Atypical Effect of Salts on the Thermodynamic Stability of Human Prion Protein*

Adrian C. Apetri and Witold K. Surewicz {ddagger}

From the Department of Physiology and Biophysics and Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106

Received for publication, February 28, 2003 , and in revised form, March 31, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prion diseases are associated with the conversion of cellular prion protein, PrPC, into a misfolded oligomeric form, PrPSc. Previous studies indicate that salts promote conformational conversion of the recombinant prion protein into a PrPSc-like form. To gain insight into the mechanism of this effect, here we have studied the influence of a number of salts (sodium sulfate, sodium fluoride, sodium acetate, and sodium chloride) on the thermodynamic stability of the recombinant human prion protein. Chemical unfolding studies in urea show that at low concentrations (below ~50 mM), all salts tested significantly reduced the thermodynamic stability of the protein. This highly unusual response to salts was observed for both the full-length prion protein as well as the N-truncated fragments huPrP90–231 and huPrP122–231. At higher salt concentrations, the destabilizing effect was gradually reversed, and salts behaved according to their ranking in the Hofmeister series. The present data indicate that electrostatic interactions play an unusually important role in the stability of the prion protein. The abnormal effect of salts is likely because of the ion-induced destabilization of salt bridges (Asp144–Arg148 and/or Asp147–Arg151) in the extremely hydrophilic helix 1. Contrary to previous suggestions, this effect is not due to the interaction of ions with the glycine-rich flexible N-terminal region of the prion protein. The results of this study suggest that ionic species present in the cellular environment may control the PrPC to PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transmissible spongiform encephalopathies or prion diseases are fatal neurological disorders that afflict both animals and humans. The best known animal forms of the disease are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk, whereas the human variants include Creutzfeld-Jacob disease, Gerstmann-Straussler-Scheinker disease, fatal familial insomnia, and kuru (13). The main histopathological marker of these diseases is the accumulation in brain of a misfolded form of the prion protein. The latter species, known as PrPSc,1 accumulates as a result of a conformational conversion of the normal prion protein, PrPC. The PrPC -> PrPSc conversion, which occurs as a post-translational process without any detectable covalent modifications to the protein, likely represents a key molecular event in the pathogenesis of prion disorders. It is also believed that PrPSc acts as an infectious agent that self-propagates by catalyzing the conversion of the endogenous PrPC into the pathogenic PrPSc isoform. Although still controversial (4), the notion that transmissible spongiform encephalopathies can be transmitted by a mechanism involving self-perpetuating changes in protein conformation is supported by a wealth of experimental data (13), including experiments with transgenic animals (5, 6) and the observations that PrPSc can induce the conversion of native PrPC into PrPSc-like conformers in vitro (7, 8). Additional support for the idea that proteins can act as infectious agents has been provided by recent studies on prion-like phenomena in yeast (911).

The mature human PrPC is a 209-residue glycoprotein that is N-glycosylated, contains a disulfide bond between Cys179 and Cys214, and is tethered to the plasma membrane by the C-terminal glycosylphosphatidylinositol anchor (12, 13). Despite an apparent identity of covalent structures, the PrPC and PrPSc isoforms have profoundly different biochemical and biophysical properties. PrPC is a monomeric protein characterized by a high proportion of {alpha}-helical conformation and susceptibility to degradation by proteolytic enzymes such as proteinase K. By contrast, PrPSc forms large insoluble oligomers that are rich in {beta}-sheet structure and partially resistant to proteinase K treatment (13, 14, 15). NMR studies have shown that the monomeric form of the recombinant prion protein (a structural model of PrPC) consists of a largely unordered N-terminal part and the folded C-terminal domain encompassing three {alpha}-helices and two short {beta}-strands (1618). Recent crystallographic studies have captured the folded domain as a domain-swapped dimer with an intermolecular disulfide bridge (19). This dimer, which is only marginally populated in solution but selectively crystallizes, is also {alpha}-helical, and its overall fold is similar to that of the monomer. In contrast to PrPC, little structural data are available for the oligomeric PrPSc conformer.

