Oligomerization of the Human Prion Protein Proceeds via a Molten Globule Intermediate
2007; Elsevier BV; Volume: 282; Issue: 9 Linguagem: Inglês
10.1074/jbc.m608926200
ISSN1083-351X
AutoresRemo Gerber, Abdessamad Tahiri‐Alaoui, P. J. Hore, William James,
Tópico(s)Neurological diseases and metabolism
ResumoThe conformational transition of the human prion protein from an α-helical to a β-sheet-rich structure is believed to be the critical event in prion pathogenesis. The molecular mechanism of misfolding and the role of intermediate states during this transition remain poorly understood. To overcome the obstacle of insolubility of amyloid fibrils, we have studied a β-sheet-rich misfolded isoform of the prion protein, the β-oligomer, which shares some structural properties with amyloid, including partial proteinase resistance. We demonstrate here that the β-oligomer can be studied by solution-state NMR spectroscopy and obtain insights into the misfolding mechanism via its transient monomeric precursor. It is often assumed that misfolding into β-sheet-rich isoforms proceeds via a compatible precursor with a β-sheet subunit structure. We show here, on the contrary, evidence for an almost natively α-helix-rich monomeric precursor state with molten globule characteristics, converting in vitro into the β-oligomer. We propose a possible mechanism for the formation of the β-oligomer, triggered by intermolecular contacts between constantly rearranging structures. It is concluded that the β-oligomer is not preceded by precursors with β-sheet structure but by a partially unfolded clearly distinguishable α-helical state. The conformational transition of the human prion protein from an α-helical to a β-sheet-rich structure is believed to be the critical event in prion pathogenesis. The molecular mechanism of misfolding and the role of intermediate states during this transition remain poorly understood. To overcome the obstacle of insolubility of amyloid fibrils, we have studied a β-sheet-rich misfolded isoform of the prion protein, the β-oligomer, which shares some structural properties with amyloid, including partial proteinase resistance. We demonstrate here that the β-oligomer can be studied by solution-state NMR spectroscopy and obtain insights into the misfolding mechanism via its transient monomeric precursor. It is often assumed that misfolding into β-sheet-rich isoforms proceeds via a compatible precursor with a β-sheet subunit structure. We show here, on the contrary, evidence for an almost natively α-helix-rich monomeric precursor state with molten globule characteristics, converting in vitro into the β-oligomer. We propose a possible mechanism for the formation of the β-oligomer, triggered by intermolecular contacts between constantly rearranging structures. It is concluded that the β-oligomer is not preceded by precursors with β-sheet structure but by a partially unfolded clearly distinguishable α-helical state. The misfolding of the prion protein is the cause of several fatal neurodegenerative diseases in humans and animals (1Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 2Weissmann C. FEBS Lett. 1996; 389: 3-11Crossref PubMed Scopus (145) Google Scholar). Among these are scrapie in sheep, bovine spongiform encephalopathy in cattle and, in humans, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, and kuru. These diseases are associated with the misfolding of the α-helix-rich cellular prion PrPC 3The abbreviations used are: PrP, prion protein; PrPC, cellular isoform of PrP; PrPSc, scrapie (proteinase-resistant) PrP isoform; HuPrP90–231, recombinant prion protein fragment 90–231; αN, native state of PrP; βO, β-oligomer state of PrP; αi, intermediate state formed during oligomerization of βO; HPLC, high performance liquid chromatography; sec-HPLC, size exclusion HPLC; ANS, 1-anilinonaphthalene-8-sulfonate; PFG, pulsed field gradient; HSQC, heteronuclear single quantum coherence. protein to the β-sheet-rich, PrPSc form (1Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Abstract Full Text PDF PubMed Scopus (262) Google Scholar, 3Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4106) Google Scholar). The native state of PrPC is represented by that of the 19.8-kDa protein (residues 89–231), which consists of three α-helices and a short anti-parallel β-sheet (4Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar) and will be denoted here as αN. The mechanism of conversion of αN to the pathogenic PrPSc form remains unclear, although it is now known that it occurs post-translationally without detectable covalent modifications (5Stahl N. Baldwin M.A. Teplow D.B. Hood L. Gibson B.W. Burlingame A.L. Prusiner S.B. Biochemistry. 