NMR characterization of the pH 4 {beta}-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility

Denis Bd O'Sullivan , Christopher E Jones , Salama R Abdelraheim , Andrew R Thompsett , Marcus W Brazier , Harold Toms , David R Brown , John H Viles (1)

1 Biological and Chemical Sciences

Prion diseases are associated with the misfolding of the prion protein (PrP) from a largely {alpha}-helical isoform to a {beta}-sheet rich oligomer. Circular dichroism (CD) has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an {alpha}-helical protein to one that contains a high proportion of {beta}-sheet like conformation. Here we characterise this soluble pH 4 folding intermediate using NMR. 15}N HSQC studies with mPrP(23-231) show that a total of ~150 dispersed amide signals are resolved in the native form, whereas only ~ 65 amide signals with little chemical shift dispersion are observable in the pH 4 form. 3D 15}N HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus; residues (23-118). 15}N transverse relaxation measurements indicate these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP(113-231), which shows only ~sixteen 15}N HSQC amide peaks at pH 4. The loss of signals from the C-terminus can be attributed to line-broadening due to an increase in the molecular size of the oligomer or exchange broadening in a molten globule state.

Mots clés: Life Sciences

Denis Bd O'Sullivan, Christopher E Jones, Salama R Abdelraheim, Andrew R Thompsett, Marcus W Brazier, et al.. NMR characterization of the pH 4 {beta}-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility. Biochemical Journal, 2006, 401 (2), pp.533-540. ⟨10.1042/BJ20060668⟩. ⟨hal-00478589⟩