Biochem. J. (2007) 401
(533–540) (Printed in Great Britain)
NMR characterization of the pH 4 b-intermediate of the prion protein: the N-terminal half of the protein remains unstructured and retains a high degree of flexibility
Denis B. D. O'SULLIVAN*, Christopher E. JONES*1, Salama R. ABDELRAHEIM†, Andrew R. THOMPSETT†, Marcus W. BRAZIER†, Harold TOMS*, David R. BROWN† and John H. VILES*2
*School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS, U.K., and †Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U.K.
Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely a-helical isoform to a b-sheet-rich oligomer. CD has shown that lowering the pH to 4 under mildly denaturing conditions causes recombinant PrP to convert from an a-helical protein into one that contains a high proportion of b-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.
Key words: CD spectroscopy, folding intermediate, prion protein (PrP), secondary structure, translational diffusion, T2 relaxation.
Abbreviations used: CJD, Creutzfeldt–Jacob disease; HSQC, heteronuclear single-quantum correlation; NOE, nuclear Overhauser effect; PrP, prion protein; huPrP, human PrP; mPrP, mouse PrP; PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; STE, stimulated echo.
1Present address: Center for Metals in Biology, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.
2To whom correspondence should be addressed (email j.viles@qmul.ac.uk).
Received 5 May 2006/17 August 2006; accepted 8 September 2006
Published as BJ Immediate Publication 8 September 2006, doi:10.1042/BJ20060668
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