Distinct Structures of Scrapie Prion Protein (PrPSc)-seeded Versus Spontaneous Recombinant Prion Protein Fibrils Revealed by Hydrogen/Deuterium Exchange
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.036558
ISSN1083-351X
AutoresVytautas Smirnovas, Jae-Il Kim, Xiaojun Lu, Ryuichiro Atarashi, Byron Caughey, Witold K. Surewicz,
Tópico(s)Alcoholism and Thiamine Deficiency
ResumoThe detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrPSc) are largely unknown. PrPSc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrPC). One manifestation of PrPSc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrPSc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrPSc. Here, we compare the structures of PrPSc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrPSc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrPSc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrPSc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP. The detailed structures of prion disease-associated, partially protease-resistant forms of prion protein (e.g. PrPSc) are largely unknown. PrPSc appears to propagate itself by autocatalyzing the conformational conversion and oligomerization of normal prion protein (PrPC). One manifestation of PrPSc templating activity is its ability, in protein misfolding cyclic amplification reactions, to seed the conversion of recombinant prion protein (rPrP) into aggregates that more closely resemble PrPSc than spontaneously nucleated rPrP amyloids in terms of proteolytic fragmentation and infrared spectra. The absence of posttranslational modifications makes these rPrP aggregates more amenable to detailed structural analyses than bona fide PrPSc. Here, we compare the structures of PrPSc-seeded and spontaneously nucleated aggregates of hamster rPrP by using H/D exchange coupled with mass spectrometry. In spontaneously formed fibrils, very slow H/D exchange in region ∼163–223 represents a systematically H-bonded cross-β amyloid core structure. PrPSc-seeded aggregates have a subpopulation of molecules in which this core region extends N-terminally as far as to residue ∼145, and there is a significant degree of order within residues ∼117–133. The formation of tightly H-bonded structures by these more N-terminal residues may account partially for the generation of longer protease-resistant regions in the PrPSc-seeded rPrP aggregates; however, part of the added protease resistance is dependent on the presence of SDS during proteolysis, emphasizing the multifactorial influences on proteolytic fragmentation patterns. These results demonstrate that PrPSc has a distinct templating activity that induces ordered, systematically H-bonded structure in regions that are dynamic and poorly defined in spontaneously formed aggregates of rPrP. Transmissible spongiform encephalopathies (TSEs), 2The abbreviations used are: TSEtransmissible spongiform encephalopathyBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolHPLChigh pressure liquid chromatographyHXMSH/D exchange combined with mass spectrometryPKproteinase KPMCAprotein misfolding cyclic amplificationPrPprion proteinPrPCcellular PrPPrPScscrapie PrPrPrPrecombinant PrPPrPSpspontaneously formed amyloid fibrilsPrPPMCAPMCA-generated PrP aggregate(s)ShaSyrian hamster. or prion diseases, are a group of infectious neurodegenerative disorders that affect many mammalian species and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, chronic wasting disease in cervids, and bovine spongiform encephalopathy ("mad cow" disease) (1.Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 2.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1113) Google Scholar, 3.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 4.Caughey B. Baron G.S. Chesebro B. Jeffrey M. Annu. Rev. Biochem. 2009; 78: 177-204Crossref PubMed Scopus (269) Google Scholar, 5.Cobb N.J. Surewicz W.K. Biochemistry. 2009; 48: 2574-2585Crossref PubMed Scopus (157) Google Scholar, 6.Chien P. Weissman J.S. DePace A.H. Annu. Rev. Biochem. 2004; 73: 617-656Crossref PubMed Scopus (288) Google Scholar, 7.Wickner R.B. Edskes H.K. Roberts B.T. Baxa U. Pierce M.M. Ross E.D. Brachmann A. Genes Dev. 2004; 18: 470-485Crossref PubMed Scopus (64) Google Scholar). All of these diseases appear to be intimately associated with conformational conversion of the normal host-encoded prion protein, termed PrPC, to a pathological isoform, PrPSc (1.Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 2.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1113) Google Scholar, 3.Aguzzi A. Polymenidou M. Cell. 2004; 116: 313-327Abstract Full Text Full Text PDF PubMed Scopus (508) Google Scholar, 4.Caughey B. Baron G.S. Chesebro B. Jeffrey M. Annu. Rev. Biochem. 2009; 78: 177-204Crossref PubMed Scopus (269) Google Scholar, 5.Cobb N.J. Surewicz W.K. Biochemistry. 2009; 48: 2574-2585Crossref PubMed Scopus (157) Google Scholar). According to the "protein-only" model, PrPSc itself represents the infectious prion agent (1.Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 8.Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4124) Google Scholar); it is believed to self-propagate by an autocatalytic mechanism involving binding to PrPC and templating the conversion of the latter protein to the PrPSc state (9.DebBurman S.K. Raymond G.J. Caughey B. Lindquist S. Proc. Natl. Acad. Sci. 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Biochem. 2004; 73: 617-656Crossref PubMed Scopus (288) Google Scholar, 7.Wickner R.B. Edskes H.K. Roberts B.T. Baxa U. Pierce M.M. Ross E.D. Brachmann A. Genes Dev. 2004; 18: 470-485Crossref PubMed Scopus (64) Google Scholar). transmissible spongiform encephalopathy 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol high pressure liquid chromatography H/D exchange combined with mass spectrometry proteinase K protein misfolding cyclic amplification prion protein cellular PrP scrapie PrP recombinant PrP spontaneously formed amyloid fibrils PMCA-generated PrP aggregate(s) Syrian hamster. PrPC is a monomeric glycophosphatidylinositol-linked glycoprotein that is highly protease-sensitive and soluble in nonionic detergents. High resolution NMR data show that the recombinant PrP (rPrP), a nonglycosylated model of PrPC, consists of a flexible N-terminal region and a folded C-terminal domain encompassing three α-helices and two short β-strands (11.Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wüthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1130) Google Scholar, 12.Riek R. Hornemann S. Wider G. Glockshuber R. Wüthrich K. FEBS Lett. 1997; 413: 282-288Crossref PubMed Scopus (666) Google Scholar, 13.Donne D.G. Viles J.H. Groth D. Mehlhorn I. James T.L. Cohen F.E. Prusiner S.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 13452-13457Crossref PubMed Scopus (637) Google Scholar). Conversely, the PrPSc isoform is aggregate in nature, rich in β-sheet structure, insoluble in nonionic detergents, and partially resistant to proteinase K (PK) digestion, with a PK-resistant core encompassing the C-terminal ∼140 residues (1.Prusiner S.B. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar, 2.Collinge J. Annu. Rev. Neurosci. 2001; 24: 519-550Crossref PubMed Scopus (1113) Google Scholar, 3.Aguzzi A. Polymenidou M. 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Thus, the structure of PrPSc conformer(s) associated with prion infectivity remains one of the best guarded mysteries, hindering efforts to understand the molecular basis of TSE diseases. Many efforts have been made over the years to recapitulate PrPSc formation and prion propagation in vitro. Early studies have shown that PrPC can be converted with remarkable species and strain specificities to a PrPSc-like conformation (as judged by PK resistance) simply by incubation with PrPSc from prion-infected animals (16.Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 17.Caughey B. Trends Biochem. Sci. 2001; 26: 235-242Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The yields of these original cell-free conversion experiments were low, and no new infectivity could be attributed to the newly converted material (18.Hill A.F. Antoniou M. Collinge J. J. Gen. Virol. 1999; 80: 11-14Crossref PubMed Scopus (176) Google Scholar). An important more recent study showed that both PrPSc and TSE infectivity can be amplified indefinitely in crude brain homogenates using successive rounds of sonication and incubation (19.Castilla J. Saá P. Hetz C. Soto C. Cell. 2005; 121: 195-206Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar), a procedure called protein misfolding cyclic amplification (PMCA) (20.Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). Similar amplification of the TSE infectivity was also accomplished by PMCA employing purified PrPC as a substrate, although only in the presence of polyanions such as RNA and copurified lipids (21.Deleault N.R. Harris B.T. Rees J.R. Supattapone S. