Influence of pH on NMR Structure and Stability of the Human Prion Protein Globular Domain

The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121–230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three α-helices, comprising residues 144–156, 174–194, and 200–228, and a short anti-parallel β-sheet, comprising residues 128–131 and 161–164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix α1 is elongated at the C-terminal end with residues 153–156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix α2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121–230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154–157 at the C-terminal end of helix α1 and residues 161–164 of β-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a “starting point” for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopaties. The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121–230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three α-helices, comprising residues 144–156, 174–194, and 200–228, and a short anti-parallel β-sheet, comprising residues 128–131 and 161–164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix α1 is elongated at the C-terminal end with residues 153–156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix α2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121–230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154–157 at the C-terminal end of helix α1 and residues 161–164 of β-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a “starting point” for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopaties. The prion protein (PrP), 1The abbreviations used are: PrP, prion protein; hPrP, human prion protein; HSQC, heteronuclear 1H-15H single-quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy.1The abbreviations used are: PrP, prion protein; hPrP, human prion protein; HSQC, heteronuclear 1H-15H single-quantum coherence; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy. a predominantly synaptic protein present in all higher organisms (1Oesch 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. 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The normal cellular isoform of the protein, PrPC, is soluble and protease-sensitive, whereas the disease-associated β-sheet-rich form, PrPSc, is insoluble, partially resistant to protease digestion (7Prusiner S.B. Groth D.F. Bolton D.C. Kent S.B. Hood L.E. Cell. 1984; 38: 127-134Abstract Full Text PDF PubMed Scopus (372) Google Scholar, 8Pan K.M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z.W. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar), and thought to propagate by converting PrPC molecules into an alternative conformation (9Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5088) Google Scholar, 10Riesner D. Contrib. Microbiol. 2001; 7: 7-20Crossref PubMed Google Scholar, 11Weissmann C. Enari M. Klohn P.C. Rossi D. Flechsig E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16378-16383Crossref PubMed Scopus (102) Google Scholar). Recently, it has been shown that the accumulation of even small quantities of misfolded PrP in the cytosol is strongly neurotoxic in cultured cells and transgenic mice (12Ma J.Y. Wollmann R. Lindquist S. Science. 2002; 298: 1781-1785Crossref PubMed Scopus (427) Google Scholar, 13Ma J.Y. Lindquist S. Science. 2002; 298: 1785-1788Crossref PubMed Scopus (266) Google Scholar). However, the subcellular localization of the conformational transition of PrPC into PrPSc is controversial (14Horwich A.L. Weissman J.S. Cell. 1997; 89: 499-510Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). There are indications that it takes place either at the cell surface, where the average interstitial milieu of the brain (15Chesler M. Kaila K. Trends Neurosci. 1992; 15: 396-402Abstract Full Text PDF PubMed Scopus (474) Google Scholar, 16Chesler M. Prog. Neurobiol. 1990; 34: 401-427Crossref PubMed Scopus (417) Google Scholar) has a pH of 7.3, or after internalization of PrPSc into endosomes (17Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 18Arnold J.E. Tipler C. Laszlo L. Hope J. Landon M. Mayer R.J. J. Pathol. 