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Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding
J Safar1, P P Roller, D C Gajdusek
1Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Abstract:
The scrapie amyloid (prion) protein (PrP27-30) is a host-derived component of the infectious scrapie agent; the potential to replicate, propagate, and form amyloid is a result of the posttranslational event or conformational abnormality. In low concentrations of guanidine hydrochloride (Gdn.HCl), PrP27-30 dissociates into a compact equilibrium intermediate with a substantial portion of secondary structure, partially denatured tertiary structure, and tryptophan residues in an apolar environment [Safar, J., Roller, P. P., Gajdusek, D. C., & Gibbs, C. J., Jr. (1993) J. Biol. Chem. 27, 20276-20284]. Here we describe the characteristics of this metastable form as monitored by 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectroscopy and circular dichroism (CD) spectroscopy, and we propose a mechanism for scrapie amyloid association. The Gdn.HCl-induced equilibrium intermediate of PrP27-30 had multiple high-affinity hydrophobic binding sites for ANS, some close to the Trp residues. The amide CD spectrum of an acid-induced intermediate (A-form), in equilibrium at pH < 2.0, was similar to the Gdn.HCl-induced intermediate and suggested the presence of a significant portion of an alpha-helical or beta-turn secondary structure. In contrast, the PrP27-30 associated into aggregates in an all beta-sheet conformation with less ordered and more exposed hydrophobic side chains. The noncooperative unfolding of the Gdn.HCl-induced intermediate at high temperature was irreversible and correlated with the loss of infectivity. The results demonstrate that PrP27-30 associates through a compact, metastable hydrophobic intermediate with an nonnative, nondenatured secondary structure and a tertiary structure close to the unfolded form.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Scrapie amyloid protein (PrP27-30) forms a compact, metastable intermediate with nonnative structure. This intermediate is crucial for amyloid formation and its unfolding irreversibly correlates with loss of infectivity.
Area of Science:
- Biochemistry
- Neuroscience
- Structural Biology
Background:
- Scrapie amyloid protein (PrP27-30) is a host-derived component of the infectious scrapie agent.
- Amyloid formation is linked to posttranslational events or conformational abnormalities.
Purpose of the Study:
- To characterize the metastable intermediate of PrP27-30.
- To propose a mechanism for scrapie amyloid association.
Main Methods:
- 8-anilino-1-naphthalenesulfonate (ANS) fluorescence spectroscopy.
- Circular dichroism (CD) spectroscopy.
- Guanidine hydrochloride (Gdn.HCl) denaturation.
Main Results:
- Gdn.HCl-induced intermediate exhibits high-affinity hydrophobic binding sites for ANS.
- Acid-induced intermediate (A-form) shares structural similarities with the Gdn.HCl intermediate, suggesting alpha-helical or beta-turn structures.
- PrP27-30 aggregates into beta-sheet conformations with exposed hydrophobic chains.
- Irreversible unfolding of the intermediate at high temperatures correlates with loss of infectivity.
Conclusions:
- PrP27-30 associates via a compact, metastable hydrophobic intermediate.
- This intermediate possesses nonnative secondary and near-unfolded tertiary structures.
- The findings provide insights into the mechanism of scrapie amyloid formation.