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9741-9746Crossref PubMed Scopus (520) Google Scholar). Unfortunately, the quantities of infectious PrPSc generated by PMCA using purified brain-derived PrPC are very small, precluding most structural studies. In contrast to brain-derived PrPC, large scale purification can be readily accomplished for bacterially expressed rPrP, a form of PrP lacking glycosylation and the glycophosphatidylinositol anchor. The latter protein can spontaneously polymerize into amyloid fibrils, and much insight has been gained into mechanistic and structural aspects of this reaction (22.Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, 23.Baskakov I.V. J. Biol. Chem. 2004; 279: 7671-7677Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24.Apetri A.C. Vanik D.L. Surewicz W.K. Biochemistry. 2005; 44: 15880-15888Crossref PubMed Scopus (74) Google Scholar, 25.Surewicz W.K. Jones E.M. Apetri A.C. Acc. Chem. Res. 2006; 39: 654-662Crossref PubMed Scopus (50) Google Scholar, 26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar, 27.Cobb N.J. Sönnichsen F.D. McHaourab H. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 18946-18951Crossref PubMed Scopus (270) Google Scholar, 28.Stöhr J. Weinmann N. Wille H. Kaimann T. Nagel-Steger L. Birkmann E. Panza G. Prusiner S.B. Eigen M. Riesner D. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 2409-2414Crossref PubMed Scopus (114) Google Scholar). However, although rPrP fibrils were shown to cause or accelerate a transmissible neurodegenerative disorder in transgenic mice overexpressing a PrPC variant encompassing residues 89–231, the infectivity titer of these "synthetic prions" was extremely low (29.Legname G. Baskakov I.V. Nguyen H.O. Riesner D. Cohen F.E. DeArmond S.J. Prusiner S.B. Science. 2004; 305: 673-676Crossref PubMed Scopus (908) Google Scholar) or absent altogether (4.Caughey B. Baron G.S. Chesebro B. Jeffrey M. Annu. Rev. Biochem. 2009; 78: 177-204Crossref PubMed Scopus (269) Google Scholar). This low infectivity coincides with much shorter PK-resistant core of rPrP amyloid fibrils compared with brain-derived PrPSc (26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar, 30.Bocharova O.V. Breydo L. Salnikov V.V. Gill A.C. Baskakov I.V. Protein Sci. 2005; 14: 1222-1232Crossref PubMed Scopus (83) Google Scholar), raising questions regarding the relationship between these fibrils and the authentic TSE agent. In this context, an important recent development was the finding that the PrPSc-seeded PMCA method can be extended to rPrP, yielding protease-resistant recombinant PrP aggregates (rPrPPMCA or rPrP-res(Sc)) (31.Atarashi R. Moore R.A. Sim V.L. Hughson A.G. Dorward D.W. Onwubiko H.A. Priola S.A. Caughey B. Nat. Methods. 2007; 4: 645-650Crossref PubMed Scopus (283) Google Scholar). These aggregates display a PK digestion pattern that is much more closely related to PrPSc than that of previously studied spontaneously formed rPrP fibrils, offering a potentially more relevant model for biochemical and biophysical studies. Here, we provide, for the first time, a direct insight into the structure of rPrPPMCA. H/D exchange data coupled with MS analysis (HXMS) allowed us to identify systematically H-bonded core region(s) of these aggregates, shedding a new light on the mechanisms underlying formation of PK-resistant structures. Recombinant Syrian hamster full-length prion protein (rShaPrP-(23–231)) and its fragment (rShaPrP-(90–231)) were expressed and purified as described previously (32.Morillas M. Swietnicki W. Gambetti P. Surewicz W.K. J. Biol. Chem. 1999; 274: 36859-36865Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar); they were stored frozen in 10 mm sodium acetate buffer, pH 4. In addition to the ShaPrP sequence, the proteins contain a 4-residue N-terminal extension (GSDP) and an extra C-terminal Ser residue. Spontaneously formed amyloid fibrils were prepared as described previously (24.Apetri A.C. Vanik D.L. Surewicz W.K. Biochemistry. 2005; 44: 15880-15888Crossref PubMed Scopus (74) Google Scholar, 26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar). PrPSc-seeded PMCA of the recombinant Syrian hamster protein was performed essentially as described by Atarashi et al. (31.Atarashi R. Moore R.A. Sim V.L. Hughson A.G. Dorward D.W. Onwubiko H.A. Priola S.A. Caughey B. Nat. Methods. 