Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

Abstract

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.

Core Sequence of PAPf39 Amyloid Fibrils and Mechanism of pH-Dependent Fibril Formation: The Role of Monomer Conformation

“…Hydrogen-Deuterium Exchange Mass Spectrometry (HDXMS) was used to probe the fibrillar core of the PAPf39 fibrils (18, 20, 21). In these experiments, the deuterium incorporation in the in-exchanged monomer, exchanged monomer, and exchanged fibrils was measured by liquid chromatography-mass spectrometry (LC-MS) and used to determine the percent HDX protection over the sequence of the fibril.…”

Determination of amyloid core structure using chemical shifts

“…24,25 Instead, a variety of studies have investigated the structure of amyloid fibrils produced in vitro from recombinant PrP. [26][27][28][29][30][31][32][33][34][35] Hereditary prion diseases include C-terminally truncated variants of the PrP, Y145X, Q160X, Y226X, and Q227X. Previously, we showed that the b-sheet content is highly similar in amyloid fibrils of the Y145X and Q160X stop mutants of human PrP.…”

Hydrogen-exchange mass spectrometry for the study of intrinsic disorder in proteins

“…HDX approaches are becoming widely used to discover which regions of IDPs are buried in various oligomeric states of prion proteins. Wemmer’s group used both HXMS and exchange-quenched NMR to probe the regions of the prion protein, PrP (89–143, P101L) that participate in fibril formation 65 . They found two regions, residues 102–109 and 117–136 that were highly protected (with a half-life of exchange longer than one week!)…”