ACCELERATED PUBLICATION
Folding of Prion Protein to Its Native alpha -Helical Conformation Is under Kinetic Control*

Ilia V. BaskakovDagger §, Giuseppe LegnameDagger §, Stanley B. PrusinerDagger §, and Fred E. CohenDagger ||**

From the Dagger  Institute for Neurodegenerative Diseases and Departments of § Neurology,  Biochemistry and Biophysics, and || Cellular and Molecular Pharmacology, Pharmaceutical Chemistry, and Medicine, University of California, San Francisco, California 94143

Received for publication, April 10, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric alpha -helical or oligomeric beta -sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the beta -rich isoform of MoPrP is thermodynamically more stable than the native alpha -helical isoform. The conformational transition from the alpha -helical to beta -rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the alpha -helical isoform and folds directly to the thermodynamically more stable beta -rich isoform. Our data demonstrate that the folding of the prion protein to its native alpha -helical monomeric conformation is under kinetic control.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Although protein folding is commonly thought to be controlled by thermodynamic preferences, it has been understood by many, including Anfinsen and others (1,2), that kinetic issues can alter the folding landscape. Whereas most small globular proteins will refold spontaneously in vitro to a native conformation, in vivo folding often exploits auxiliary molecules and defined subcellular compartments to avoid the deposit of misfolded forms (3). Increasingly, a role for protein misfolding in a variety of neurodegenerative diseases has emerged. A common thread joining prion-based diseases and Alzheimer's disease, and possibly Parkinson's disease and frontotemporal dementia, is the conversion of a normal, cellular, monomeric isoform of a protein into a beta -sheet-rich, polymeric form (4-6). When the deposited polymeric form is sufficiently ordered to bind Congo red and exhibit birefringence to polarized light, the pathologic term amyloid is used to cluster these and other maladies (7).

Recent studies by Dobson and others (8-12) have demonstrated that a broad variety of proteins that rapidly fold into monomeric or oligomeric cellular forms under native-like conditions can also be refolded into beta -rich, amyloid forms under conditions that destabilize the native state. So far, these proteins have not been associated with human deposition diseases. This finding has led to the suggestion that the ability to adopt alternative beta -rich folds capable of forming amyloid is not a unique property of specific proteins associated with conformational diseases but reflects a general property of polypeptide chains (13). The interplay between protein concentration and the conformational preferences of the monomeric chain in driving the transition to a beta -rich multimeric isoform remains to be more fully explored.

Glockshuber and colleagues (14) have shown that a fragment of the mouse prion protein folds very rapidly into the alpha -helix-rich conformation with a half-life of 170 µs as measured at 4 °C. Here, we report that a beta -sheet-rich conformation of the mouse prion protein (MoPrP)1 is thermodynamically more stable than its native alpha -helix-rich conformation. The conformational transition from the alpha -helical to a beta -sheet-rich isoform is controlled by a large energetic barrier that is associated with partial unfolding and oligomerization of an intermediate state. Under partially denaturing conditions, it is possible to avoid the kinetic trap that leads to the normal cellular isoform, PrPC, and fold the prion protein directly to a thermodynamically more stable, non-native beta -isoform. Our data demonstrate that folding the prion protein to its native alpha -conformation is under kinetic, not thermodynamic, control.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Protein Preparation-- The expression and purification of recombinant MoPrP 89-231 was performed as described by Mehlhorn et al. (15).

Circular Dichroism-- CD spectra were recorded with a J-720 CD spectrometer (Jasco, Easton, MD) scanning at 20 nm/min, with a bandwidth of 1 nm and data spacing of 0.5 nm using a 0.1-cm cuvette. Each spectrum represents the accumulation of three individual scans after subtracting the background spectra. To monitor the refolding curves, MoPrP was diluted from 10 M to various concentrations of urea in 20 mM sodium acetate in the absence or in the presence of 0.2 M NaCl, pH 3.6, and then incubated at room temperature for different periods of time. No change in pH value was detected during the time course of incubation. To monitor the kinetic trace of the conformational transition, alpha -MoPrP was rapidly mixed with 10 M urea in a 1:1 volume ratio, whereas to monitor the kinetics of refolding to the beta -MoPrP, MoPrP unfolded in 10 M urea was mixed with buffer, again at a 1:1 volume ratio. All kinetic experiments were carried out in 20 mM sodium acetate and 0.2 M NaCl, pH 3.6.