The molecular mechanism of the self-propagating PrPC -> PrPSc conversion remains enigmatic. Nonetheless, certain clues regarding this mechanism have been provided by the cell-free conversion experiments of Caughey and co-workers (7, 8, 20, 21) and recent studies on conformational transitions of the recombinant prion protein (2224). One intriguing finding of these studies is that the conversion of the recombinant PrP to the oligomeric {beta}-sheet-rich structure (PrPSc-like form) is strongly promoted by the presence of common salts (23), suggesting the importance of electrostatic interactions in prion protein folding and stability. The role of electrostatic interactions is also indicated by theoretical considerations of Morrissey and Shakhnovich (25). These authors have noted that helix 1 of PrP is unique among all naturally occurring {alpha}-helices with respect to its high polarity and lack of hydrophobic contacts with other parts of the molecule. This helix appears to be stabilized almost exclusively by electrostatic interactions such as intrahelical salt bridges. To gain further insight into the role of electrostatic effects in prion protein conformational transitions, here we have performed systematic studies on the effect of salts on the thermodynamic stability of the recombinant PrP. Our data reveal a highly atypical behavior of prion protein with respect to salt-dependent folding/unfolding equilibria in urea, indicating that electrostatic interaction may play an unusually important role in the stability of PrPC.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction and Protein Purification—Plasmids encoding the full-length human prion protein (huPrP23–231) and the N-terminally truncated fragment huPrP90–231 (with N-terminal linkers containing a His6 tail and a thrombin cleavage site) have been described previously (26). The construct for the expression of huPrP122–231 was obtained by deleting the segment coding for residues 90–121 in the vector encoding huPrP90–231. This was accomplished using the QuikChange kit (Stratagene) and the primers 5'-GCGTGGTTCGGTGGGGGGCC-3' and 5'-GGCCCCCCACCGAACCACGC-3'. The DNA sequence of the final construct was verified by automatic sequencing. The proteins huPrP23–231 and huPrP90–231 were expressed in Escherichia coli, purified, and cleaved with thrombin as described previously (26, 27). A similar procedure was used for the purification of the N-terminally truncated fragment huPrP121–231, with the exception that the poly-His tail was cleaved using 0.5 unit of biotinylated thrombin (Novagen; 0.5 unit/mg of protein). Following cleavage, the biotinylated thrombin was quantitatively removed using streptavidin immobilized on agarose beads. The purity of the proteins was better than 97% as determined by SDS-polyacrylamide gel electrophoresis. The concentration of prion protein variants was determined spectrophotometrically using the molar extinction coefficients, {epsilon}276, of 56,650, 21,640, and 15,950 M–1 cm1 for huPrP23–231, huPrP90–231, and huPrP122–231, respectively.

Circular Dichroism Spectroscopy—The far-UV CD spectra (between 190 and 250 nm) were obtained on a Jasco J-810 spectropolarimeter. The measurements were performed at 25 °C using a 1-mm path length cell at a protein concentration of 5 µM. Typically, five spectra were averaged to improve the signal-to-noise ratio, and the spectra were corrected for small buffer contributions.

Equilibrium Unfolding in Urea—The urea-induced unfolding curves of the proteins were obtained at 25 °C using a model JWATS-489 automatic titrator interfaced to a spectropolarimeter. In a typical equilibrium unfolding experiment, two stock solutions of PrP at identical protein concentration (1.4 µM) were prepared: one in buffer alone (native protein) and one in an appropriate buffer containing 9 M urea (unfolded protein). The buffers were composed of 10 mM sodium acetate (pH 4) or 10 mM Tris-HCl (pH 7) and appropriate concentrations of salts studied. For each unfolding/refolding curve, the sample in 9 M urea was titrated (in ~0.1 M urea increments) to a sample of native protein in a 1-cm path length cell. Upon each urea addition, the mixture was incubated for 10 s, and the ellipticity at 222 nm was recorded for 32 s. In the control experiments, it was verified that the incubation time of less than 1 s is sufficient for the system to reach equilibrium (the unfolding/refolding of the prion protein is very fast, occurring on the millisecond time scale (28, 29)) and that the unfolding and refolding reactions are fully reversible. Prior to the unfolding experiments, urea was deionized using a mixture of anion exchange (trimethylbenzylammonium) and cation exchange (Dowex MR-3) resins. The concentration of the denaturant was determined by measuring the refractive index.