2007; 4: 645-650Crossref PubMed Scopus (283) Google Scholar). Briefly, monomeric rShaPrP-(90–231) or rShaPrP-(23–231) was diluted to a concentration of 0.1 mg/ml in phosphate-buffered saline containing 0.1% SDS and 0.1% Triton X-100, pH 7.4. One hundred-microliter aliquots of this solution were placed in 0.2-ml tubes, and 100 ng of PK-treated PrPSc purified from 263K-infected hamster brain (33.Rubenstein R. Carp R.I. Ju W. Scalici C. Papini M. Rubenstein A. Kascsak R. Arch. Virol. 1994; 139: 301-311Crossref PubMed Scopus (18) Google Scholar) was added as a seed. The mixture was then subjected to nine rounds of PMCA, each of them consisting of 18 cycles of 40-s sonication followed by a 1-h incubation (Misonix S3000 bath-type sonicator equipped with an adaptor for 96 PCR tubes). After each round, an aliquot of the amplified samples was taken and diluted 100-fold into PMCA buffer (phosphate-buffered saline plus 0.1% SDS, 0.1% Triton X-100, pH 7.4) containing monomeric protein (0.1 mg/ml) as a substrate. Before H/D exchange experiments, pepsin digestion fragments of the PrP were identified by a standard procedure involving separation on a C18 high pressure liquid chromatography (HPLC) column coupled to a Finnigan LTQ mass spectrometer, sequencing by tandem MS, and analysis using the SEQUEST search algorithm (ThermoFisher Scientific, San Jose, CA) and manual inspection. The identification was confirmed by exact mass measurements using Fourier transform ion cyclotron resonance MS (LTQ-ICR, ThermoFisher Scientific). Spontaneously formed rShaPrP amyloid fibrils and rShaPrP aggregates produced by PrPSc-seeded PMCA were collected by centrifugation (10,000 × g; 10 min) and washed four or five times in 10 mm phosphate buffer, pH 7. To initiate deuterium labeling, pellets (∼50 μl) were resuspended in 1 ml of the buffer (10 mm phosphate, pH 7) prepared in D2O. After different times of incubation at room temperature, samples were collected by centrifugation (16,000 × g, 5 min) and rapidly dissociated into rShaPrP monomers by adding an ice-cold solution of 7 m guanidine hydrochloride in an exchange quench buffer (0.1 m phosphate, pH 2.4) containing a reducing agent (0.1 m tris(2-carboxyethyl)phosphine hydrochloride)). After a 5-min incubation on ice, the samples were diluted 10 times with ice-cold 0.05% trifluoroacetic acid in H2O and digested for 5 min with agarose-immobilized pepsin (ThermoFisher Scientific) using 100 μl of the slurry/100 μl of protein solution (∼100 μg/ml). The peptic fragments were collected in a peptide microtrap, washed to remove salts, and eluted on a C18 HPLC column using a gradient of 2–35% acetonitrile at a flow rate of 50 μl/min. Peptides separated on the column were analyzed by a Finnigan LTQ mass spectrometer. The trap and the column were immersed in ice. Peptide masses were calculated from the centroid of the isotopic envelope using MagTran software, and the extent of deuterium incorporation at each time point was determined from the shift in mass of labeled peptides relative to unlabeled peptides. To correct for the 5% H2O present during the exchange-in and for deuterium back-exchange during proteolysis, HPLC separation, and MS analysis, control experiments were performed by using fully deuterated protein prepared by a 2-h incubation in 6.6 m guanidine deuterochloride in D2O/H2O (95:5, v/v) buffered with 10 mm potassium phosphate, pH 7. The extent of deuterium incorporation (corrected for back-exchange) was calculated as % D = (m(t) − m(0%))/(m(100%) − m(0%)) × 100, where % D is the relative amount of amide deuterium atoms incorporated in each peptic fragment, m(t) is the observed centroid mass of the peptide at time point t, m(0%) is the measured mass of an undeuterated reference sample, and m(100%) is the observed mass of a fully deuterated reference sample. Isotopic envelopes showing bimodal-like distribution were analyzed mathematically to estimate the fraction of deuterated species. To this end, the envelopes were fitted as a sum of Gaussian curves corresponding to each isotopic peak. Peaks with the best signal-to-noise ratio that do not overlap with the isotopic envelope for the fully protonated species were selected, and the areas of these peaks were divided by the areas