Analysis of the Kinetic Data-- The rate constant and apparent rate order of refolding were calculated from Equation 1,


1/C<SUP>n−1</SUP>=1/C<SUB>0</SUB><SUP>n−1</SUP>+(n−1) k t (Eq. 1)
in which C0 is concentration of the monomer MoPrP at zero time, C is concentration of monomer MoPrP at time t, and n is apparent order of the process. Delta EDagger was calculated from the Arrhenius relation (1) with kobs measured experimentally and k0 determined from the equation for diffusion-controlled reaction, assuming that the reaction follows fifth-order kinetics.

Size-exclusion Chromatography-- All separations were performed at 23 °C with a flow rate of 1 ml/min using TSK-3000 high pressure liquid chromatography gel filtration column (300 mm × 7.80 mm) equilibrated in 20 mM sodium acetate, 0.2 M NaCl, pH 3.6, and the corresponding concentration of urea.

Thioflavin T Assay-- To follow the kinetics of amyloid formation, 0.64 mg/ml beta -MoPrP was incubated in 20 mM sodium acetate and 0.2 M NaCl, pH 5.5, constantly shaken at 36 °C. In the time course of incubation, aliquots of MoPrP were diluted 8 times by phosphate-buffered saline, pH 7.0, and the fluorescence was measured using a LS50B fluorimeter (PerkinElmer Life Sciences) at 482 nm (excitation at 450 nm, excitation slit is 5 nm, emission slit is 10 nm, 0.4-cm rectangular cuvettes) with 5 µM thioflavin T.

Congo Red Binding-- Congo red (Sigma) was dissolved in 5 mM potassium phosphate, 150 mM NaCl, filtered 5 times with a 0.22-mm filter (Millipore, Bedford, MA), and adjusted to 0.2 mM. The difference spectra were obtained by subtracting the Congo red spectra in the absence of MoPrP from the Congo red spectra in the presence of 1.5 µM MoPrP amyloid, corrected for MoPrP scattering.

Electron Microscopy-- Samples were absorbed on carbon-coated, 600-mesh copper grids for 30 s, stained with freshly filtered 2% ammonium molybdate or 2% uranyl acetate, and were viewed in a JEOL JEM 100CX II electron microscope at 80 kV at standard magnifications of 40,000.

    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

To estimate the thermodynamic stability of alpha -MoPrP, its urea-induced unfolding and refolding was measured using the far-UV CD as a probe of its secondary structure. In a low salt buffer, pH 3.6, the urea-induced unfolding profile of the molar ellipticity at 222 nm shows a single cooperative transition between the alpha -isoform and the unfolded state (Fig. 1a). When alpha -MoPrP is unfolded in 10 M urea and then refolded by diluting the urea concentration, its refolding curve expresses hysteresis, a phenomenon indicative of a non-two-state process (Fig. 1a). Both the unfolding and refolding limbs of the curve remain stable for at least 5 weeks when MoPrP is kept in a low salt buffer (20 mM sodium acetate). However, when refolding of MoPrP at 10 µM concentration is performed in a high salt buffer (0.2 M NaCl, 20 mM sodium acetate), the refolding curve undergoes a gradual time-dependent transformation from a single cooperative transition to a transition with local intermediates (Fig. 1b). If a similar experiment is performed at 30 µM MoPrP, the migration of refolding curve occurs more rapidly (Fig. 1c).


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Fig. 1.   Urea-induced unfolding and refolding transitions of MoPrP monitored by CD at pH 3.6. a, unfolding (squares) and refolding (circles) curves monitored upon incubation of 10 µM MoPrP for 10 min (filled symbols) and for 5 weeks (open symbols) at various concentrations of urea. Data were analyzed according to a two-state transition model; the result of the fitting is represented by solid curves (18). b, refolding curves measured upon incubation of 10 µM MoPrP for 10 min (filled circles), 72 h (open circles), 1 week (filled triangles), and 5 weeks (open triangles) after dilution at a particular concentration of urea and 0.2 M NaCl. The solid line represents the unfolding curve from panel a. c, refolding curves measured upon incubation of 30 µM MoPrP for 10 min (filled circles), 72 h (open circles), 1 week (filled triangles), and 5 weeks (open triangles) after dilution at a particular concentration of urea and 0.2 M NaCl. The solid line represents the unfolding curve from panel a. d, far-UV CD spectra of original alpha -MoPrP (solid line), recorded immediately after dilution from 10 M urea (long-dashed line) and after a 5-week incubation following dilution (short-dashed line). e, SEC profile of original alpha -MoPrP (1) and profiles obtained upon incubation for 10 min (2), 72 h (3), 1 week (4), and 5 weeks (5) after dilution from 10 to 1 M urea.

