期刊
BIOCHEMICAL JOURNAL
卷 401, 期 -, 页码 533-540出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20060668
关键词
CD spectroscopy; folding intermediate; prion protein (PrP); secondary structure; translational diffusion; T(2) relaxation
资金
- Biotechnology and Biological Sciences Research Council [BB/D005027/1] Funding Source: Medline
- Medical Research Council [MC_U117533887] Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [BB/D005027/1] Funding Source: researchfish
- Medical Research Council [MC_U117533887] Funding Source: researchfish
- BBSRC [BB/D005027/1] Funding Source: UKRI
- MRC [MC_U117533887] Funding Source: UKRI
Prion diseases are associated with the misfolding of the PrP (prion protein) from a largely a-helical isoform to a P-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 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 pH4 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.
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