Formation of Critical Oligomers Is a Key Event during Conformational Transition of Recombinant Syrian Hamster Prion Protein

Abstract

We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90–232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90–232 showed an infrared absorbance spectrum typical of proteins with a predominant α-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3–2 m guanidine hydrochloride), the secondary structure of SHaPrP90–232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel β-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in α-helical structures and the increase in β-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No β-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90–232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90–232. A new model for the structural transition and aggregation process of the prion protein is described. We have investigated the conformational transition and aggregation process of recombinant Syrian hamster prion protein (SHaPrP90–232) by Fourier transform infrared spectroscopy, circular dichroism spectroscopy, light scattering, and electron microscopy under equilibrium and kinetic conditions. SHaPrP90–232 showed an infrared absorbance spectrum typical of proteins with a predominant α-helical structure both at pH 7.0 and at pH 4.2 in the absence of guanidine hydrochloride. At pH 4.2 and destabilizing conditions (0.3–2 m guanidine hydrochloride), the secondary structure of SHaPrP90–232 was transformed to a strongly hydrogen-bonded, most probably intermolecularly arranged antiparallel β-sheet structure as indicated by dominant amide I band components at 1620 and 1691 cm-1. Kinetic analysis of the transition process showed that the decrease in α-helical structures and the increase in β-sheet structures occurred concomitantly according to a bimolecular reaction. However, the concentration dependence of the corresponding rate constant pointed to an apparent third order reaction. No β-sheet structure was formed within the dead time (190 ms) of the infrared experiments. Light scattering measurements revealed that the structural transition of SHaPrP90–232 was accompanied by formation of oligomers, whose size was linearly dependent on protein concentration. Extrapolation to zero protein concentration yielded octamers as the smallest oligomers, which are considered as "critical oligomers." The small oligomers showed spherical and annular shapes in electron micrographs. Critical oligomers seem to play a key role during the transition and aggregation process of SHaPrP90–232. A new model for the structural transition and aggregation process of the prion protein is described. The prion protein (PrP) 1The abbreviations used are: PrP, prion protein; BSE, bovine spongiform encephalopathy; DLS, dynamic light scattering; FTIR, Fourier transform infrared; GdnHCl, guanidine hydrochloride; HPLC, high performance liquid chromatography; huPrP91–231, fragment comprising amino acids 91–231 of the human prion protein; PrPC, cellular isoform of prion protein; SEC, size exclusion chromatography; PrP-res, proteinase K-resistant core of PrPSc; PrPSc, scrapie isoform of prion protein; SHaPrP90–232, fragment comprising amino acids 90–232 of the Syrian hamster prion protein; SLS, static light scattering.1The abbreviations used are: PrP, prion protein; BSE, bovine spongiform encephalopathy; DLS, dynamic light scattering; FTIR, Fourier transform infrared; GdnHCl, guanidine hydrochloride; HPLC, high performance liquid chromatography; huPrP91–231, fragment comprising amino acids 91–231 of the human prion protein; PrPC, cellular isoform of prion protein; SEC, size exclusion chromatography; PrP-res, proteinase K-resistant core of PrPSc; PrPSc, scrapie isoform of prion protein; SHaPrP90–232, fragment comprising amino acids 90–232 of the Syrian hamster prion protein; SLS, static light scattering. is, following the protein-only hypothesis, the sole agent causing a group of neurodegenerative disorders (1Prusiner S.B. Science. 1982; 216: 136-144Crossref PubMed Scopus (4073) Google Scholar, 2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5101) Google Scholar), the so-called prion diseases or prionoses (3Wisniewski T. Aucouturier P. Soto C. Frangione B. Amyloid. 1998; 5: 212-224Crossref PubMed Scopus (41) Google Scholar). The most important ones among them are bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and Creutzfeldt-Jakob disease in humans. The crucial step in transmission and manifestation of prion diseases is the conversion of benign monomeric cellular prion protein (PrPC), which has a mainly α-helical secondary structure, to pathogenic multimeric scrapie prion protein (PrPSc), which is predominantly folded into β-sheets (4Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar, 5Pan K.