Comparison of the conformation, hydrophobicity, and model membrane interactions of diphtheria toxin to those of formaldehyde-treated toxin (diphtheria toxoid): formaldehyde stabilization of the native conformation inhibits changes that allow membrane insertion.
Toxoids are inactivated protein toxins that are used in vaccines. The behavior of diphtheria toxin reacted with formaldehyde (diphtheria toxoid) was compared to that of diphtheria toxin in order to understand the nature of the changes that occur in toxoids upon protein reaction with formaldehyde. Despite the intramolecular cross-links in the toxoid, the conformations of the toxoid and the toxin were very similar in both the native and low pH-induced membrane-penetrating states as judged by fluorescence and hydrophobicity properties. However, the toxoid underwent thermal-, low-pH-, and guanidinium chloride-induced conformational changes only at more extreme conditions than needed to induce such changes in the toxin. This implies that formaldehyde modification stabilizes the native conformation relative to several conformations that involve different degrees of unfolding. The stabilization to conformational changes induced by low pH is particularly interesting because low pH induces partial unfolding of the toxin to a molten globule-like state. It was found that the toxoid only gained the ability to interact with model membrane vesicles at a lower pH than the toxin. Because low-pH-induced unfolding and membrane interaction are critical steps in the entry of diphtheria toxin into cells, the resistance of the toxoid to these changes may be linked to its lack of toxicity. The implications of these results for the construction of toxoids are discussed.
Paliwal
R
R
Department of Biochemistry and Cell Biology, State University of New York at Stony Brook 11794-5215, USA.
London
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E
D001665:Binding Sites; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism; D004168:Diphtheria Toxoid / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde / Q000737:chemistry*; D019791:Guanidine; D006146:Guanidines; D006358:Hot Temperature; D008567:Membranes, Artificial; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
D004167:Diphtheria Toxin; D004168:Diphtheria Toxoid; D006146:Guanidines; D008567:Membranes, Artificial; D005557:Formaldehyde; D019791:Guanidine