatory trigger of the entry pathway. In this report we describe the effect of pH upon the conformation of PTx. We have found that the intrinsic fluorescence of PTx is strongly dependent on pH, decreasing between pH 7.4 and 4.0 with a red shift in the emission lambda max. The changes are reversible and associated with the acquisition of a binding site for the fluorescent dye 1-anilino-8-naphthalenesulfonic acid (ANS). The fluorescence intensity of ANS in the presence of PTx increases with decreasing pH and is accompanied by a blue shift in emission spectra, indicative of exposure of hydrophobic surfaces. These changes are also reversible. Both the intrinsic fluorescence and ANS binding profiles show a dramatic dependence on pH, with the transitions centered on pH 5.0 and 4.5, respectively. Circular dichroism studies reveal a 9% decrease in alpha-helicity between pH 7.7 and 4. The susceptibility of toxin to trypsin cleavage is also a function of pH, increasing with decreasing pH. The pH 7.4 cleavage profile is regained when the acid-treated samples are brought back to pH 7.4. The conformational changes observed in these pH shift experiments are likely to be physiologically significant because the conditions closely resemble those that the toxin would encounter if entry into the cytoplasm of a cell involves escape from an endosomal compartment.
D036002:ADP Ribose Transferases; D000817:Anilino Naphthalenesulfonates; D001427:Bacterial Toxins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005098:Exotoxins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000817:Anilino Naphthalenesulfonates; D001427:Bacterial Toxins; D005098:Exotoxins; D014357:Trypsin; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C027132:1-anilino-8-naphthalenesulfonate