1995; 176: 403-411Crossref PubMed Scopus (149) Google Scholar, 19Borchelt D.R. Taraboulos A. Prusiner S.B. J. Biol. Chem. 1992; 267: 16188-16199Abstract Full Text PDF PubMed Google Scholar), where pH values range between 4.7 and 5.8 (20Lee R.J. Wang S. Low P.S. Biochim. Biophys. Acta-Mol. Cell Res. 1996; 1312: 237-242Crossref PubMed Scopus (215) Google Scholar).The in vitro conversion of human brain PrPC to a PrPSc-like form is enhanced at acidic pH (21Zou W.Q. Cashman N.R. J. Biol. Chem. 2002; 277: 43942-43947Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Biophysical studies have shown that the free energy of unfolding of hPrP-(90–231) is lower at acid pH than at neutral pH (22Swietnicki W. Petersen R. Gambetti P. Surewicz W.K. J. Biol. Chem. 1997; 272: 27517-27520Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and that in acidic guanidinium chloride hPrP-(90–231) forms a folding intermediate that contains a large amount of β-sheet secondary structure. A β-sheet-rich folding intermediate has also been observed for mouse PrP-(121–231) at low pH in urea but is not seen at neutral pH (23Hornemann S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google Scholar). NMR structures are available for several recombinantly expressed mammalian prion proteins (24Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wüthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1117) Google Scholar, 25James T.L. Liu H. Ulyanov N.B. FarrJones S. Zhang H. Donne D.G. Kaneko K. Groth D. Mehlhorn I. Prusiner S.B. Cohen F.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10086-10091Crossref PubMed Scopus (428) Google Scholar, 26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar, 27Garcia F.L. Zahn R. Riek R. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8334-8339Crossref PubMed Scopus (364) Google Scholar) but only in acidic solution conditions between pH 4.5 and 5.5. A crystal structure of human PrP-(90–231) has recently been determined from crystals grown in pH 8 solution, where two globular domains are linked through interchain disulfide bonds (28Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (457) Google Scholar).In an attempt to investigate the possible effects of pH on the structure of PrPC, we have studied the recombinant human prion protein globular domain of residues 121–230 in pH 7.0 solution. We describe a high quality NMR structure of monomeric hPrP-(121–230), amide hydrogen exchange experiments monitored by NMR, and thermal unfolding experiments monitored by CD. These results are compared with the previously published structural and thermodynamic data of hPrP-(121–230) obtained at pH 4.5 (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar) and with the crystal structure of hPrP-(90–231) determined at pH 8 (28Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (457) Google Scholar).EXPERIMENTAL PROCEDURESSample Preparation— 15N- and 13C,15N-labeled recombinant hPrP-(121–230) was expressed and purified as described previously (29Zahn R. von Schroetter C. Wüthrich K. FEBS Lett. 1997; 417: 400-404Crossref PubMed Scopus (247) Google Scholar). NMR samples were 0.5–1.2 mm in protein concentration in buffer solution containing 10 mm sodium phosphate at pH 7.0 and 0.05% sodium azide. Dynamic light scattering and size exclusion chromatography measurements show that under these conditions samples are homogenous and monomeric. Samples were prepared either in 95% H2O, 5% D2O or in 99.9% D2O.Circular Dichroism and Thermal Denaturation Experiments—Circular dichroism spectra were recorded with a Jasco J720 spectropolarimeter interfaced with a Peltier-type temperature control unit with 1-mm path length cuvette. Thermal denaturation experiments were performed by monitoring the circular dichroism at 222 nm while changing the temperature from 10 to 90 °C or vice versa with a constant temperature gradient of 50 °C/h. Reference spectra were collected at 10 °C before and after each experiment. Denaturation curves were analyzed assuming a two-state unfolding model (30Santoro M.M. Bolen D.W. Biochemistry. 