Resolution of sub-nanosecond motions in botulinum neurotoxin endopeptidase: An evidence of internal flexibility

• BoNT/A endopeptidase is holistically a flexible protein structure.
• Protein structure is readily modulated by factors like temperature and viscosity.
• Flexibility in internal peptide segments plays a role in BoNT/A biological function.
• BoNT/A protein dynamics may be critical for the design of inhibitors against botulism.

Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind, which act on the peripheral nervous system leading to flaccid paralysis. Although co-crystal structure of BoNT/A light chain (LC) reveals some unique features of the biological function of this molecule, structural characteristics in solution reveal its dynamic features, not available through the published crystal structures. In this study, we have examined internal flexibility of this molecule by measuring rotational correlation time as a function of viscosity, using frequency domain fluorescence anisotropy decay technique. Fluorescence anisotropy decay of BoNT/A LC resolved sub-nanosecond local motion (faster component), interpreted as internal flexibility of the molecule was affected significantly with viscosity. Both local and global movements were affected by viscosity, which indicates the accessibility of protein core and flexibility of overall structure. In conclusion, this work demonstrates the presence of flexibility in the internal peptide segments, which appears to play a significant role in BoNT/A LC biological function.