-M. Baldwin M.A. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2064) Google Scholar). It is noteworthy that PrPC and PrPSc do not differ in their amino acid sequence. Similar mechanisms play an essential role in a number of other neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases. Therefore, the coupled processes of protein misfolding and aggregation, the kinetics of these processes, and the molecular species involved are of fundamental interest. Late products of the conversion are amyloid fibrils and amyloid plaques, which are widely considered to be direct effectors of the above mentioned disorders. However, evidence is accumulating that intermediates or by-products of the assembly process could be the pathogenic form of PrP (6Chiesa R. Harris D.A. Neurobiol. Dis. 2001; 8: 743-763Crossref PubMed Scopus (146) Google Scholar, 7Ma J. Wollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (427) Google Scholar) and other disease-related proteins (8Lashuel H.A. Hartley D. Petre B.M. Walz T. Lansbury Jr., P.T. Nature. 2002; 418: 291Crossref PubMed Scopus (1117) Google Scholar). In vitro transition of unglycosylated recombinant fragments of PrPC into aggregated, β-rich, yet not infectious isoforms has been reported for a number of sequences from different species (9Morillas M. Vanik D.L. Surewicz W.K. Biochemistry. 2001; 40: 6982-6987Crossref PubMed Scopus (154) Google Scholar, 10Baskakov 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 (387) Google Scholar, 11Lu B.-Y. Chang J.-Y. Biochem. J. 2002; 364: 81-87Crossref PubMed Scopus (29) Google Scholar). A particularly interesting β-rich oligomer has recently been reported (10Baskakov 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 (387) Google Scholar, 11Lu B.-Y. Chang J.-Y. Biochem. J. 2002; 364: 81-87Crossref PubMed Scopus (29) Google Scholar). This oligomer was described as consisting of at least eight monomers and appearing as a transient species under conditions favoring the formation of mature fibrillar structures (acidic pH, 2–4 m urea, incubation at 37 °C, constant agitation) (10Baskakov 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 (387) Google Scholar) or as a relatively stable end product under conditions optimized for the formation of the oligomer (pH 4 and 2 m GdnHCl or pH 2 and 5 m urea, incubation at 23 °C) (11Lu B.-Y. Chang J.-Y. Biochem. J. 2002; 364: 81-87Crossref PubMed Scopus (29) Google Scholar). The structural formation of the β-rich oligomer takes place with PrP exhibiting the intact disulfide bridge and comprises the important initial reorganization of the secondary structure, namely a decrease of α-helical structure and an increase of β-sheet structure. Since the β-rich oligomer could be both an important precursor of later stages of the assembly process and a toxic species in itself, we have studied its formation, structural properties, and subsequent structural transitions in more detail using Fourier transform infrared (FTIR) spectroscopy, static (SLS) and dynamic light scattering (DLS), CD spectroscopy, and electron microscopy. The combination of all of these techniques provided solid information on secondary, tertiary, and quaternary structural changes and morphological properties of PrP. Since our expression and purification system differed from those published earlier (12Hornemann S. Korth C. Oesch B. Riek R. Wider G. Wüthrich K. Glockshuber R. FEBS Lett. 1997; 413: 277-281Crossref PubMed Scopus (165) Google Scholar, 13Jackson G.S. Hill A.F. Joseph C. Hosszu L. Power A. Waltho J.P. Clarke A.R. Collinge J. Biochim. Biophys. Acta. 1999; 1431: 1-13Crossref PubMed Scopus (113) Google Scholar, 14Zahn R. von Schroetter C. Wüthrich K. FEBS Lett. 1997; 417: 400-404Crossref PubMed Scopus (248) Google Scholar, 15Mehlhorn I. Groth D. Stöckel J. Moffat B. Yansura D.R.D. Willett W.S. Baldwin M. Fletterick R. Cohen F.E. Vandlen R. Henner D. Prusiner S.B. Biochemistry. 1996; 35: 5528-5537Crossref PubMed Scopus (194) Google Scholar, 16Alvarez-Martinez M.T. Torrent J. Lange R. Verdier J.-M. Balny C. Liautard J.