pH-dependent Stability and Conformation of the Recombinant Human Prion Protein PrP(90–231)

A recombinant protein corresponding to the human prion protein domain encompassing residues 90–231 (huPrP(90–231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity. Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90–231) are strongly pH-dependent. Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein. At pH between 7 and 5, the unfolding of hPrP(90–231) in guanidine hydrochloride occurs as a two-state transition. This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate. While the secondary structure of the native huPrP(90–231) is largely α-helical, the stable intermediate is rich in β-sheet structure. These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein. A recombinant protein corresponding to the human prion protein domain encompassing residues 90–231 (huPrP(90–231)) was expressed in Escherichia coli in a soluble form and purified to homogeneity. Spectroscopic data indicate that the conformational properties and the folding pathway of huPrP(90–231) are strongly pH-dependent. Acidic pH induces a dramatic increase in the exposure of hydrophobic patches on the surface of the protein. At pH between 7 and 5, the unfolding of hPrP(90–231) in guanidine hydrochloride occurs as a two-state transition. This contrasts with the unfolding curves at lower pH values, which indicate a three-state transition, with the presence of a stable protein folding intermediate. While the secondary structure of the native huPrP(90–231) is largely α-helical, the stable intermediate is rich in β-sheet structure. These findings have important implications for understanding the initial events on the pathway toward the conversion of the normal into the pathological forms of prion protein. Prion diseases comprise a group of transmissible neurodegenerative disorders. The best known animal forms of the disease are scrapie and bovine spongiform encephalopathy; the human versions include kuru, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker disease, and fatal familial insomnia (1Prusiner S.B. DeArmond S.J. Annu. Rev. Neurosci. 1994; 17: 311-339Crossref PubMed Scopus (160) Google Scholar, 2Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Abstract Full Text PDF PubMed Scopus (263) Google Scholar, 3Weissmann C. FEBS Lett. 1996; 389: 3-11Crossref PubMed Scopus (145) Google Scholar, 4Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 181-187Crossref Scopus (2) Google Scholar). All of these disorders are characterized by cerebral accumulation of an abnormal protein, designated PrPres, which has a strong tendency to aggregate into insoluble fibrils. According to the “protein only hypothesis” (5Griffith J.S. Nature. 1967; 215: 1043-1044Crossref PubMed Scopus (899) Google Scholar, 6Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1749) Google Scholar), PrPres constitutes the sole component of the infectious pathogen responsible for the transmission of prion disease. PrPres is derived from a host-encoded glycoprotein, the prion protein (PrP). 1The abbreviations used are: PrP, prion protein; GdnHCl, guanidine hydrochloride; huPrP(90–231), human prion protein domain 90–231; bis-ANS, 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid. The transition between the cellular form of PrP, designated PrPC, and PrPres occurs by a post-translational mechanism and appears to take place without any detectable covalent modifications to the protein molecule (7Stahl 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 (537) Google Scholar). One of the main characteristics distinguishing PrPC from PrPresis the resistance of the latter to proteolytic digestion (8Meyer R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (518) Google Scholar, 9Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. 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). Furthermore, recent spectroscopic studies indicate that the two isoforms have profoundly different conformation: while the secondary structure of PrPC consists largely of α-helices (10Pan K.M. Baldwin M. Nguyen J. Gasset M. Servban 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 (2084) Google Scholar), PrPres appears to be rich in β-sheet structure (10Pan K.M. Baldwin M. Nguyen J. Gasset M. Servban 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 (2084) Google Scholar, 11Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (744) Google Scholar, 12Gasset M. Baldwin M.A. Fletterick R.