Thermal Unfolding Experiments—Thermal unfolding experiments were performed at a protein concentration of 5 µM by monitoring changes in ellipticity at 222 nm as a function of temperature. The measurements were carried out in 100 mM Tris-HCl buffer, pH 7.5, in the presence of 50–250 mM NaCl or Na2SO4. These experiments were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control system.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Equilibrium Unfolding Studies in Urea—In the initial experiments, we have focused on the effect of salts on the thermodynamic stability of the recombinant protein corresponding to human PrP fragment 90–231. This region of the protein is of special interest because it encompasses the entire proteinase-resistant sequence found in prion-infected brain and appears to be sufficient for the propagation of the disease (1). The stability of the protein was studied at acidic and neutral pH by measuring the ellipticity at 222 nm as a function of urea concentration. Under the present experimental conditions, the unfolding/refolding curves were fully reversible, and the protein remained monomeric throughout the time course of the experiments. Representative unfolding curves for huPrP90–231, at pH 4, in the absence and presence of NaCl and Na2SO4 are shown in Fig. 1. All these curves are indicative of a cooperative transition between the native and unfolded states. At low concentrations, both sodium chloride and sodium sulfate shifted the unfolding curves to considerably lower urea concentrations. This effect was reversed at high concentrations of Na2SO4 but not NaCl.



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FIG. 1.
Normalized urea-induced unfolding curves for huPrP90–231 in the presence of increasing concentrations of sodium chloride (left panel) and sodium sulfate (right panel). The unfolding curves were obtained in 10 mM acetate buffer, pH 4. The solid lines represent the best fit of experimental data to a two-state unfolding model.

 

To obtain a thermodynamic description of salt effect on huPrP90–231 stability, we have used two complementary approaches. In the first approach, the individual denaturation curves were fitted according to a two-state model (30), yielding the free energy of unfolding extrapolated to zero denaturant concentration, {Delta}GUH2O, and the m values.2 The above parameters for protein unfolding in the presence of varying concentrations of NaCl are shown in Table I. Inspection of the {Delta}GUH2O values indicates that sodium chloride induces a significant decrease in the thermodynamic stability of huPrP90–231. However, the drawback of this type of analysis is that the {Delta}GUH2O values are subject to relatively large error because of data extrapolation from the transition region to water. To overcome this problem, we have used an alternative procedure originally proposed by Fersht and co-workers (31, 32). In the latter approach, individual unfolding curves were analyzed in terms of the midpoint unfolding concentration of the denaturant, [D]50%, and the m values. These parameters were then used to obtain {Delta}{Delta}GU[D]50%, the difference in the free energy of denaturation in the absence and presence of salt at the denaturant concentration at which 50% of the protein is unfolded. The {Delta}{Delta}GU[D]50% values were calculated according to the formula {Delta}{Delta}GU[D]50% = <m>{Delta}[D]50%, where {Delta}[D]50% is the difference between the value of [D]50% for the protein under the reference conditions (no salt) and in the presence of a given salt, and <m> is the average value of m under different salt conditions studied. Because the [D]50% values are subject to a very small error, the {Delta}{Delta}GU[D]50% values provide a more reliable measure of relatively small differences in protein stability than the absolute {Delta}GUH2O values obtained by extrapolation of experimental data to zero denaturant concentration. It should be noted that the m values for prion protein unfolding in NaCl (Table I) and other salts (data not shown) show no systematic dependence on salt concentration. Therefore, the more accurate approach based on the {Delta}{Delta}GU[D]50% values is fully applicable to our system. The latter approach has been used throughout the remainder of this study.


