Circular dichroic and fluroescence spectroscopic study of the conformation of botulinum neurotoxin types A and E

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Abstract
Botulinum neurotoxin (NT) is synthesized byClostridium botulinum in any of seven antigenically distinct forms, called types A through G. Protease(s) endogenous to the bacteria, or trypsin, nicks the single chain protein to a dichain molecule which generally is more toxic. The conformation of dichain type A (nicked by endogenous protease), single chain type E, and dichain type E NT (nicked by trypsin) have been determined using circular dichroism (CD) and fluorescence spectroscopy. The high degree of ordered secondary structure (α helix 28%, β sheet 42%, total 70%) found in type A NT at pH 6.0 was similar to that found at pH 9.0 (α 22%, β 47%, total 69%). The secondary structure of the single chain type E NT at pH 6.0 (α 18%, β 37%, total 55%) differed somewhat from these values at pH 9.0 (α 22%, β 43%, total 65%). The dichain type E NT at pH 6.0 assumed a secondary structure (α 20%, β 47%, total 67%) more similar to that of dichain type A than the single chain type E NT. Examination with the fluorogenic probe toluidine napthalene sulfonate revealed that the hydrophobicity of the type A and E NTs were higher at pH 9.0 than at pH 6.0. Also, the hydrophobicity of the dichain type E NT was higher than its precursor the single chain protein and appeared similar to that of the dichain type A NT. The CD and fluorescence studies indicate that conversion of the single chain type E NT to the dichain form (i.e. nicking by trypsin) induced changes in conformation. The ordered secondary structure (a + β contents) of botulinum NT, 70% for type A and 67% for dichain type E, agree well with 65% of α + β contents of tetanus toxin [21] that is produced byClostridium tetani.