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.
Highlights
Infectious and neurotoxic form PrPSc (PrP scrapie) [2]
Later work reconstituted prion infectivity from a synthetic protein identified as a 55-residue PrP fragment, PrP(89 –143, P101L), that readily associated into amyloid fibrils
Hydrogen Exchange Mass Spectrometry of PrP(89 –143, P101L) Fibrils—Synthetic PrP(89 –143, P101L) fibrils that had been grown in H2O were incubated in D2O and aliquots removed after incubation for 1, 6, and 21 h and 1, 3, and 6 weeks
Summary
Amyloid Fibril Formation—Amyloid fibrils of PrP(89 –143, P101L) were formed by dissolving 10 mg of peptide into 500 l of 20 mM sodium acetate, 100 mM NaCl, pH 5, and adding 500 l of acetonitrile. Fibrils used in hydrogen exchange experiments were collected after 3 weeks of incubation in the acetonitrile solution. Exchange samples were prepared for MS analysis by dilution with 55 l of ice-cold 10 mM sodium phosphate buffer and rapid addition of 90 l of quench buffer (6.8 M guanidine hydrochloride (GdnHCl), 16.6% glycerol, and 0.8% formic acid), yielding a final pH* of 2.1, followed by 5 min of incubation over thawing ice to fully dissolve the fibrils. Expression, and Purification—A synthetic gene for PrP(89 –143, P101L) with an N-terminal six residue histidine tag and tobacco etch virus cleavage site was designed based on Escherichia coli optimum codon usage and synthesized by GenScript (Piscataway, NJ) in a pUC19 plasmid. The supernatant was discarded, and the inclusion body was resolubilized by applying three rounds of resuspension in 6 M GdnHCl, 200 mM NaPi, pH
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