A phosphorylation-induced major structural change in the N-terminal domain of the P protein of Chandipura virus.

It has previously been shown that phosphorylation of P protein of vesicular stomatitis virus as well as Chandipura (CHP) virus is required for transcription activation and replication switch. The structural nature of this crucial conformational change, however, is largely unknown. We have studied the phosphorylation-associated conformational change in the P protein of Chandipura (CHP) virus using chemical modification, fluorescence, and circular dichroism spectroscopy. Sulfhydryl groups of unphosphorylated CHP-P protein are unreactive to DTNB under nondenaturing conditions. Upon phosphorylation, one sulfhydryl group becomes reactive. We have identified this sulfhydryl group as cysteine 57. The two tryptophan residues (105 and 135) become significantly more buried in the phosphorylated protein. Circular dichroism spectra show significant enhancement in the far-UV region upon phosphorylation. Anisotropy decay of AEDANS-labeled C57 CHP-P protein shows rapid rotation of the probe, suggesting significant mobility of the N-terminal domain in the phosphorylated P protein. The results suggest a global conformational change in the N-terminal domain of the P protein is induced by phosphorylation and yet the phosphorylated N-terminal domain shows significant flexibility.