-P. Biochim. Biophys. Acta. 2003; 1645: 228-240Crossref PubMed Scopus (24) Google Scholar), the procedure is described here in detail. It is worth mentioning that it led to a product with >99% high performance liquid chromatography (HPLC) purity. SHaPrP90–232 was expressed in Escherichia coli strain BL21(DE3) (Novagen) transformed by a pET-15b vector (Novagen) in which the sequence corresponding to amino acids 90–232 of the prion protein of the Syrian hamster was cloned. huPrP91–231 was expressed in an equivalent system. The pET-15b vector contains the sequences for a His6 tag and a thrombin cleavage site directly C-terminal to this tag. For cultivation, 100 μl of bacteria suspension were grown for 7–8 h at 37 °C in 10 ml of Luria-Bertoni medium containing 100 μg/ml ampicillin (LB + Amp). This culture was used to inoculate 1 liter of LB + Amp. After growth for 16 h, the culture was centrifuged for 10 min at 2350 × g. The pellet was resuspended in 5 ml of LB medium and used to inoculate 1 liter of LB + Amp. After growth for 30 min at 37 °C, the overexpression of PrP was induced by 1 mm isopropyl-β-D-thiogalactoside. After an additional 5 h at 37 °C, the cells were harvested by centrifugation at 2350 × g. The cell pellet was resuspended in 50 ml of lysis buffer (100 mm Tris, 200 mm NaCl, 10 mm Na-EDTA, 0.2% Triton X-100, pH 7.2) and stirred for 30 min. 1 mmol of MgCl2 (final concentration 19 mm) and 500 units of Benzonase (Roche Applied Science) were added. After an additional 5 min, the lysate was centrifuged at 10,240 × g. The pellet was washed with 50 mm Tris, pH 7.4. For purification, PrP was extracted from cell lysate equivalent to 2 liters of cell culture and reduced by resuspending the pellet in 40 ml of reduction buffer (50 mm Tris, 6 m GdnHCl, 50 mm dithiothreitol, pH 7.4). After centrifugation (30 min at 25,280 × g) and vacuum filtration through a 0.45-μm filter, the protein solution was applied to 10 ml of nickel-nitrilotriacetic acid-agarose (Qiagen), which was prewashed in guanidine buffer (50 mm Tris, 6 m GdnHCl, pH 7.4) and incubated for 30 min under shaking. Nonspecifically bound proteins were removed by washing three times with guanidine buffer and once with guanidine buffer plus 1 mm imidazole. The agarose with bound PrP was filled into an "Econo-Pac" column (Bio-Rad). PrP was eluted by 20 ml of guanidine buffer plus 250 mm imidazole. To prevent formation of oligomers, eluted PrP was collected in 50 ml of guanidine buffer. The concentration of PrP was determined by HPLC analysis (ET 250/4 Nucleosil 300–7 C8 column (Macherey-Nagel); HP 1050 device (Hewlett-Packard)). Elution was performed by a linear gradient of 30–60% eluent B (0.1% trifluoroacetic acid in acetonitrile) in eluent A (0.1% trifluoroacetic acid in water) over 12 min. The solution was diluted to 0.25 mg/ml with guanidine buffer and oxidized by adding 2 μm CuSO4 and stirring overnight at room temperature. Completion of oxidation was monitored by HPLC analysis. After concentration to 1–2 mg/ml, the PrP solution was diluted 1:10 into Tris buffer (20 mm Tris, pH 8.0) to allow refolding of the protein. After 1–2 h, precipitated PrP was removed by filtration. The solution was concentrated to a final volume of 100 ml and dialyzed against Tris buffer. During dialysis, 10 units of thrombin per mg PrP were added. Cleavage of the His6 tag was allowed to take place overnight at room temperature. Completion of cleavage was checked by SDS-PAGE. Due to the vector construction, the expressed and cleaved PrP contained N-terminally four additional amino acids (Gly-Ser-His-Met). The PrP solution was filtrated, 10 μl of protease inhibitor (Complete, Mini, EDTA-free; Roche Applied Science) were added, and the solution was concentrated to a final volume of 25 ml. After dialysis overnight against phosphate buffer (20 mm NaH2PO4, pH 7.0), PrP was analyzed by HPLC, sterile filtrated, and stored at -20 °C until final purification. For final purification, GdnHCl was added to the prepurified PrP samples to obtain a concentration of 6 m. 10–15 mg of PrP were loaded on an EP 250/16 Nucleosil 300–7 C8 column (Macherey-Nagel). A solvent port had to be used to load the column with the sample. To prevent precipitation of the protein in the pump, the HPLC system was prewashed thoroughly with 6 m GdnHCl at a flow rate of 8 ml/min. Elution was performed by a linear gradient of 30–40% eluent B in eluent A over 20 min at a flow rate of 10 ml/min. PrP-containing fractions were pooled, lyophilized, resuspended in guanidine/phosphate buffer (20 mm NaH2PO4,6 m GdnHCl, pH 7.0), and dialyzed against the same buffer to remove residual trifluoroacetic acid, which has a very intense C=O stretching band at 1680 cm-1 in the amide I region. Afterward, PrP was refolded by diluting 1:10 with phosphate buffer. After 1–2 h, precipitated protein was removed by filtrating. The solution was dialyzed against phosphate buffer. For examination at pH 4.2, PrP was furthermore dialyzed against acetate buffer (20 mm sodium acetate, pH 4.2). The purity of the protein was determined by HPLC and SDS-PAGE; identity of the protein was verified by mass spectroscopic analysis. Stopped Flow Device—All measurements were performed using a novel stopped flow device, linked to an IFS 28/B FTIR spectrometer (Bruker Optics, Germany) equipped with a rapid scan option. The stopped flow device was designed specifically for high precision FTIR kinetic and difference spectroscopy of biological macromolecules in 1H2O and is described in detail elsewhere (17Masuch R. Moss D.A. Appl. Spectroscopy. 