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TABLE I
Thermodynamic parameters for equilibrium unfolding of huPrP90-231 in the presence of NaCl

{Delta}GUH2O is the free energy of unfolding extrapolated to zero urea concentration. The parameter m represents the urea concentration dependence of the free energy of unfolding, and [D]50% is the concentration of urea at the midpoint of denaturation. The {Delta}{Delta}GU[D]50% values represent the difference in the free energy of denaturation in the absence and presence of NaCl at the urea concentration at which 50% of protein is unfolded. These values were calculated according to the formula: {Delta}{Delta}GU[D]50% = <m>{Delta}[D]50%, where {Delta}[D]50% is the difference between the [D]50% values under the reference conditions (no NaCl) and in the presence of a given concentration of NaCl, and <m> is the average of m values derived from individual unfolding curves at different concentrations of NaCl.

 

Fig. 2 shows the effect of different salts (NaF, Na2SO4, CH3COONa, NaCl) on the thermodynamic stability of huPrP90–231 at pH 4 as assessed by the {Delta}{Delta}GU[D]50% values. At low concentrations, all salts studied induced a marked protein destabilization (decrease in {Delta}{Delta}GU[D]50%). For sodium fluoride, sodium sulfate, and sodium acetate, the destabilizing effect was gradually reversed upon increasing salt concentration between ~50 and 200 mM. No such reversal was observed for sodium chloride. Accurate measurements at salt concentrations above ~200 mM were not feasible because of practical limitations of our experimental setup (the use of an automatic titrator) and potential problems resulting from protein aggregation at very high salt concentration.



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FIG. 2.
The effect of different salts (NaF, Na2SO4, sodium acetate, NaCl) on the thermodynamic stability of huPrP90–231 in 10 mM acetate buffer, pH 4. The {Delta}{Delta}GU[D]50% values represent the difference in stability of huPrP90–231 under the reference conditions (10 mM acetate buffer) and in the presence of a given salt. The {Delta}{Delta}GU[D]50% values were calculated according to the formula: {Delta}{Delta}GU[D]50% = <m>{Delta}[D]50%, where {Delta}[D]50% represents the concentration of urea at which 50% of the protein is unfolded and <m> is the average of individual m values at different salt concentrations. {Delta}GUH2O for urea unfolding of huPrP90–231 at pH 4 in the absence of salts is 14.5 kJ/mol, and the <m> parameter is 3.5 kJ/mol/M.

 

The observed decrease in the thermodynamic stability of huPrP90–231 in the presence of sodium fluoride, sodium sulfate, sodium acetate, and sodium chloride is highly unusual because these salts are generally known to have a stabilizing effect on proteins (3336). To gain insight into the molecular basis of this effect, measurements similar to those described above were performed at neutral pH. Also in this case, a significant destabilization of the protein was observed at low salt concentrations (Fig. 3). However, somewhat higher concentrations of salts were required to produce the maximum destabilizing effect at pH 7 than at pH 4. It should also be noted that, at salt concentrations corresponding to maximum protein destabilization, the absolute decrease in stability was very similar (2–3 kJ/mol) at pH 4 and 7. However, in relative terms, the effect was significantly stronger at acidic pH because {Delta}GUH2O for huPrP90–231 unfolding (in the absence of salt) is 14.5 and 26.5 kJ/mol at pH 4 and 7, respectively. Similar to the behavior observed under acidic conditions, the destabilizing effect at pH 7 was gradually reversed at higher salt concentrations.



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FIG. 3.
The effect of different salts (NaF, Na2SO4, NaCl) on the thermodynamic stability of huPrP90–231 in 10 mM Tris buffer, pH 7. The {Delta}{Delta}GU[D]50% values represent the difference in stability of huPrP90–231 under the reference conditions (10 mM Tris buffer) and in the presence of a given salt. {Delta}GUH2O for urea unfolding of huPrP90–231 at pH 7 in the absence of salts is 26.5 kJ/mol, and the <m> parameter is 4.6 kJ/mol/M.

