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

Publisher Website
Google Scholar
Abstract
Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely α-helical isoform to a β-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an α-helical protein into one that contains a high proportion of β-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. 15N-HSQC (heteronuclear single-quantum correlation) studies with mPrP (mouse PrP)-(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. Three-dimensional 15N-HSQC-TOCSY and NOESY spectra indicate that the observable residues are all assigned to amino acids in the N-terminus: residues 23–118. 15N transverse relaxation measurements indicate that these N-terminal residues are highly flexible with additional fast motions. These observations are confirmed via the use of truncated mPrP-(112–231), which shows only 16 15N-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.