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1-5Crossref PubMed Scopus (239) Google Scholar, 13Safar J. Roller P.P. Gajdusek D.C. Gibbs C.J. Protein Sci. 1993; 2: 2206-2216Crossref PubMed Scopus (174) Google Scholar). In line with these observations, the current view is that prion diseases may be classified as disorders resulting from abnormal protein folding and that the key event in the pathogenic process is the transition between the “benign” conformation of PrPCand the “pathological” conformation of PrPres. It is believed that the propagation of the disease can be described according to nucleation-dependent polymerization and/or template-assisted models (6Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1749) Google Scholar, 14Gajdusek D.C. J. Neuroimmunol. 1988; 20: 95-110Abstract Full Text PDF PubMed Scopus (123) Google Scholar, 15Jarrett J.T. Lansbury Jr., P.T. Cell. 1993; 73: 1055-1058Abstract Full Text PDF PubMed Scopus (1933) Google Scholar, 16Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 17Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). However, the molecular mechanism and potential intermediate forms of PrP underlying the conformational transition between the normal and pathogenic isoforms of the protein remain unknown. Recent studies have provided an insight into the three-dimensional structure of the recombinant mouse prion protein fragment 121–231 (18Hornemann S. Glockshuber R. J. Mol. Biol. 1996; 262: 614-619Crossref Scopus (74) Google Scholar,19Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1130) Google Scholar). However, bacterial expression of larger PrP fragments proved to be more difficult, resulting in an aggregated protein that could only be solubilized using refolding procedures (20Mehlhorn I. Groth D. Stockel J. Moffat B. Reilly D. Yansura 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, 21Zhang H. Stockel J. Mehlhorn I. Groth D. Baldwin M.A. Prusiner S.B. James T.L. Cohen F.E. Biochemistry. 1997; 36: 3543-3553Crossref PubMed Scopus (168) Google Scholar). In the present study, we have developed an expression system that produces soluble protein corresponding to human prion protein domain encompassing residues 90–231 (huPrP(90–231)). Spectroscopic data show that the stability and conformation of the protein are strongly pH-dependent and suggest that conditions of acidic pH may be conducive to the transition between the conformational states characteristic of PrPC and PrPres. The DNA sequence corresponding to the huPrP(90–231) (Met129 variant) was amplified by polymerase chain reaction from a clone containing the coding region (22Petersen R.B. Parchi P. Richardson S.L. Ulrig C.B. Gambetti P. J. Biol. Chem. 1996; 271: 12661-12668Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) using the primers 5′-CAGCTGGAGAGGAATTCTCAGCTCGATCCT-3′ and 5′-CATGGTGGTGGATCCGGGTCAAGGAGG-3′. The amplified DNA was digested with EcoRI and BamHI (sites indicated in bold in the primer sequences) and cloned into the pET22b(+) vector (Novagen). The cloned protein contained the vector sequence MDIGINSDP fused to the N terminus of the huPrP(90–231). The final construct was confirmed by sequencing the coding region using a Sequenase 2.0 kit (Amersham Corp.) and transformed into an Escherichia coliexpression strain, B834(DE3) (Novagen). DNA manipulations were performed as described by Sambrook et al. (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A midlog phase liquid culture of B834(DE3) cells in 2 × YT medium containing 0.1 mg/ml ampicillin was diluted 9-fold with a fresh medium (to a total volume of 2 liters) and grown at 37 °C until the optical density at 600 nm reached a value of approximately 2. At that time, protein expression was induced by the addition of 0.3 mmisopropyl-β-d-thiogalactoside, and the cultures were grown for an additional hour. Cells from four liters of medium were harvested by centrifugation and sonicated in 200 ml of buffer A (50 mm potassium phosphate, 5 mm EDTA, pH 7.8). After removal of cellular debris, the nucleic acids and some acidic proteins were removed by batch-mode chromatography (in buffer A), using DEAE-Sephadex A-50 (Pharmacia Biotech Inc.) followed