Unfolded MoPrP folds first to the alpha -helical form upon dilution from 10 M urea (Fig. 1d). During incubation for 5 weeks in the high salt buffer, it undergoes a slow conformational transition to the beta -rich form as illustrated by the change in the overall CD spectra, as well as by reduction of the CD signal at 222 nm (Fig. 1, b and d). The conformational transition from the alpha -helical to a beta -sheet-rich isoform is accompanied by oligomerization as judged by size-exclusion chromatography (SEC) (Fig. 1e). Immediately after dilution from 10 M urea, a new peak corresponding to an oligomer appears, in addition to the peak that represents a monomer. During the conformational transition, the population of monomer decreases whereas the fraction of oligomer grows. Although the square variance analysis of the oligomer peak indicates that there is certain heterogeneity of the oligomer species, electrospray mass spectrometry suggests that an octamer is the dominant multimeric assembly (data not shown).

The unfolding and refolding behavior of MoPrP demonstrates hysteresis, a time-dependent transformation of the single transition curve into a double transition curve, and a concentration-dependence for this process. These observations challenge the application of either of the two possible classical three-state models used previously to estimate the thermodynamic parameters for PrP unfolding (16, 17). In contrast, a model with two independent transitions, one between the alpha -isoform and unfolded and the other between the beta -isoform and unfolded, can be used to fit the data. We have observed that the refolding to the alpha -isoform is much faster than the refolding to a beta -isoform, whereas the time-dependent accumulation of a beta -isoform indicates that it is thermodynamically more stable than the alpha -isoform. Thus, MoPrP diluted out of urea folds predominantly to the alpha -isoform with little beta -isoform present. The presence of a beta -isoform would account for the hysteresis between the unfolding and the refolding curves (Fig. 1b). With time, the refolding curve transforms from an apparent single transition to the double transition, demonstrating equilibration of the alpha - and the beta -isoforms.

Direct comparison of the thermodynamic stability of the alpha -and the beta -isoforms illustrate that the alpha -isoform is not the lowest energy state. First, we estimated the thermodynamic parameters for the alpha -isoform using the urea-induced unfolding curve and applying a classical two-state model (see Fig. 1a and Table I) (18). To evaluate the thermodynamic stability of the beta -isoform, two parameters, the molar ellipticity at 222 nm and the fraction of the oligomer, were monitored in parallel as a function of urea concentration after re-equilibration of MoPrP for 5 weeks. Despite the fact that a small fraction of MoPrP remains trapped in the alpha -helical conformation even after 5 weeks, we have exploited the fraction of the beta -oligomer as directly measured by SEC to analyze the "unfolded left-right-arrow beta -isoform" equilibrium using the two-state model (Fig. 2a). The unfolding curve measured by CD requires deconvolution, because it is composed of signals from the beta -isoform, the unfolded state, and the alpha -isoform. Using the population of the monomer measured by SEC as a function of urea concentration and the thermodynamic parameters estimated previously for the "alpha -isoform left-right-arrow unfolded" equilibrium, we calculated the contribution of the alpha -isoform to the CD curve (Fig. 2b). When this contribution is subtracted from the original curve, a curve reflecting the unfolded left-right-arrow beta -isoform transition results. As shown in Fig. 2c, the transition curves for the beta -isoform are superimposible, with Delta G, m, and C1/2 determined from the two techniques equal within the uncertainty of the experiment (Table I). Both Delta G and C1/2 demonstrate that the beta -isoform is thermodynamically more stable than the alpha -isoform (see Fig. 2c and Table I). Because both isoforms can be refolded directly from the unfolded state, we have used the unfolded state as a reference in the free energy diagram (Fig. 2d).