1993; 32: 1991-2002Crossref PubMed Scopus (536) Google Scholar). The transformation requires a substantial change of conformation from an α-helix-rich monomer to a β-sheet-rich amyloid structure. In vitro, αN can be converted into a variety of stable non-native structures with high β-sheet content (6Baskakov 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 (388) Google Scholar, 7Lu B.Y. Chang J.Y. Biochemistry. 2001; 40: 13390-13396Crossref PubMed Scopus (28) Google Scholar). Different solution conditions can induce different structures, one of which is the β-oligomer, βO, first described by Baskakov et al. (6Baskakov 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 (388) Google Scholar). The βO state is formed under mildly denaturing and acidic conditions similar to those found in endocytic vesicles in humans. Some of the biophysical properties of βO are similar to PrPSc: high β-sheet content, protease K resistance with slightly different cleavage sites (8Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1253) Google Scholar), and high binding affinity to 1-anilinonaphthalene-8-sulfonate (ANS). In contrast to PrPSc, βO is soluble, monodisperse, and does not bind thioflavin T (6Baskakov 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 (388) Google Scholar). Importantly, βO is not a template for the formation of PrPSc;on the contrary, it is a very stable long-lived macromolecular assembly, the formation of which proceeds through a pathway that competes with amyloid formation (6Baskakov 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 (388) Google Scholar). In recent publications (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 10Tahiri-Alaoui A. Sim V.L. Caughey B. James W. J. Biol. Chem. 2006; 281: 34171-34178Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), the polymorphism at codon 129 was shown to exhibit a measurable effect on the kinetics of formation of βO. It was further shown that βO formed from an equimolar mixture of valine 129 and methionine 129 PrP is very refractory to amyloid formation when compared with homogeneous protein of either allelic form. As heterozygosity at this position is associated epidemiologically with lower rates of prion disease, it was inferred that βO might have an adaptive role by sequestering PrP away from the pathway of amyloid formation. The key to understanding the mechanism of the conformational transition from α-helical to β-sheet structure is the identification of precursors. Among the hypotheses concerning the nature of the possible precursors of PrPSc, an early model due to Cohen et al. (11Cohen F.E. Pan K.M. Huang Z. Baldwin M. Fletterick R.J. Prusiner S.B. Science. 1994; 264: 530-531Crossref PubMed Scopus (440) Google Scholar) discusses the possibility of a partially unfolded intermediate whose formation and conversion into PrPSc could be promoted by an irreversible reaction with insoluble PrPSc. Similarly, the formation of the off pathway βO involves a dramatic change of secondary structure (6Baskakov 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 (388) Google Scholar). Others have hypothesized about the existence of weakly populated intermediates during the oligomerization process of βO but could not enrich or stabilize such a species (12Sokolowski F. Modler A.J. Masuch R. Zirwer D. Baier M. Lutsch G. Moss D.A. Gast K. Naumann D. J. Biol. Chem. 2003; 278: 40481-40492Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). One of the key questions is the structural nature of such an intermediate and whether at an early stage it adopts a β-sheet structure or remains α-helicalrich. To this end, we have studied the oligomerization process during the formation of βO where we have identified and partially characterized a precursor state, αi. Protein Expression and Purification—Escherichia coli expression of recombinant human PrP was performed as described previously (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, the 90–231 fragment of the PRNP gene was cloned into the pTrcHis2B vector incorporating a C-terminal His tag (Invitrogen, Paisley, UK) and was expressed in the E. coli strain BL21(DE3) (Novagen). Cells were grown in a minimal medium with 15NH4Cl as the sole source of nitrogen for fully 15N-labeled protein. The purification was performed as described previously (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), and stocks of highly purified proteins were kept in a storage buffer (6 m guanidine hydrochloride containing 50 mm Tris-HCl, pH 7.2). The protein was fully oxidized as judged by reverse phase chromatography, and the integrity of the samples was further analyzed by mass spectrometry (data not shown). The oxidation state is of particular importance with respect to previously reported and potentially controversial observations of reversible monomeric β-sheet-rich structures (13Jackson G.S. Hosszu L.L. Power A. Hill A.F. Kenney J. Saibil H. Craven C.J. Waltho J.P. Clarke A.R. Collinge J. Science. 