of corresponding peaks in the mass spectrum of the fully deuterated peptide (normalized to the same area). rPrP aggregates were collected by centrifugation at 10,000 × g for 10 min and, unless indicated differently, used directly for PK digestion analysis. In some experiments, samples were washed four or five times with phosphate-buffered saline and resuspended in the same buffer with or without detergents employed in the PMCA buffer (0.1% SDS, 0.1% Triton X-100). Samples were then treated with PK (1 h, 37 °C, PK:rShaPrP ratio of 1:10, w/w). A fraction of PK-digested samples was used for Western blot analysis and another fraction for MS (after dissociation into monomers as described above). For Western blotting, samples were separated by SDS-PAGE using 12% NuPAGE BisTris gels (Invitrogen), electrotransferred onto nitrocellulose membrane, and analyzed using monoclonal 3F4 antibody (1:20,000) (34.Kascsak R.J. Rubenstein R. Merz P.A. Tonna-DeMasi M. Fersko R. Carp R.I. Wisniewski H.M. Diringer H. J. Virol. 1987; 61: 3688-3693Crossref PubMed Google Scholar) or polyclonal anti-PrP antibody 78295 (1:5,000) (35.Kascsak R.J. Tonna-DeMasi M. Fersko R. Rubenstein R. Carp R.I. Powers J.M. Dev. Biol. Stand. 1993; 80: 141-151PubMed Google Scholar). Previous studies have shown that the PMCA procedure of Soto and coworkers (19.Castilla J. Saá P. Hetz C. Soto C. Cell. 2005; 121: 195-206Abstract Full Text Full Text PDF PubMed Scopus (690) Google Scholar, 20.Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar) can be adapted to convert bacterially expressed recombinant Syrian hamster full-length prion protein to the PK-resistant form, with a 16–17-kDa PK-resistant fragment similar to that found in deglycosylated brain-derived PrPSc (31.Atarashi R. Moore R.A. Sim V.L. Hughson A.G. Dorward D.W. Onwubiko H.A. Priola S.A. Caughey B. Nat. Methods. 2007; 4: 645-650Crossref PubMed Scopus (283) Google Scholar). This conversion has been accomplished using a buffer containing a mixture of nonionic and acidic detergents (0.1% Triton X-100, 0.1% SDS). Because, for technical reasons, certain aspects of structural studies involving mass spectrometric analysis are more practical using the N-terminally truncated rPrP-(90–231) fragment (see below), here we have adapted the PMCA protocol to rShaPrP-(90–231). Upon completion of the reaction, the product was treated with PK (PK:rPrP ratio of 1:10) and analyzed by gel electrophoresis and Western blotting. As shown in Fig. 1, Western blots probed with 3F4 antibody (which recognizes the epitope at residues 109–112) revealed a single band with a molecular mass of ∼16 kDa (previously described as an ∼17 kDa band (31.Atarashi R. Moore R.A. Sim V.L. Hughson A.G. Dorward D.W. Onwubiko H.A. Priola S.A. Caughey B. Nat. Methods. 2007; 4: 645-650Crossref PubMed Scopus (283) Google Scholar)). However, when probed with silver staining (data not shown) or polyclonal anti-PrP antiserum 78295 (which recognizes both the N- and C-terminal epitopes), electrophoretic gels revealed additional PK-resistant fragments with molecular masses between ∼10 and 14 kDa (Fig. 1). Importantly, this electrophoretic profile for PMCA-converted rShaPrP-(90–231) is very similar to that of PMCA-converted full-length rShaPrP, indicating essentially identical PK-resistant fragments. The first step of HXMS analysis involves identification of peptic fragments that can be separated under the conditions of the rapid HPLC gradient required for hydrogen exchange experiments. Tandem MS experiments with peptic digest of rShaPrP-(90–231) allowed us to identify >60 fragments, 43 of which had a signal-to-noise ratio sufficient for reliable analysis of deuterium incorporation. The number of these fragments (covering ∼96% of the entire sequence with multiple overlaps in some regions) compares favorably with peptic coverage in the previous study with human rPrP-(90–231) (26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar). This improvement is due largely to better MS instrumentation available for the present experiments and the use of resin-immobilized pepsin. Peptide mapping for rShaPrP-(23–231) identified the same fragments as in the case of rShaPrP-(90–231) for all peptides starting at and C-terminal to residue 117. However, no peptic fragments could be detected with a sufficient signal-to-noise-ratio within the entire 23–90 region, and