                              
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Table I
Thermodynamic parameters for the urea-induced unfolding of the alpha - and beta -isoforms of recombinant MoPrP 89-231


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Fig. 2.   Estimated thermodynamic stability of beta -MoPrP. MoPrP unfolded at 10 M urea was diluted to various concentrations of urea at pH 3.6 in the presence of 0.2 M NaCl and incubated for 5 weeks at 23 °C before SEC and CD measurements. a, examples of SEC profiles monitored at different concentrations of urea, from bottom to top: 1, 2, 3, 3.5, 4, 5, 6, 7, 8, and 9 M. To apply a two-state model, the population of the beta -oligomer versus urea concentration was normalized relative to the amount of the beta -oligomer observed with 1 M urea taken as 100%. b, CD measurements of the original refolding curve (filled circles) and the transition curve after subtracting the contribution of the alpha -isoform (open circles). The solid line represents the result of the fitting to the two-state model. c, normalized transition of the alpha -isoform (filled circles) and the beta -isoform (open circles) as determined by SEC (solid line) and by CD (dashed line) against varying concentrations of urea. d, free energy diagram for the alpha - and the beta -isoform of MoPrP determined at pH 3.6. U represents the unfolded state.

Although the beta -isoform is thermodynamically more stable than the alpha -isoform, it might be not a true global energy minimum state, because the beta -isoform can undergo an additional time-dependent transition to a polymeric amyloid form. Incubation of beta -MoPrP at 37 °C and constant shaking lead to the formation of higher molecular weight aggregates that possess amyloid properties. The process of amyloid formation monitored by thioflavin T binding displays an apparent latent period and then an exponential accumulation of the aggregate (Fig. 3a). In addition to thioflavin T, the amyloid of MoPrP binds Congo red in a specific manner as judged by birefringence of polarized light and typical red shift of absorbance spectra (Fig. 3b). Aggregated MoPrP forms numerous twisted fibrilar filaments as seen by electron microscopy (Fig. 3c).


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Fig. 3.   beta -MoPrP assembles into amyloid fibrils. a, the kinetics of assembly of 40 µM MoPrP monitored by thioflavin T binding. b, difference spectra obtained at 1.7 (solid line), 3.4 (dotted line), 5.1 (dashed line), and 6.8 µM (dotted-dashed line) Congo red in the presence of 1.5 µM MoPrP taken after 160 h. c, electron micrograph of fibrils negatively stained with ammonium molybdate.

Why is the thermodynamically more stable beta -isoform not accessible during folding under native conditions? Previously, it has been shown that the folding of PrP to the alpha -isoform is an extremely fast, first-order process (14). Folding to the beta -isoform is slower by several orders of magnitude and is concentration-dependent. To prevent the conformational conversion, the alpha -isoform has to be separated by a large energetic barrier from the beta -isoform. Although the free energy diagram does not provide a view of the actual kinetic pathway for the conformational transition, several important observations can be made concerning the origin of the energetic barrier. First, the alpha -isoform has to unfold substantially on route to the beta -isoform. As we have seen before, the alpha -isoform converts very slowly to the beta -isoform at pH 3.6 in the absence of urea (Fig. 1b). This process can be accelerated by shifting the alpha -isoform left-right-arrow unfolded equilibrium toward the unfolded state. After jumping the urea concentration from 0 to 5 M, we observed a very fast loss of secondary structure by the alpha -monomer within the dead time of manual mixing, followed by an accumulation of a beta -sheet-rich conformation (Fig. 4a). This result illustrates that a substantial portion of the energetic barrier requires partial unfolding of the alpha -isoform. The connection between the structural complexity of the pretransition state and the energetic barrier is demonstrated by previous observations that conversion of PrP-derived peptides with low structural complexity into beta -rich isoforms occurs spontaneously and does not require partially denaturing conditions (19-21). Whether the transition state on the way from the alpha - to the beta -isoform is predominantly unfolded under native conditions or whether it has residual beta -sheet or alpha -helical structure remains to be established.


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Fig. 4.   a, the kinetic trace of transition from the alpha -isoform to the beta -isoform (10 µM MoPrP) induced by jumping the urea concentration from 0 to 5 M at pH 3.6, as monitored simultaneously by SEC (open squares) and CD (filled circles). b, the kinetic trace of folding the beta -isoform induced by jumping the urea concentration from 10 to 5 M at pH 3.6 monitored by CD. In the inset, the linearity of the fifth-order (1/theta normalized - 1 versus time) plot suggests that the process of folding may follow an apparent fifth-order kinetics. c, free energy diagram of the conformational transition, representing the activation energy (kcal/mol) estimated at pH 3.6 and 10 µM of MoPrP. TS represents the transitional state. The beta -MoPrP undergoes an additional transition to the amyloid form, represented by the dotted line. d, the activation energy versus the concentration of MoPrP shown at different pH values, as calculated from the Arrhenius relation applying the diffusion-controlled reaction rates. The physiological concentration range of MoPrP is shown in the shaded bar.