1999; 283: 1935-1937Crossref PubMed Scopus (365) Google Scholar, 14Maiti N.R. Surewicz W.K. J. Biol. Chem. 2001; 276: 2427-2431Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The data presented here are solely for the allelomorph HuPrP90–231 Val129. Studies of direct refolding show that the Val129 allelomorph oligomerizes more slowly to βO than Met129 (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Other than that, the mutation at position 129 has no measurable effect on the folding, dynamics, or stability of PrPC (15Hosszu L.L. Jackson G.S. Trevitt C.R. Jones S. Batchelor M. Bhelt D. Prodromidou K. Clarke A.R. Waltho J.P. Collinge J. J. Biol. Chem. 2004; 279: 28515-28521Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Size-exclusion HPLC Preparation of αN and αi—We used sec-HPLC to refold the protein to either the native state αN or the precursor αi. To form αN and αi, 0.8 mg of protein denatured in 100 μl of storage buffer was injected onto a TSK®-Gel SWXL G3000 HPLC column (7.8 × 300 mm, Phenomenex, Maccles-field, UK), equilibrated with either αN buffer (20 mm sodium acetate, pH 5.5, 150 mm sodium chloride, 1 m urea, and 0.02% azide) or buffer A (20 mm sodium acetate, pH 4.0, 200 mm sodium chloride, 1 m urea, and 0.02% azide), as appropriate. The peak corresponding to monomeric proteins at 9.04 min of retention time was manually collected (see also Fig. 1, a and b). For NMR studies, up to five runs were collected, pooled, and concentrated (Amicon Ultra-4, 10,000 molecular weight cut-off, Millipore, Carrigtwohill, County Cork, Ireland) to final protein concentrations of 0.8 and 0.2 mm for αN and αi, respectively. All sec-HPLC separations were performed at room temperature with a flow rate of 1 ml min-1 by means of a PerkinElmer Life Sciences HPLC system composed of a Series 200 pump and a diode array detector 235C controlled by Total Chrome software version 6.2 (PerkinElmer Life Sciences, Seer Green, UK), through a PE Nelson 600 series link. The eluent was monitored by UV absorption at 280 nm. β-Oligomer (βO) Formation by Dialysis—As described previously (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), βO was formed by dialyzing 0.5 ml of GuHCl-denatured protein (14.8 mg ml-1) at room temperature for 24 h, twice against 2 liters of 2 m urea, 0.2 m NaCl, 20 mm sodium acetate, pH 3.6, and once against βO buffer (1 m urea, 0.2 m NaCl, 20 mm sodium acetate, pH 3.6, 0.02% sodium azide). We used a Slide-A-Lyser dialysis cassette (Perbio Science UK Ltd., Tattenhall, UK) with a 10-kDa cut-off. 1-Anilinonaphthalene-8-sulfonate Fluorescence Spectroscopy—Fluorescence spectra were recorded with a Fluorolog®-3 spectrofluorimeter (Jobin Yvon Ltd., Stanmore, Middlesex, UK) in 5-mm path length quartz cells (Hellma Cells Inc., Jamaica, NY). The fluorescence of ANS was monitored between 400 and 600 nm with excitation at 385 nm as described previously (6Baskakov 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 (388) Google Scholar). All samples were measured with protein and ANS concentrations of 1 and 100 μm, respectively. Circular Dichroism Spectroscopy—CD spectra were recorded using a Jasco-720 spectrometer at room temperature with the following parameters for far UV and near UV spectra, respectively: cell path 0.1 cm, speed 50 or 100 nm min-1, bandwidth 2.0 or 1.0 nm, resolution 1.0 or 0.5 nm, and a response time of 4 or 1 s, averaged over 4 or 16 scans. Far and near UV spectra were measured in βO buffer (protein concentration 40 and 550 μm), αN buffer (protein concentration 50 and 780 μm), and buffer A (protein concentration 22 and 440 μm) for βO, αN, and αi, respectively (the buffer compositions are specified above). The amount of helical structure was calculated using the Chen algorithm (16Chen Y.H. Yang J.T. Martinez H.M. Biochemistry. 