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.
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.
O'Sullivan
Denis B D
DB
School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, UK.
Jones
Christopher E
CE
Abdelraheim
Salama R
SR
Thompsett
Andrew R
AR
Brazier
Marcus W
MW
Toms
Harold
H
Brown
David R
DR
Viles
John H
JH
D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry; D011328:Prions / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary
D010446:Peptide Fragments; D011328:Prions