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)
Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
J
J
Safar
P P
PP
Roller
D C
DC
Gajdusek
C J
CJ
Gibbs
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