by QA resin (Sigma) (in each step, about 2 g of preequilibrated resin was used to treat material recovered from a 4-liter cell culture). The protein was dialyzed overnight at 4 °C against 10 mm phosphate buffer, pH 6.0, and purified by cation-exchange chromatography on a prepacked 5-ml FPLC SP HiTrap column (Pharmacia). About 100 ml of crude material was loaded on the column, and the protein was eluted using a linear gradient of NaCl (0–0.2 m) and a flow rate of 5 ml/min. Individual fractions were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting; those containing huPrP(90–231) (between approximately 5 and 50 ml of gradient volume) were pooled, dialyzed against 50 mm potassium phosphate buffer, pH 7.0, and concentrated to a volume of approximately 6 ml. After this step, the huPrP(90–231) preparation was 80–85% pure. Final purification of the protein was accomplished by size-exclusion chromatography on a 1.6 × 60-cm HiLoad Superdex 75 column (Pharmacia). Two ml of a concentrated material was applied on the column (preequilibrated in 50 mm potassium phosphate, pH 7), and the protein was eluted at a flow rate of 1 ml/min. The protein was finally dialyzed against 10 mm potassium phosphate, pH 7.0, concentrated to 1 mg/ml and stored in small aliquots at −70 °C. To study the unfolding of huPrP(90–231) in GdnHCl, native protein was diluted (to a final concentration of approximately 0.1 mg/ml) in a 50 mm buffer (sodium phosphate at pH 7.2 or sodium acetate at pH 5 and below) containing different concentrations of GdnHCl. Samples were incubated for 24 h at room temperature, and the ellipticity at 222 nm was measured in a 1-mm cell by averaging the signal over 2 min. The denaturation curves were analyzed according to the two- or three-state protein unfolding models (24Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1609) Google Scholar, 25Jackson S.E. Moracci M. elMasry N. Johnson C.M. Fersht A.R. Biochemistry. 1993; 32: 11259-11269Crossref PubMed Scopus (279) Google Scholar). Full far-UV CD spectra of huPrP(90–231) in the presence of 1 mGdnHCl were obtained in a 0.2-mm cell at a protein concentration of 0.8 mg/ml. All CD measurements were carried out at room temperature on a Jasco 600 spectropolarimeter. Protein concentration was determined by using a molar extinction coefficient at 280 nm of 20,800m−1 cm−1, as calculated based on the extinction coefficients of aromatic residues (26Gill S.C. von Hippel P.H. Anal. Biochem. 1989; 182: 319-326Crossref PubMed Scopus (5073) Google Scholar). Protein from a stock solution was diluted to a concentration of 0.05 mg/ml in 50 mm Tris, pH 8.8 and 8.0, or phosphate-citric acid, pH 7–2.6, buffers containing 8 μm bis-ANS (Molecular Probes). After 1-h incubation in the dark, fluorescence of bis-ANS was measured on an SLM 8100 spectrofluorometer using excitation and emission wavelengths of 395 and 495 nm, respectively. To minimize photobleaching of the dye, the excitation slit was set at 1 nm. In this study, we developed an efficient system for expression of soluble recombinant human prion protein 90–231. The protein was secreted into the periplasmic space of E. coli. A simple chromatographic procedure yielded a homogenous protein of a purity greater than 98% as judged by SDS-polyacrylamide gel electrophoresis (Fig. 1). The identity of huPrP(90–231) was confirmed by Western blotting (Fig. 1), mass spectroscopy, and N-terminal sequencing. The CD spectrum of the native protein at neutral pH (Fig. 2) exhibits a double minimum at 208 and 222 nm (with a mean residue ellipticity at 222 nm of −10,000 degree cm2 dmol−1) typical of highly α-helical proteins (27Yang J.T. Wu C.S. Martinez H.M. Methods 1986; PubMed Scopus Google Scholar). The CD spectrum characteristic of α-helical structure was the pH between 7.2 and However, at pH was a in the of the ellipticity at 222 nm to that at 208 nm, a small and/or of α-helical structure. unfolding of huPrP(90–231) in was by the ellipticity at 222 nm as a of in at pH 7.2 the protein a highly two-state transition characterized by a unfolding concentration m) of and a between the native and of A two-state transition unfolding of huPrP(90–231) was at pH at this and to 10 and respectively. A different protein unfolding was at pH unfolding curves at pH and show two transition and indicate the presence of a stable folding intermediate (Fig. The unfolding at pH could be by a three-state transition S.E. Moracci M. elMasry N. Johnson C.M. Fersht A.R. Biochemistry. 