Probing the conformation of a prion protein fibril with hydrogen exchange.
A fragment of the prion protein, PrP(89-143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89-143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102-109 and 117-136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.
Damo
Steven M
SM
Department of Chemistry, University of California, Berkeley, California 94720, USA.
Phillips
Aaron H
AH
Young
Anisa L
AL
Li
Sheng
S
Woods
Virgil L
VL
Wemmer
David E
DE
D000595:Amino Acid Sequence; D000682:Amyloid / Q000648:ultrastructure*; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D006859:Hydrogen / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011328:Prions / Q000235:genetics / Q000648:ultrastructure*; D017433:Protein Structure, Secondary
D000682:Amyloid; D011328:Prions; D006859:Hydrogen