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.

FIGURE 1.

Exchange behavior of PrP(89–143, P101L) fibrils observed by mass spectrometry. A, shows the mass envelope of the 1+ charge state of the proteolytic fragment of PrP(89–143, P101L) corresponding to residues 133–139. B, mass envelope of the same peptide obtained from PrP fibrils that had been incubated in D₂O for 1 week. C, exchange behavior of proteolytic fragments of PrP(89–143, P101L) after 6 weeks of incubation in D₂O. The extent of exchange is depicted as a percent protection of exchangeable backbone amides. Each primary peptide is numbered. The identity of the two primary peptides used to obtain each secondary peptide is indicated to the left of the secondary peptides. D, consensus exchange behavior calculated according to Equation 1.

FIGURE 2.

Exchange behavior of PrP(89–143, P101L) fibrils observed with NMR. A, shows the assigned ¹H,¹⁵N HSQC spectrum of PrP(89–143, P101L) under the conditions of a typical 0-h time point, dissolving fibrils that had not been exposed to deuterium in DMSO containing 5% D₂O and 0.03% TFA-D, pH* 5. The four ambiguous glycine residues are denoted with an asterisk. B, summary of the exchange behavior of PrP(89–143, P101L) fibrils.