1993; 32: 11259-11269Crossref PubMed Scopus (279) Google Scholar), for the transition and of and respectively. The for the transition are of of To insight into the conformational properties of huPrP(90–231) conditions corresponding to the presence of a stable folding we measured the far-UV CD spectra of the protein at pH in the presence of 1 GdnHCl. In the pH between 7.2 and 5, the spectra in 1 are from those in the of and indicate a largely α-helical structure of the protein. These spectra from those obtained in the presence of at pH 4 and conditions to the of a stable folding intermediate of huPrP(90–231). In the latter spectra the double minimum at 222 and 208 nm is by a minimum at approximately nm, a characteristic of proteins rich in β-sheet structure (27Yang J.T. Wu C.S. Martinez H.M. Methods 1986; PubMed Scopus Google Scholar). It be that at a protein concentration to that used in CD the of the folding intermediate on a column is from that of native huPrP(90–231), in a of the protein for The fluorescence of bis-ANS is strongly on the of the is in and to hydrophobic of This has used to conformational in proteins as as to the exposure of hydrophobic patches on the protein surface S. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar, J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). in is fluorescence of bis-ANS in the presence of huPrP(90–231) in the pH between and However, in pH in a dramatic of the fluorescence which at pH the fluorescence of bis-ANS is the fluorescence in the presence of huPrP(90–231) of the to the protein as a of an exposure of hydrophobic patches on the surface of the protein The of this conformational transition in huPrP(90–231) is about The key molecular event in the of prion diseases appears to be a conformational in PrP, resulting in the conversion of a of the protein molecule from an α-helical conformation to a structure (1Prusiner S.B. DeArmond S.J. Annu. Rev. Neurosci. 1994; 17: 311-339Crossref PubMed Scopus (160) Google Scholar, 2Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Abstract Full Text PDF PubMed Scopus (263) Google Scholar, 3Weissmann C. FEBS Lett. 1996; 389: 3-11Crossref PubMed Scopus (145) Google Scholar, 4Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 181-187Crossref Scopus (2) Google Scholar, R.K. McKinley M.P. Bowman K.A. Braunfeld M.B. Barry R.A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2310-2314Crossref PubMed Scopus (518) Google Scholar, 9Oesch B. Westaway D. Walchli M. McKinley M.P. Kent S.B.H. 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, K.M. Baldwin M. Nguyen J. Gasset M. Servban 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 (2084) Google Scholar, 11Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (744) Google Scholar, 12Gasset M. Baldwin M.A. Fletterick R.J. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1-5Crossref PubMed Scopus (239) Google Scholar, 13Safar J. Roller P.P. Gajdusek D.C. Gibbs C.J. Protein Sci. 1993; 2: 2206-2216Crossref PubMed Scopus (174) Google Scholar). Recent studies of the domain of mouse PrP have that this of the protein forms an folding of α-helices and two (18Hornemann S. Glockshuber R. J. Mol. Biol. 1996; 262: 614-619Crossref Scopus (74) Google Scholar, R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wuthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1130) Google Scholar). However, is the molecular of the conformational that conversion of PrPC to PrPres. In is known about the of the N-terminal in the folding of different PrP To these we developed a bacterial expression system that to a recombinant protein corresponding to human PrP domain 90–231. This region of PrP appears to be of the sequence of protein in prion known with human prion and is for the propagation of the disease (1Prusiner S.B. DeArmond S.J. Annu. Rev. Neurosci. 