STRUCTURAL STABILIZATION OF BOTULINUM NEUROTOXINS BY TYROSINE PHOSPHORYLATION

Tyrosine phosphorylation of botulinum neurotoxins augments their prote olytic activity and thermal stability, suggesting a substantial modifi cation of the global protein conformation. We used Fourier-transform i nfrared (FTIR) spectroscopy to study changes of secondary structure an d thermostability of tyrosine phosphorylated botulinum neurotoxins A ( BoNT A) and E (BoNT E), Changes in the conformationally-sensitive amid e I band upon phosphorylation indicated an increase of the alpha-helic al content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in beta-sheet content. These changes in secondary structure:e were accompanied by an increas e in the residual amide ii absorbance band remaining upon H-D exchange , consistent with a tighter packing of the phosphorylated proteins, FT IR and differential scanning calorimetry (DSC) analyses of the denatur ation process show that phosphorylated neurotoxins denature at tempera tures higher than those required by non-phosphorylated species, These findings indicate that tyrosine phosphorylation induced a transit-ion to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and t hermostability. (C) 1998 Federation of European Biochemical Societies.