1972; 11: 4120-4131Crossref PubMed Scopus (1907) Google Scholar). Pulsed Field Gradient NMR Diffusion Spectroscopy—PFG NMR diffusion measurements were performed at 20 °C on a home-built spectrometer with a 1H operating frequency of 600.20 MHz. The pulsed gradient stimulated echo longitudinal encode-decode pulse sequence was used (17Jones J.A. Wilkins D.K. Smith L.J. Dobson C.M. J. Biomol. NMR. 1997; 10: 199Crossref Scopus (205) Google Scholar), incorporating composite sine gradient pulses (18Merrill M.R. J. Magn. Reson. Series A. 1993; 103: 223Crossref Scopus (32) Google Scholar), as described elsewhere (19Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (825) Google Scholar). The lengths of all pulses and delays in this sequence were held constant, and 20 spectra were acquired with the strength of the diffusion gradient varying between 5 and 100% of its maximum value. To increase the signal-to-noise ratio and to estimate the experimental error (quoted as ±1 standard deviation), measurements were repeated five, six, and four times for αN, αi, and βO, respectively. The duration of the pulsed gradients (δ) and the stimulated echo delay time (τ) were optimized for each sample to give a total decay in the protein signal of between 80 and 90% for the strongest field gradients. The following values were used for αN, αi, and βO, respectively: δ = 5.5, 5.5, and 6.5 ms and τ = 75, 100, and 150 ms. The protein concentrations were 300, 100, and 150 μm for αN, αi, and βO, respectively. 0.15% dioxan was added as a viscosity probe of known hydrodynamic radius (17Jones J.A. Wilkins D.K. Smith L.J. Dobson C.M. J. Biomol. NMR. 1997; 10: 199Crossref Scopus (205) Google Scholar). The buffer conditions were αN, A, and βO buffer, as appropriate, except that water was exchanged for D2O. The intensities of the signals (S) from the protein and dioxan as a function of the gradient strength (g) were fitted to the Gaussian function S(g)=A exp(−dg2) (Eq. 1) enabling determination of the decay rate d, which is proportional to the diffusion coefficient, D. The hydrodynamic radius of the protein was obtained from that of dioxan, rref, as rref (dref/dprot). NMR Spectroscopy—One-dimensional 1H and two-dimensional heteronuclear HSQC NMR experiments were carried out on a Varian Inova 600 spectrometer with a 1H operating frequency of 600 MHz. The buffers used were as described above except that 10% D2O was added without correcting for the volume effect. All 1H-15N HSQC spectra were recorded at 293 K over a period of about 12 h with a data size of 512 × 256 complex points; the one-dimensional 1H data comprised 1024 complex points. The spectra were internally referenced to 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt. The spectra were processed with NMRPipe (20Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11566) Google Scholar) and analyzed with CARA (21Keller R. The Computer Aided Resonance Assignment Tutorial. CANTINA Verlag, Goldau, Switzerland2004Google Scholar) (freely available via Computer Aided Resonance Assignment). Preparation and Characterization of αN, βO, and αi—Recombinant HuPrP90–231 Val129 was metabolically labeled with 15N, purified, and refolded in vitro, as described previously for unlabeled protein (Ref. 9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar and "Experimental Procedures"). The αN and βO states so formed were identical to those of the unlabeled protein as judged by sec-HPLC, far-UV CD, and ANS binding (Fig. 1, a, c, and e, respectively). The native αN and oligomeric βO states can be clearly differentiated by sec-HPLC with elution times of 9.04 min for αN and 6.4 min for βO, respectively (Fig. 1a). The stability of βO is remarkably high, as judged by reverse phase-HPLC of samples matured for more than a year (9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In an attempt to observe intermediates between αN and βO, the oligomerization process was monitored with sec-HPLC by injecting samples previously refolded into buffer A (see "Experimental Procedures") and incubated at room temperature for different times (Fig. 1b). The transient species appearing in these chromatograms at an elution time of ∼9 min is referred to as αi; it elutes with the same retention time as αN. Although αi and αN cannot be resolved by HPLC, they will be shown by optical and NMR spectroscopy (see below) to be distinct species. Detectable amounts of βO are formed from αi after about 2 h. The reaction takes more than 100 days to come to equilibrium under the conditions chosen (buffer A). Reducing pH and/or raising the urea or protein concentrations accelerates the oligomerization. The first and second HPLC chromatograms in Fig. 1b correspond to the initial state for all the experiments on αi; a fresh preparation was used for each experiment. In the far UV CD measurements (Fig. 1c), the αN state exhibits its α-helical spectrum, whereas βO shows the expected β-sheet character (22Morillas M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Crossref PubMed Scopus (154) Google Scholar). Interestingly, the αi state exhibits a clear α-helix secondary structure, although it was directly refolded in buffer A, which should favor the formation of a β-sheet structure. Judging from the signals at 222 nm, αi has about 45% α-helix structure when compared with 48% in αN and 28% in βO. A clear difference between αN and αi can be seen in the near-UV CD spectra (Fig. 1d). These spectra, which provide a qualitative measure of tertiary structure, display signal loss for αi and βO, whereas αN is clearly highly organized. The dye, ANS, preferentially binds to hydrophobic surfaces of proteins with a substantial increase in fluorescence intensity (23Stryer L. Science. 