1994; 17: 311-339Crossref PubMed Scopus (160) Google Scholar, 2Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Abstract Full Text PDF PubMed Scopus (263) Google Scholar, 3Weissmann C. FEBS Lett. 1996; 389: 3-11Crossref PubMed Scopus (145) Google Scholar, 4Parchi P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 181-187Crossref Scopus (2) Google Scholar). of the CD data for huPrP(90–231) with those for mouse PrP fragment 121–231 (18Hornemann S. Glockshuber R. J. Mol. Biol. 1996; 262: 614-619Crossref Scopus (74) Google Scholar) that the of α-helical structure in the protein is residue ellipticity at 222 nm, of and degree cm2 for mouse and huPrP(90–231), However, the for proteins the data for huPrP(90–231) were that the N-terminal fragment to the ellipticity at 222 nm. This with stability of mouse and huPrP(90–231) for unfolding at pH 7 of and strongly the recent that the domain residues 121–231 constitutes the only PrP with a and structure M. R. Wider G. Hornemann S. Glockshuber R. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) and against the a between residues and Z. Baldwin M.A. Fletterick R.J. Prusiner S.B. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). The of structure in the N-terminal of huPrP(90–231) is indicated by the fluorescence properties of the sole residue at R. P. and K. While with data for the corresponding to mouse the properties of huPrP(90–231) to in some denaturation curves at pH from those for the recombinant protein corresponding to H. Stockel J. Mehlhorn I. Groth D. Baldwin M.A. Prusiner S.B. James T.L. Cohen F.E. Biochemistry. 1997; 36: 3543-3553Crossref PubMed Scopus (168) Google Scholar). It is that these in the structure of However, be that the corresponding to PrP was expressed as an insoluble protein purification chromatography in an followed by a refolding from a concentrated solution of (20Mehlhorn I. Groth D. Stockel J. Moffat B. Reilly D. Yansura 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, 21Zhang H. Stockel J. Mehlhorn I. Groth D. Baldwin M.A. Prusiner S.B. James T.L. Cohen F.E. Biochemistry. 1997; 36: 3543-3553Crossref PubMed Scopus (168) Google Scholar). This may in a structure that the native conformation of expression system produces a secreted protein that is any refolding The of a soluble recombinant protein corresponding to a of human prion protein studies on the pathway of PrP folding and the of protein folding that are to of PrPres from J. Roller P.P. Gajdusek D.C. Gibbs Jr., C.J. Biochemistry. 1994; PubMed Scopus Google Scholar, J. Semin. Virol. 1996; 7: Scopus Google Scholar). The key of the present study is that the conformational properties of huPrP(90–231) are strongly pH-dependent. One of this pH-dependent is the conformational transition at pH between 6 and This transition a increase in the exposure of hydrophobic without the secondary structure of huPrP(90–231). Furthermore, acidic pH induces a in the unfolding pathway of the protein. in the pH the denaturation of huPrP(90–231) in occurs as a simple two-state transition typical for small In the unfolding curves at lower pH indicate a three-state transition, with the presence of protein folding intermediate. This intermediate appears to have a the of unfolding in is at as as that of the native protein at pH while the secondary structure of the native huPrP(90–231) is largely α-helical, spectroscopic properties of the intermediate are characteristic of a protein rich in β-sheet structure. It be that the folding intermediate described is soluble and to the infectious has in the present However, the that the recombinant huPrP(90–231) can in two stable forms on have different secondary has important implications for understanding the pathway in the PrPC PrPres In this we that the properties of the intermediate hydrophobic this as a for the of a soluble PrP folding intermediate that may the of an insoluble PrPres In the present in the transition to an intermediate was by acidic This is of potential the conversion of PrPC to PrPres appears to take at in the pathway which acidic B. Ernst D. J. Virol. 1991; PubMed Google Scholar, A. Prusiner S.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). However, can that conditions the of a PrP folding intermediate to that in this study may be by with proteins P. M. G. Cohen F.E. Prusiner S.B. 1997; PubMed Scopus Google Furthermore, the properties of the protein may be by the to forms of prion disease P. Petersen R.B. Gambetti P. Semin. Virol. 1996; 7: 181-187Crossref Scopus (2) Google Scholar). The to of soluble PrP in a bacterial expression system a for the of these pathogenic on the folding pathway and conformational stability of