1988; 27: 8063-8068Crossref PubMed Scopus (1593) Google Scholar), where the temperature dependence of the Gibbs free energy change of denaturation, ΔGU–N,is given by Equation 1 (31Privalov P.L. Adv. Protein Chem. 1979; 33: 167-241Crossref PubMed Scopus (2194) Google Scholar), ΔGU-N=ΔHm(1-T/Tm)-ΔCp(Tm-T+Tln(T/Tm))(Eq. 1) where U is unfolded protein, N is native protein, ΔHm is the change in enthalpy at the midpoint of the denaturation temperature, Tm ; ΔCp is the difference in heat capacity between denatured and native protein (ΔCp of hPrP-(121–230) was estimated by assuming 12 cal mol–1 deg–1/amino acid (32Kim D. Kim C. Park C. J. Mol. Biol. 1994; 240: 385-395Crossref PubMed Scopus (5) Google Scholar); T is the absolute temperature in kelvin; R is the gas constant.NMR Measurements and Structure Determination—NMR spectra were recorded on Bruker DRX600 and DRX750 spectrometers. The programs Prosa (33Güntert P. Dötsch V. Wider G. Wüthrich K. J. Biomol. NMR. 1992; 2: 619-629Crossref Scopus (276) Google Scholar) and Xeasy (34Bartels C. Xia T.H. Billeter M. Güntert P. Wüthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1593) Google Scholar) were used for data processing and spectral analysis, respectively. Sequence-specific resonance assignments were derived by adapting the assignments at pH 4.5 (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar) to pH 7.0 and confirmed by standard triple-resonance NMR experiments (35Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (787) Google Scholar). Distance constraints for the structure calculation were obtained from three NOESY spectra recorded at a proton frequency of 750 MHz with a mixing time of 40 ms: a three-dimensional 13C-resolved [1H, 1H]NOESY, a three-dimensional 15N-resolved [1H,1H]NOESY, and a two-dimensional [1H,1H]NOESY spectrum. The program DYANA (36Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar) was used to convert NOE intensities into upper distance bounds according to an inverse sixth power volume-to-distance relationship (37Wüthrich K. NMR of Proteins and Nucleic Acids. Wiley, New York1986Crossref Google Scholar). Final structure calculations using the torsion angle dynamics protocol of DYANA with 8′000 steps were started from 100 randomized conformers. The 20 conformers with the lowest final DYANA target function value were energy-minimized in a water shell with the program Opalp (38Koradi R. Billeter M. Güntert P. Comput. Phys. Commun. 2000; 124: 139-147Crossref Scopus (148) Google Scholar) using the Amber force field (39Cornell W.D. Cieplak P. Bayly C.I. Gould I.R. Merz K.M. Ferguson D.M. Spellmeyer D.C. Fox T. Caldwell J.W. Kollman P.A. J. Am. Chem. Soc. 1996; 118 (2309): 2309Crossref Google Scholar). Figures of molecules were prepared with the program Molmol (40Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-58Crossref PubMed Scopus (6469) Google Scholar).Amide Proton Protection Factors—The exchange rate of amide protons in proteins with deuterium is generally analyzed in terms of a two-state equilibrium between the protected, closed form of the protein and the unprotected, open form. The exchange of amide protons with deuterium takes place from the open form with the intrinsic exchange rate constant k intr, where ko is the first order rate constant for the opening of the folded protein, kc the rate constant for its return to the closed state, and k intr the intrinsic rate constant for exchange with solvent for an unprotected amide proton that is known from model-peptide studies (41Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins. 1993; 17: 75-86Crossref PubMed Scopus (1743) Google Scholar). Under folding conditions kc ≫ ko ; thus the observed exchange rate k obs becomes kobs=(kokintr)/(kc+kintr)SCHEME 1 In the limiting case of kc ≫ k intr (EX2 exchange regime), Equation 1 can be simplified to kobs=(ko/kc)kintr(Eq. 2) In the EX2 regime the exchange data can be used to measure the local equilibrium constant between “open” and “closed” backbone hydrogen bonds. This equilibrium constant is given by the ratio kc /ko , which defines the protection factor (P) of each amide proton. P=kintr/kobs(Eq. 3) The protection factors for each individual residue can be related to the free energy of the apparent opening reaction that dominates exchange under EX2 conditions according to the equation, ΔGHX=RTlnP(Eq. 4) x003C;INLINE-FIGx003E;x003C;LINKLOCATOR=“355922”x003E;x003C;/INLINE-FIGx003E;(Eq. 5) The amide proton exchange rates were calculated following the decrease of two-dimensional [15N,1H]HSQC cross-peak integrals over time after dissolving the lyophilized protein in D2O. The resulting decay curves were fitted to a single exponential decay equation. The intrinsic exchange rates k intr were calculated taking into account the effect of neighboring side chains and corrected for temperature and pH effects (41Bai Y. Milne J.S. Mayne L. Englander S.W. Proteins. 