Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier-transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally-sensitive amide I band upon phosphorylation indicated an increase of the alpha-helical 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 were accompanied by an increase in the residual amide II absorbance band remaining upon H-D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non-phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability.
        
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Encinar JA, Fernandez A, Ferragut JA, Gonzalez-Ros JM, DasGupta BR, Montal M, Ferrer-Montiel A (1998) Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation FEBS Letters429: 78-82
Encinar JA, Fernandez A, Ferragut JA, Gonzalez-Ros JM, DasGupta BR, Montal M, Ferrer-Montiel A (1998) FEBS Letters429: 78-82