A significant contribution to the energetic barrier seems to be associated with the process of oligomerization. As shown on Fig. 4b, the accumulation of a beta -rich conformation is accompanied by oligomerization. The fact that both kinetic curves are superimposible illustrates that the two processes are coupled (Fig. 4a). MoPrP can be refolded directly to the beta -isoform if the unfolded protein is diluted first to 5 M urea (Fig. 4b). When dialyzed out of urea and salt, beta -MoPrP is stable for months at room temperature with no detectable conversion to the alpha -isoform. Analysis of the kinetic traces indicates that the process of folding to the beta -isoform represents a single transition with apparent reaction order of 5, regardless of whether the refolding is initiated by dilution of urea from 10 to 5 M, a jump of the urea concentration from 0 to 5 M, or if the conformational transition occurs in the absence of urea. Such a high order of reaction suggests that the conformational transition will depend upon the concentration of the transition state.

To estimate the energy of activation (Delta EDagger ) of the conformational transition, the Arrhenius relation,
k<SUB>obs</SUB>=k<SUB><IT>0</IT></SUB> <UP>exp</UP>(<UP>−</UP>&Dgr;E<SUP>‡</SUP>/<UP>RT</UP>) (Eq. 2)
can be used, in which kobs is the constant rate of the conformational transition measured experimentally, and k0 is the rate of the process under diffusion control. Under experimental conditions employed (pH 3.6 and 10 µM MoPrP), we found that the alpha -isoform is separated from the beta -isoform by an energy barrier of 20 kcal/mol (Fig. 4c). The energetic barrier is predicted to be much higher under physiological conditions because of the lower concentration of PrP and the higher thermodynamic stability of the alpha -isoform at pH 5-7 (Fig. 4d). For wild-type MoPrP, the calculated energy barrier of 35-45 kcal/mol is sufficient to prevent the process of conformational transition over the functional lifetime of the protein. Hence, a large energetic barrier prevents the conversion of the alpha -isoform to the thermodynamically more stable beta -isoform. From the kinetic perspective, the process of conformational transition can be facilitated by the reduction of the energetic barrier (22). Thus, single point mutations associated with inherited forms of prion diseases might reduce the energetic barrier by stabilizing the transition state. Additionally, if PrPSc provides a template for the conversion of PrPC to PrPSc by binding and stabilizing the transition state, this would also speed up the conformational conversion.

Our results clearly indicate that the folding of native PrPC is under kinetic control. The observations that many proteins are able to adopt alternative amyloid-like folds require us to revisit the role of kinetic traps in protein folding (8-12). If a beta -rich amyloid competent structure is an intrinsic preference especially at a high protein concentration, then compartmentalization of partially folded intermediates and proteins that mediate unfolding and clearance of misfolded proteins play critical roles in cellular health. In addition, side-chain patterns that favor the formation of amyloid, such as alternating polar and non-polar amino acid residues, will be avoided (23). Despite these strategies, some proteins, including PrP, Abeta , alpha -synuclein, parkin and tau, find a route to a beta -rich, multimeric structure with unfortunate consequences.

    FOOTNOTES

* This work was supported in part by grants from the National Institutes of Health (AG0Z132, AG10770, and NS14069), as well as by a gift from the G. Harold and Leila Y. Mathers Foundation. I.B. was supported by the John Douglas French Foundation for Alzheimer's Disease Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Dept. of Cellular and Molecular Pharmacology, University of California at San Francisco, Box 0450, San Francisco, CA 94143. Tel.: 415-476-8519; Fax: 415-476-6515; E-mail: cohen@cmpharm.ucsf.edu.

Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.C100180200

    ABBREVIATIONS

The abbreviations used are: MoPrP, mouse prion protein; PrP, prion protein; PrPC, cellular isoform; PrpSc, scrapie isoform; SEC, size-exclusion chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Anfinsen, C. B. (1973) Science 181, 223-230[Medline] [Order article via Infotrieve]
2. Baker, D., and Agard, D. A. (1994) Biochemistry 33, 7505-7509[Medline] [Order article via Infotrieve]
3. Thomas, P. J., Qu, B.-H., and Pederson, P. L. (1995) Trends Biochem. Sci. 20, 456-459[CrossRef][Medline] [Order article via Infotrieve]
4. Prusiner, S. B. (1997) Science 278, 245-251[Abstract/Free Full Text]
5. Kelly, J. W. (1998) Curr. Opin. Struct. Biol. 8, 101-106[CrossRef][Medline] [Order article via Infotrieve]
6. Cohen, F. E. (1999) J. Mol. Biol. 293, 313-320[CrossRef][Medline] [Order article via Infotrieve]
7. Klunk, W. E., Jacob, R. F., and Mason, R. P. (1999) Methods Enzymol. 309, 285-305[Medline] [Order article via Infotrieve]
8. Chiti, F., Webster, P., Taddei, N., Clark, A., Stefani, M., Ramponi, G., and Dobson, C. M. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3590-3594[Abstract/Free Full Text]
9. Grob, M., Wilkins, D. K., Pitkeathly, M. C., Chung, E. W., Higham, C., Clark, A., and Dobson, C. M. (1999) Protein Sci. 8, 1350-1357[Abstract]
10. Ramirez-Alvarado, M., Merkel, J. S., and Regan, L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8979-8984[Abstract/Free Full Text]
11. Chiti, F., Taddei, N., Bucciantini, M., White, P., Ramponi, G., and Dobson, C. M. (2000) EMBO J. 19, 1441-1449[Abstract/Free Full Text]
12. Yutani, K., Takayama, G., Goda, S., Yamagata, Y., Maki, S., Namba, K., Tsunasawa, S., and Ogasahara, K. (2000) Biochemistry 39, 2769-2777[CrossRef][Medline] [Order article via Infotrieve]
13. Guijarro, J. I., Sunde, M., Jones, J. A., Campbell, I. D., and Dobson, C. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4224-4228[Abstract/Free Full Text]
14. Wildegger, G., Liemann, S., and Glockshuber, R. (1999) Nat. Struct. Biol. 6, 550-553[CrossRef][Medline] [Order article via Infotrieve]
15. Mehlhorn, I., Groth, D., Stöckel, J., Moffat, B., Reilly, D., Yansura, D., Willett, W. S., Baldwin, M., Fletterick, R., Cohen, F. E., Vandlen, R., Henner, D., and Prusiner, S. B. (1996) Biochemistry 35, 5528-5537[CrossRef][Medline] [Order article via Infotrieve]
16. Hornemann, S., and Glockshuber, R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 6010-6014[Abstract/Free Full Text]
17. Swietnicki, W., Petersen, R., Gambetti, P., and Surewicz, W. K. (1997) J. Biol. Chem. 272, 27517-27520[Abstract/Free Full Text]
18. Santoro, M. M., and Bolen, D. W. (1988) Biochemistry 27, 8063-8068[Medline] [Order article via Infotrieve]
19. Supattapone, S., Bosque, P., Muramoto, T., Wille, H., Aagaard, C., Peretz, D., Nguyen, H.-O. B., Heinrich, C., Torchia, M., Safar, J., Cohen, F. E., DeArmond, S. J., Prusiner, S. B., and Scott, M. (1999) Cell 96, 869-878[Medline] [Order article via Infotrieve]
20. Baskakov, I. V., Aagaard, C., Mehlhorn, I., Wille, H., Groth, D., Baldwin, M. A., Prusiner, S. B., and Cohen, F. E. (2000) Biochemistry 39, 2792-2804[CrossRef][Medline] [Order article via Infotrieve]
21. Zhang, H., Kaneko, K., Nguyen, J. T., Livshits, T. L., Baldwin, M. A., Cohen, F. E., James, T. L., and Prusiner, S. B. (1995) J. Mol. Biol. 250, 514-526[CrossRef][Medline] [Order article via Infotrieve]
22. Cohen, F. E., and Prusiner, S. B. (1998) Annu. Rev. Biochem. 67, 793-819[CrossRef][Medline] [Order article via Infotrieve]
23. Broome, B. M., and Hecht, M. H. (2000) J. Mol. Biol. 296, 961-968[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.