1968; 162: 526-533Crossref PubMed Scopus (620) Google Scholar). Although its affinity to αN is low, it binds strongly to misfolded isoforms of PrP, in particular to PrPSc and βO (6Baskakov 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 (388) Google Scholar, 24Safar J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. Biochemistry. 1994; 33: 8375-8383Crossref PubMed Scopus (112) Google Scholar). In addition to a significant increase in ANS fluorescence in the presence of βO, we also found an increase for αi (Fig. 1e), which probably reflects the accessibility of the hydrophobic core of this partially unfolded state (25Semisotnov G.V. Rodionova N.A. Razgulyaev O.I. Uversky V.N. Gripas A.F. Gilmanshin R.I. Biopolymers. 1991; 31: 119-128Crossref PubMed Scopus (1237) Google Scholar). Hydrodynamic Radius Measurements with PFG NMR—To assess the aggregation state and compactness of αi, we performed NMR diffusion experiments to measure the hydrodynamic radii (Rh) of αN, αi, and βO (Fig. 2) as described under "Experimental Procedures." The obtained radii were 28.3 ± 1.56, 31.9 ± 0.34, and 57.4 ± 3.4 Ä for αN, αi, and βO, respectively. Wilkins et al. (19Wilkins D.K. Grimshaw S.B. Receveur V. Dobson C.M. Jones J.A. Smith L.J. Biochemistry. 1999; 38: 16424-16431Crossref PubMed Scopus (825) Google Scholar) established empirical relationships between Rh and the number of residues in the polypeptide chain (N): for globular folded proteins, Rh/Ä = 4.75 N0.29, whereas for highly denatured states without disulfide bridges, Rh/Ä = 2.21 N0.57. It was shown that predictions based on these equations agree well with data from dynamic light scattering. HuPrP90–231 comprises 169 residues including the unstructured 26-residue histidine tag construct. The overall fold of αN comprises a globular domain between residues Tyr128 and Tyr226 and two unstructured segments at the N and C termini comprising 70 residues in total. For n = 169, the equations above give Rh = 21.1 and 41.3 Ä, respectively, for a fully folded and a fully unfolded state (with the disulfide bond reduced). A very approximate estimate of Rh can be obtained as follows; considering that 41% of the native protein is naturally unstructured, a simple weighted average of these two values of Rh gives a radius of 29.4 Ä, which compares well with the 28.3 Ä measured for αN. A similar analysis can be performed for βO taking into account its decameric nature (10Tahiri-Alaoui A. Sim V.L. Caughey B. James W. J. Biol. Chem. 2006; 281: 34171-34178Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Assuming no change in the percentage of the monomer unit that is unstructured (an assumption supported by the HSQC spectra discussed below), the radius of βO can be estimated as 10⅓ 29.4 = 63.3 Ä, which is reasonably close to the measured value of 57.4 Ä. In comparison, flow field-flow fractionation measurements and multiple angle light scattering gave a radius of 56 Ä for βO with a mass of about 200 kDa (10Tahiri-Alaoui A. Sim V.L. Caughey B. James W. J. Biol. Chem. 2006; 281: 34171-34178Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and a Stokes radius of 65 Ä was measured with dynamic light scattering (6Baskakov 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 (388) Google Scholar). Interestingly, the radius of αi is 10% larger than that of αN (Fig. 2c). This is in good agreement with the observation of a limited loss of tertiary structure (Fig. 1d) and thus a partial unfolding retaining a high degree of compactness and a monomeric state. One- and Two-dimensional NMR Spectroscopy—The effects of oligomerization can be studied with NMR spectroscopy. The spectrum in Fig. 3a is of the native state and shows good chemical shift dispersion and narrow line widths (4Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar). Given their very different molecular masses, αi and βO can be clearly distinguished as βO shows very broad lines, whereas αi retains relatively sharp ones. The spectrum of αi is clearly distinguishable from the two other states, exhibiting intermediate characteristics between the αN and βO