1993; 17: 75-86Crossref PubMed Scopus (1743) Google Scholar).RESULTSResonance Assignment and Structure Determination of hPrP-(121–230) at pH 7.0 —The combination of standard triple-resonance NMR experiments for backbone assignment (35Bax A. Grzesiek S. Acc. Chem. Res. 1993; 26: 131-138Crossref Scopus (787) Google Scholar) allowed nearly complete assignment of the backbone resonances with the exception of the backbone amide protons of Tyr169, Ser170, Asn171, and Phe175. The side chains were assigned based on chemical shift comparisons with hPrP-(121–230) at pH 4.5 (29Zahn R. von Schroetter C. Wüthrich K. FEBS Lett. 1997; 417: 400-404Crossref PubMed Scopus (247) Google Scholar) and have been confirmed using three-dimensional 15N-resolved [1H,1H]NOESY and three-dimensional 15N-resolved [1H,1H]TOCSY spectra. No assignments were obtained for the Hα and Hβ of Tyr169 and Phe175 and the Hγ of Glu168. The labile side chain protons of all seven asparagine and glutamine residues have been assigned as well as the ϵ-proton resonances of five of the eight arginine residues and the η-proton resonances of Arg156 and Arg220. The detection of the NHη of Arg156 and Arg220 indicates that they are involved in salt bridges. The ability to assign the hydroxyl protons of threonines 183, 188, 192, and 199 as well as Tyr157 is indicative of a high level of protection. The upfield-shifted chemical shifts of Hϵ2 and Hδ1 of His140, His155, and His177 indicate that the histidine side chains are predominantly deprotonated.The structure calculation was performed with the program DYANA (36Güntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2545) Google Scholar), and the relevant parameters are given in Table I. The small residual constraint violations show that the structure is consistent with the experimental constraints, and the global root-mean-square deviation values among the bundle of 20 energy-minimized conformers is representative of a high quality structure determination (Fig. 1). The analysis of the family of conformers, the NOE constraints, and the 13Cα chemical shifts (Fig. 2a) indicate the presence of the following secondary structure elements: β-strand 1 with residues 128–131, helix α1 with residues 144–156, β-strand 2 with residues 161–164, helix α2 with residues 174–194, and helix α3 with residues 200–228. Although helix α2 and helix α3 are regular α-helices, helix α1 consists of a regular α-helix comprising residues 144–152 followed by a short 310-helix of residues 153–156. The amide proton of Met134 forms a hydrogen with the of that be with an of the β-sheet the first α-helix with a at residue The of residues which β-strand 2 and helix is and its resonance show of in the time which in the detection of NMR L. D.A. Güntert P. von Schroetter C. Riek R. Zahn R. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google of the energy-minimized NMR structure of at pH for the two the average for the 20 conformers with the lowest residual DYANA target function values and the standard deviation among are upper distance angle DYANA target function distance constraint angle constraint deviation from deviation to the for the two the average for the 20 conformers with the lowest residual DYANA target function values and the standard deviation among are in a of 13Cα chemical shift the hPrP-(121–230) acid hPrP-(121–230) at pH 7.0 the shifts A. J. Biomol. NMR. 1995; PubMed Scopus Google Scholar). hPrP-(121–230) at pH 7.0 hPrP-(121–230) at pH 4.5 (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar). The chemical shifts were using a two-dimensional constant time collected protein solution in 10 mm sodium phosphate at pH The of the regular secondary structure in hPrP-(121–230) at pH 7.0 are given at the and Proton Protection The thermal stability of hPrP-(121–230) was following circular dichroism at 222 nm heat denaturation and pH hPrP-(121–230) a and two-state transition with a temperature of °C and a free energy of unfolding of This value is to mol–1 of mouse PrP-(121–231) G. S. Glockshuber R. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar) as determined from urea equilibrium of backbone amide protons deuterium was after dissolving lyophilized hPrP-(121–230) in D2O and of two-dimensional [15N,1H]HSQC spectra over a time of The data were analyzed on the of an EX2 exchange where the rate constant of is at order of larger than the intrinsic exchange rate constant of unprotected amide protons of the rate constant for of hPrP-(121–230) can be obtained from data on mouse PrP-(121–231) that folding is and in the range of G. S. Glockshuber R. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar), whereas the calculated values of k intr at 20 °C and pH 7.0 are in the range of calculated backbone amide proton protection factors (P) at pH 7.0 are shown in of the amide protons with protection factors 100 are within the regular secondary structure all of the secondary structure of hPrP-(121–230) show protection with the exception of β-strand 1 and the C-terminal of helix The lower thermodynamic stability of the C-terminal end of helix α2 is also by the small values of in free energy of exchange and protection factors (P) of hPrP-(121–230) at pH 7.0 the acid exchange was at 20 °C in 99.9% D2O containing 10 mm sodium phosphate at pH The of the regular secondary structure are given at the segments with exchange within the time for the first two-dimensional [15N,1H]HSQC within the amide protons with a high of the calculated values of the free energy of is to the value of that within these segments of secondary structure exchange from a folding that is to the denatured protein Y. Mayne L. Englander S.W. Science. 1995; PubMed Scopus Google of the Structure of hPrP-(121–230) at pH three-dimensional structures of hPrP-(121–230) at pH 7.0 and 4.5 show global and local The global structure at neutral pH is to that at acidic pH (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar), with a root-mean-square deviation value of between the backbone of residues in the structures determined at the two pH values (Fig. differences between the two structures are at the C-terminal of α1 and of the NMR structures of hPrP-(121–230) determined at pH 7.0 and pH 4.5 The shown is the of backbone of residues The of the is to the global backbone residue of the 20 energy-minimized conformers. The side chains of the residues from the pH 7.0 structure are shown in pH residues 153–156 at the end of helix α1 a 310-helix whereas at acidic pH the same residues show a regular conformation (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar). an elongated helix α1 has also been described for the structure of which was determined from crystals grown in pH 8 solution (28Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (457) Google Scholar). α1 is and has a low capacity to form M.P. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar), that its regular secondary structure be by The protonation of His155 at acidic to the of helix α1 by an to the of the global the backbone of residues at the C-terminal end of helix α2 show a for the bundle of conformers calculated at pH 7.0 than at pH 4.5 (Fig. The helical within is also more at pH as by the of 13Cα chemical shifts shown in The same helical in hPrP-(121–230) at pH 4.5 is in equilibrium with unfolded (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar). In it is to that in the crystal structure of hPrP-(90–231) residues of helix α2 as a for the of helix α3 into the neighboring in the crystal (28Knaus K.J. Morillas M. Swietnicki W. Malone M. Surewicz W.K. Yee V.C. Nat. Struct. Biol. 2001; 8: 770-774Crossref PubMed Scopus (457) Google Scholar). The stability of at low pH could be to the protonation of as histidine side chains in the of have been shown to have a effect of with the helix K.M. Baldwin Proc. Natl. Acad. Sci. U. S. A. 1993; 90: PubMed Scopus Google Scholar).The of the NMR structure of mouse PrP-(121–231) determined at pH 4.5 is by a of and residues (24Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wüthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1117) Google Scholar), and the same is seen also in human (26Zahn R. Liu A. Lührs T. Riek R. von Schroetter C. Garcia F.L. Billeter M. Calzolai L. Wider G. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 145-150Crossref PubMed Scopus (939) Google Scholar). It has been that of the of PrPC with its and also the the cell (24Riek R. Hornemann S. Wider G. Billeter M. Glockshuber R. Wüthrich K. Nature. 1996; 382: 180-182Crossref PubMed Scopus (1117) Google Scholar). The two at and be on the