Scrapie amyloid (prion) protein has the conformational characteristics of an aggregated molten globule folding intermediate.
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)
Safar
J
J
Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
Roller
P P
PP
Gajdusek
D C
DC
Gibbs
C J
CJ
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D006224:Cricetinae; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006851:Hydrochloric Acid / Q000494:pharmacology; D008647:Mesocricetus; D011328:Prions / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D006146:Guanidines; D011328:Prions; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D019791:Guanidine; D006851:Hydrochloric Acid