The Structural Transition of the Prion Protein into its Pathogenic Conformation is Induced by Unmasking Hydrophobic Sites

Leffers, Karl-Werner; Schell, Jeff; Jansen, Katja; Lucassen, Ralf; Kaimann, T.; Nagel-Steger, Luitgard; Tatzelt, Jörg; Riesner, Detlev

doi:10.1016/j.jmb.2004.09.071

Cited by 44 publications

(47 citation statements)

References 58 publications

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“…In a similar way, the second component of 35.2 kDa can be interpreted as a dimer of recPrP with 10 molecules of SDS bound. This interpretation is in good accordance with earlier data of monomeric recPrP-SDS and dimeric recPrP-SDS complexes, although those were obtained in the absence of NaCl (18). CD Spectroscopy.…”

Section: Resultssupporting

confidence: 93%

“…CD Spectroscopy. In earlier studies, the secondary structure of recPrP in 0.03% SDS but without NaCl was determined to be a soluble ␤-sheet-rich structure that is prone to form amorphous aggregates (18). The addition of NaCl leads to a conformation composed of ␣-helical and random-coil secondary structures (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we described an in vitro conversion system, in which different conformational states of recPrP could be established by varying the concentration of SDS (18). In a follow-up study, this system was optimized to produce amyloid fibrils from recombinant and natural PrP (19).…”

Section: Resultsmentioning

confidence: 99%

“…Calculation of molecular mass with bound SDS per PrP was performed by using the method of Casassa and Eisenberg (37) as described in ref. 18. Digestion with PK.…”

Section: Methodsmentioning

confidence: 99%

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Mechanisms of prion protein assembly into amyloid

Stöhr

Weinmann

Wille

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

132

112

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The conversion of the ␣-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, ␤-sheet-rich, infectious, diseasecausing isoform (PrP Sc ) is the key event in prion diseases. In an earlier study, several forms of PrP were converted into a fibrillar state by using an in vitro conversion system consisting of low concentrations of SDS and 250 mM NaCl. Here, we characterize the structure of the fibril precursor state, that is, the soluble state under fibrillization conditions. CD spectroscopy, analytical ultracentrifugation, and chemical cross-linking indicate that the precursor state exists in a monomer-dimer equilibrium of partially denatured, ␣-helical PrP, with a well defined contact site of the subunits in the dimer. Using fluorescence with thioflavin T, we monitored and quantitatively described the kinetics of seeded fibril formation, including dependence of the reaction on substrate and seed concentrations. Exponential, seed-enhanced growth can be achieved in homogeneous solution, which can be enhanced by sonication. From these data, we propose a mechanistic model of fibrillization, including the presence of several intermediate structures. These studies also provide a simplified amplification system for prions.dimer ͉ seeding ͉ fibril ͉ precursor state

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Calculation of molecular mass with bound SDS per PrP was performed by using the method of Casassa and Eisenberg (37) as described in ref. 18. Digestion with PK.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Mechanisms of prion protein assembly into amyloid

Stöhr

Weinmann

Wille

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

132

112

View full text Add to dashboard Cite

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“…A SDS-concentration as high as 0.2 % (vol/vol) only solubilises prion fibrils. 67 Prion infectivity, which depends crucially on structural features, remains even after boiling in 5 % SDS. 68 Sonication of monomeric ovrecPrP(25-233) for 45 min at 240 W changes neither ellipticity intensity nor spectral features.…”

Section: Circular Dichroism (Cd) Spectroscopymentioning

confidence: 99%

Progress towards structural understanding of infectious sheep PrP-amyloid

Müller

Brener

Andréoletti

et al. 2014

Prion

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The still elusive structural difference of non-infectious and infectious amyloid of the mammalian prion protein (PrP) is a major pending milestone in understanding protein-mediated infectivity in neurodegenerative diseases. Preparations of PrP-amyloid proven to be infectious have never been investigated with a high-resolution technique. All available models to date have been based on low-resolution data. Here, we establish protocols for the preparation of infectious samples of full-length recombinant (rec) PrP-amyloid in NMR-sufficient amounts by spontaneous fibrillation and seeded fibril growth from brain extract. We link biological and structural data of infectious recPrP-amyloid, derived from bioassays, atomic force microscopy, and solid-state NMR spectroscopy. Our data indicate a semi-mobile N-terminus, some residues with secondary chemical shifts typical of a-helical secondary structure in the middle part between »115 to »155, and a distinct b-sheet core C-terminal of residue »155. These findings are not in agreement with all current models for PrP-amyloid. We also provide evidence that samples seeded from brain extract may not differ in the overall arrangement of secondary structure elements, but rather in the flexibility of protein segments outside the b-core region. Taken together, our protocols provide an essential basis for the high-resolution characterization of non-infectious and infectious PrP-amyloid in the near future.

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Amyloids and Protein Aggregation—Analytical Methods

Li¹,

Rahimi²,

Sinha³

et al. 2009

Encyclopedia of Analytical Chemistry

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More than 30 diseases are associated with amyloid‐forming proteins, which undergo conformational alterations and “misfolding” under particular conditions. These proteins deposit as insoluble proteinaceous aggregates generating disease‐specific histopathologic lesions. The proteins contributing to each disease have dissimilar sequences and native structures, yet they share the commonality of forming insoluble amyloid aggregates characterized by fibrillar morphology and cross‐β structure. Presently, it is thought that the predominant agents causing cell toxicity and tissue damage are soluble, prefibrillar assemblies of these proteins. Because of the metastable nature of these prefibrillar assemblies and the noncrystalline nature of fibrillar protein aggregates, structural study of amyloid proteins is difficult. As a result, structural characterization of these proteins is typically done using combinations of low‐resolution analytical methods. Here, we present a compendium of analytical methods used to study the secondary, tertiary, and quaternary structures, and morphology of prefibrillar and fibrillar assemblies of amyloid‐forming proteins.

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The Structural Transition of the Prion Protein into its Pathogenic Conformation is Induced by Unmasking Hydrophobic Sites

Cited by 44 publications

References 58 publications

Mechanisms of prion protein assembly into amyloid

Mechanisms of prion protein assembly into amyloid

Progress towards structural understanding of infectious sheep PrP-amyloid

Amyloids and Protein Aggregation—Analytical Methods

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