spectra (Fig. 3, a–c). The αi and βO spectra show a loss of high field methyl proton signals when compared with αN, indicating a loss of tertiary structure (Fig. 3, boxed regions). The loss of these resonances is more pronounced for βO than for αi, in agreement with the near UV CD and ANS binding observations above and underlining the intermediate nature of the αi state. Downfield shifts of the backbone α-proton resonances, which appear at around 4.0 ppm, can be observed in both αi and βO spectra. These shifts are most noticeable for βO and correspond well with the observation of a β-sheet-rich secondary structure (26Wishart D.S. Sykes B.D. Richards F.M. J. Mol. Biol. 1991; 222: 311-333Crossref PubMed Scopus (1790) Google Scholar). Similarly, the amide protons exhibit downfield shifts that are pronounced for βO and less so for αi. Chemical shift differences among the three species also exist for the side chain protons, but these are less obvious than for the α and amide protons and somewhat obscured in the spectra by overlap of resonances. The 1H-15N HSQC spectra of αi and βO show a similar set of resonances with almost identical chemical shifts within the random coil region (Fig. 4). Of the 130 resonances expected on the basis of the native αN spectrum (Fig. 4a), only about 70 appear in the spectra of αi and βO (Fig. 4, b and c). The dispersed amide cross-peaks from the core region (residues 128–231) of αN were not observable for αi or βO. The resonances of αi and βO cover a narrow range of 1H chemical shifts in the random coil region between 7.8 and 9.0 ppm and overlap with resonances of the disordered N-terminal segment (residues 90–128) of the native state, αN (4Zahn R. Liu A. Luhrs T. Riek R. von Schroetter C. Lopez Garcia F. Billeter M. Calzolai L. Wider G. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (948) Google Scholar). Comparison of the spectra of αi and βO with the known chemical shifts of αN (15Hosszu L.L. Jackson G.S. Trevitt C.R. Jones S. Batchelor M. Bhelt D. Prodromidou K. Clarke A.R. Waltho J.P. Collinge J. J. Biol. Chem. 2004; 279: 28515-28521Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) allows assignment of 30 out of the 35 resonances in the N terminus. The other signals most probably emanate from the C-terminal His tag. The observation of an unstructured highly flexible N terminus is in good agreement with previous observations that βO and PrPSc have different proteinase K cleavage sites. The cleavage site in βO is around residue Ala117, but it is around Met90 in the authentic, infectious PrPSc (8Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B. Aebersold R. Barry R.A. Tempst P. Teplow D.B. Hood L.E. Prusiner S.B. Weissmann C. Cell. 1985; 40: 735-746Abstract Full Text PDF PubMed Scopus (1253) Google Scholar, 9Tahiri-Alaoui A. Gill A.C. Disterer P. James W. J. Biol. Chem. 2004; 279: 31390-31397Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 27Qin K. Yang D.S. Yang Y. Chishti M.A. Meng L.J. Kretzschmar H.A. Yip C.M. Fraser P.E. Westaway D. J. Biol. Chem. 2000; 275: 19121-19131Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The failure to detect a large number of the expected resonances in the HSQC spectra of αi and βO can be attributed in both cases to severe line broadening. The core of the βO structure tumbles slowly in solution, consistent with the high molecular mass (∼200 kDa (10Tahiri-Alaoui A. Sim V.L. Caughey B. James W. J. Biol. Chem. 2006; 281: 34171-34178Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar)) of the oligomer, and has broad NMR lines, whereas the N-terminal domain has a high degree of internal mobility yielding sharp resonances. The same argument applies for the N-terminal region of αi, but having clearly shown the monomeric nature of this state, we propose (below) an alternative source of line broadening for the core region of this species. To understand the molecular mechanisms underlying the misfolding process, it is generally believed that knowledge of intermediate states is essential. Such intermediates may not be stable at equilibrium, but they might be detectable as transient kinetic intermediates with preformed domains. The refolding process of PrP to its native state, αN, is best described as a sequential three-state reaction with the involvement of an intermediate state (28Apetri A.C. Surewicz W.K. J. Biol. Chem. 2002; 277: 44589-44592Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Moreover, hydrogen exchange experiments (29Nicholson E.M. Mo H. Prusiner S.B. Cohen F.E. Marqusee S. J. Mol. Biol. 2002; 316: 807-815Crossref PubMed Scopus (69) Google Scho
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