2003; 57 (in press)Crossref PubMed Scopus (23) Google Scholar). Briefly, the principle elements of the system are a two-channel high pressure syringe pump, a microstructured diffusional mixer, and a specially designed thin layer infrared flow cell. HPLC tubing and fittings are used throughout. Samples are injected into a continuous flow of distilled water via HPLC sample injection valves under computer control, and the measurement is triggered after the flow is stopped when the samples have filled the flow cell. For the measurements described in the present work, a flow rate of 3 ml/min and a sample loop volume of 15 μl were used. The dead time of the experiment (i.e. the time delay between mixing the samples and obtaining the first spectrum) was an average value of 190 ms. Kinetic measurements of the transition of PrP were performed by mixing GdnHCl and PrP solutions in the stopped flow device, and the appropriate control experiments were performed with GdnHCl or PrP alone. The particular advantages of this stopped flow device for the present work were the extremely precise and reproducible cell path length, the short time between sample and reference data acquisitions, and the very gentle mixing achieved with the diffusion micromixer. These benefits resulted in the unprecedentedly high quality of the FTIR spectra presented below. FTIR Parameters—The interferograms were recorded double sided (forward-backward) at a mirror frequency of 200 kHz. The upper and lower frequency folding limits were 7899 and 0 cm-1, respectively. A Blackman-Harris three-term function and a zero filling factor of 4 were used for Fourier transformation, resulting in spectra encoding ∼1 data point per 1 cm-1. The path length of the flow cell was ∼8 μm. The nonlinearity of the MCT detector was corrected prior to Fourier transformation. Acquisition of Protein Spectra—The initial PrP concentration was 8 mg/ml. Since all PrP solutions were diluted 1:2 in the stopped flow device, measurements were performed at a concentration of 4 mg/ml (∼0.24 mm). The GdnHCl-containing solutions were buffered by either 20 mm NaH2PO4 or 20 mm sodium acetate to ensure proper pH values of 7.0 or 4.2, respectively. Steady state spectra of either buffer or PrP plus buffer were measured independently five times with 256 scans in each case and finally averaged. Buffer absorbance was subtracted from PrP plus buffer spectra. Time-resolved spectra of buffered GdnHCl solutions and buffered PrP plus GdnHCl mixtures were measured in the rapid scan mode. All spectra were recorded continuously; the time lag between two spectra was determined by the number of scans per spectrum (see below). The acquisition processor of the FTIR spectrometer had a capacity to store 60 spectra. The early ones were recorded with a low number of scans to get spectra with high time resolution but low signal/noise ratio; the later ones were recorded with more scans to obtain spectra with a higher signal/noise ratio. Since the reaction kinetics of the α-β transition was a function of GdnHCl concentration, the distribution pattern of scans per spectrum was adjusted as needed (Table I).Table IDistribution pattern of scans per spectrum for all measured GdnHCl concentrations Since no more than 60 spectra could be stored for each rapid scan measurement, the number of scans and thus the time of measurement (larger numbers of averaged scans result in longer times of measurement) for each of the 60 spectra had to be chosen in order to observe the whole reaction with an appropriate time resolution.Measurement at 0.3 M GdnHClaTotal time of measurement 380 sMeasurement at 0.5, 0.7, and 1.0 M GdnHClbTotal time of measurement 273 sMeasurement at 1.5 and 2.0 M GdnHClcTotal time of measurement 243 sNo. of spectraNo. of scans averaged per spectrumNo. of spectraNo. of scans averaged per spectrumNo. of spectraNo. of scans averaged per spectrum10210215210410410410810810161032103210321012815128101281051255125512a Total time of measurement 380 sb Total time of measurement 273 sc Total time of measurement 243 s Open table in a new tab The pure PrP spectra were obtained by subtracting GdnHCl and buffer spectra. To obtain the time-dependent