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

• Published in: Biochemical journal 2007-01, Vol.401 (2), p.533-540
• Main Authors: O'Sullivan, Denis B D, Jones, Christopher E, Abdelraheim, Salama R, Thompsett, Andrew R, Brazier, Marcus W, Toms, Harold, Brown, David R, Viles, John H
• Format: Article
• Language: English
• Subjects: Circular Dichroism; Hydrogen-Ion Concentration; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments - chemistry; Prions - chemistry; Protein Folding; Protein Structure, Secondary
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20060668

Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely alpha-helical isoform to a beta-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an alpha-helical protein into one that contains a high proportion of beta-sheet-like conformation. In the present study, we characterize this soluble pH 4 folding intermediate using NMR. (15)N-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 (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 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 (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.