the fragments starting in rShaPrP-(90–231) at residue 90 (Fig. 2) were now replaced by corresponding fragments starting at residue 23. Because of much longer peptic fragments, the precision and resolution of H/D exchange measurements for the ∼90–120 region in rShaPrP-(23–231) were much lower than those for the same region in rShaPrP-(90–231). Thus, the C-terminally truncated rShaPrP-(90–231) is better suited for HXMS studies of PrPSc-mimicking PrP aggregates. Although the focus of the present study is on structural properties of rShaPrPPMCA, an important point of reference for interpretation of H/D exchange data is relatively well characterized spontaneously formed recombinant prion protein amyloid fibrils, rPrPSp. The extent of deuterium incorporation for peptic fragments derived from rShaPrP-(90–231)Sp after different times of isotope exchange (5 min, 2 h, and 24 h) is summarized in Fig. 2A. All fragments corresponding to the N-terminal part of rShaPrP-(90–231) up to residue 161 were found to incorporate deuterium very rapidly (with essentially complete labeling within the first 5 min of the exchange experiment), clearly indicating the lack of any ordered structures within this part of amyloid fibrils. This contrasts sharply with strong protection against deuterium labeling for peptides derived from the C-terminal part of rShaPrP-(90–231)Sp. In particular, fragments corresponding to the entire 170–213 region appear to be especially highly protected, with less than ∼35% deuterium incorporation even after 24 h of exchange. A relatively high, although slightly lower, level of protection (less than ∼50% labeling within 24 h) is also observed for peptic fragments corresponding to residues 161/162–168, 161–174/175, and 218–224. As discussed in the previous study with human rPrP amyloid (26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar), this highly protected region represents systematically H-bonded cross-β structure, defining the "core" of amyloid fibrils. On the basis of the level of deuterium labeling for the flanking peptides 162–168 and 218–224, we estimate that both of them contain 1–2 unprotected residues. This would place the N- and C-terminal boundaries of the ordered core region at residues ∼163/164 and 222/223, respectively. This H-bonded core region determined herein for rShaPrP-(90–231)Sp is similar to that previously found for human rPrP amyloid fibrils (26.Lu X. Wintrode P.L. Surewicz W.K. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1510-1515Crossref PubMed Scopus (204) Google Scholar). The only significant difference is the N-terminal boundary, which for the latter protein appears to be at residue ∼169 (as indicated by very little protection for the peptic fragments within the 161–168 region). The "protection map" for peptic fragments derived from rShaPrP-(90–231)PMCA (based on masses calculated from the overall centroids of the isotopic envelopes in mass spectra) is quite different from that for spontaneously formed rShaPrP-(90–231) amyloid fibrils (Fig. 2). The most notable of these differences is the apparent protection against deuterium incorporation for most peptic fragments corresponding to the 117–161 region in rShaPrP-(90–231)PMCA, whereas in rShaPrP-(90–231)Sp, this entire region is highly accessible to deuterium labeling. Furthermore, inspection of mass spectra for peptic fragments derived from rShaPrP-(90–231)PMCA indicates that some of them display at least two partially overlapping isotopic envelopes, indicating an apparently bimodal mass distribution. Spectra of this type are exemplified in Fig. 3 for peptides 120–133, 145–154, and 155–168. Importantly, there is no indication of such a bimodal mass distribution for any of the peptic fragments derived from rShaPrP-(90–231)Sp (Fig. 3). The bimodal mass distribution for some peptic fragments derived from rShaPrP-(90–231)PMCA indicates the presence of two different species, one that incorporates deuterium very rapidly and one that is highly protected against H/D exchange. The presence of such two different populations was observed for all peptic fragments within residues 117–133 and 145–168. Thus, rShaPrP-(90–231)PMCA contains at least two conformationally distinct populations of protein molecules: one in which the regions ∼117–133 and ∼145–168 are largely disordered (i.e
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