changes, the difference spectra between each of the 60 single spectra and the last spectrum (i.e. the 60th spectrum) were calculated. Due to small instabilities of the FTIR spectrometer, base-line shifts in the range of ±0.0002 absorbance units were observed between subsequent spectra and were offset corrected using the region from 1900 to 1750 cm-1, which is free of spectral as a were by the and with A. Chem. Scopus Google Scholar). For of structural changes of PrP place within the dead the difference between the first of the 60 spectra obtained after mixing PrP with GdnHCl and a state spectrum of PrP in the absence of GdnHCl was calculated. and were measured with and the same at a scattering of The equipped with a continuous and a high has been described in detail K. A. M. D. G. Biophys. J. 1997; Scopus Google Scholar). were from the scattering using as a reference sample and a The diffusion were obtained from the measured using either the 1982; Scopus Google Scholar) or the of J. Chem. Scopus Google Scholar). The diffusion were into via the B B is is the in and is the solvent For kinetic light scattering two were used. At high protein solutions and were in At low mixing was filtration into CD measurements were at protein concentrations between and mg/ml on a CD spectrometer, which was with acid at and Jr., PubMed Scopus Google Scholar). and path length cells were used at low and high protein respectively. were using the of Kinetic measurements were by mixing the protein by 20 mm sodium acetate, 50 mm NaCl, pH with a of GdnHCl-containing buffer to obtain a final concentration of 1 m The dead time of mixing was 20 the first spectrum was obtained 1 min after of the for electron samples were diluted to a protein concentration of 30 μg/ml with the corresponding were by with using a with a were used as were with an electron at and a of The time of FTIR spectra between and 1900 cm-1 was analyzed by value using the value of the A fundamental the of the kinetic particularly in the present is the reaction order of the process under Since aggregation is involved in the transition an apparent higher order reaction be into order higher order are by a of the A is the reaction and is the reaction A of the reaction order the of the dependence is to Scholar). This is linear for to from the of the and for 1 and higher order FTIR and CD kinetics were in this In the kinetics were directly by a function with is directly the concentration or is to the from This to the reaction order from the concentration dependence of the apparent reaction rate by of recombinant PrP of coli were obtained in >99% purity as by HPLC, and mass not to the purification The PrP was oxidized as by HPLC not FTIR stopped flow device with its flow cell the of reproducible FTIR spectra of the prion protein in with a high signal/noise of 250 at the amide I cm-1 as is in This independently measured PrP spectra the small absorbance values to the relatively low PrP concentration and the path length of the measurement For band and analysis of spectral were in order to the apparent spectral resolution were according to the (see S. J. Protein Chem. PubMed Scopus Google Scholar, of Spectroscopy. Scholar, A. C. Biophys. 2002; 35: PubMed Scopus Google Scholar). Briefly, the band at cm-1 (see is to the amide I comprising the C=O stretching of all amide the band at cm-1 is to the amide to the and stretching of the secondary amide S. J. Protein Chem. PubMed Scopus Google Scholar). are and the amide I are used to the secondary structure of proteins of Spectroscopy. Scholar, A. C. Biophys. 2002; 35: PubMed Scopus Google Scholar, M. Biochem. Biol. 30: PubMed Scopus Google Scholar). of 1 and revealed a structure of that be to different secondary structure elements and amino acid The at and cm-1 are both to α-helical and the amide I at cm-1 to loop structures of Spectroscopy. Scholar, A. C. Biophys. 2002; 35: PubMed Scopus Google Scholar). a very band is at cm-1 to β-sheet structure. The band at cm-1 is to the amino acid absorbance of and the at and cm-1 are to of and predominantly A. Biophys. Biol. PubMed Scopus Google Scholar). pH 7.0 and 4.2, no structural changes of PrP could be as from the spectra of 1 and and the difference spectrum between both The small difference cm-1 is to a small frequency and of the band between PrP at pH 7.0 and at pH 4.2, in structure. This is in with our CD and which indicated a small decrease in and of PrP on the transition from pH 7.0 to 4.2 (see below). The band at cm-1 very were observed between the FTIR spectra of hamster and human PrP at pH not This is not both proteins have a high sequence of the amino acids are and