 

To identify which specific region in the prion protein molecule is responsible for the atypical effect of salt-induced protein destabilization, the thermodynamic measurements were extended to the full-length prion protein (huPrP23–231). The glycine-rich N-terminal region (missing in huPrP90–231) has been previously suggested to modulate the effect of salts on prion protein stability (37). However, the present experiments with two selected salts, Na2SO4 and NaCl, clearly indicate that the effect of ions on the stability of the full-length PrP is very similar to that described for huPrP90–231 (Fig. 4). At low concentrations, both salts induced a destabilization of the full-length PrP. This effect was gradually reversed at higher concentrations of Na2SO4 but not NaCl. In another series of experiments, we have probed the effect of salts on the stability of the N-truncated prion protein fragment corresponding to the autonomously folded domain 122–231. Also in this case, Na2SO4 and NaCl at low concentrations induced significant protein destabilization (Fig. 5). Within the accuracy of the present measurements, the magnitude of salt-induced destabilization of huPrP122–231 was similar to that observed for huPrP90–231 and huPrP23–231.



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FIG. 4.
The effect of sodium sulfate ({blacktriangleup}) and sodium chloride ({circ}) on the thermodynamic stability of the full-length prion protein (huPrP23–231) in 10 mM Tris buffer, pH 7. The {Delta}{Delta}GU[D]50% values represent the difference in stability of huPrP23–231 under the reference conditions (10 mM Tris buffer) and in the presence of a given salt. {Delta}GUH2O for urea unfolding of huPrP23–231at pH 7 in the absence of salts is 25.2 kJ/mol, and the <m> parameter is 4.3 kJ/mol/M.

 


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FIG. 5.
The effect of sodium sulfate ({blacktriangleup}) and sodium chloride ({circ}) on the thermodynamic stability of the huPrP122–231 in 10 mM Tris buffer, pH 7. The {Delta}{Delta}GU[D]50% values represent the difference in stability of huPrP122–231 under the reference conditions (10 mM Tris buffer) and in the presence of a given salt. {Delta}GUH2O for urea unfolding of huPrP122–231 at pH 7 in the absence of salts is 28.1 kJ/mol, and the <m> parameter is 4.7 kJ/mol/M.

 

Thermal Denaturation Experiments—As a complementary approach to the urea-induced unfolding, we have attempted to probe the effect of salts on prion protein stability by the thermal denaturation method. However, under most experimental conditions tested, the thermal denaturation of PrP was irreversible and led to protein aggregation. The above effects were especially pronounced at neutral pH and in the presence of salts. Irreversible aggregation of prion protein at elevated temperatures precluded any meaningful thermodynamic analysis of the thermal denaturation curves.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of recent observations have spurred considerable interest in understanding the role of electrostatic interactions and salts in the folding, stability, and conformational conversions of the prion protein. Of particular significance in this respect is the finding that common salts such as NaCl strongly promote the oligomerization and spontaneous conversion of the recombinant prion protein to a {beta}-sheet-rich form with physicochemical properties similar to those of brain PrPSc (23). Salts have also been found to affect the PrPSc-induced conversion of PrPC in a cell-free assay (21). However, the molecular basis of these effects remains poorly understood. In addition to the experimental observations, the importance of ionic interactions has been emphasized in a recent theoretical study that points to a potentially crucial role of electrostatic effects in stabilizing the native conformation of PrPC (25).

One experimental approach to probe the role of different types of non-covalent interactions in the folding and stability of proteins is to examine the properties of these proteins in the presence of salts. The present study provides direct experimental evidence that, at relatively low concentrations, a variety of salts including sodium fluoride, sodium sulfate, sodium acetate, and sodium chloride decrease the thermodynamic stability of the recombinant human prion protein. This effect occurs for both the full-length PrP as well as its N-truncated fragments huPrP90–231 and huPrP122–231. Destabilization of native protein structure by salts such as NaF, Na2SO4 or CH3COONa is highly unusual. Indeed, the effect of different anions on protein thermodynamic stability usually follows their ranking in the Hofmeister series, the order of which is: F {approx} > CH3COO > Cl > Br > I. Anions at the beginning of the series (kosmotropes) are known as stabilizing agents, whereas those toward the end of the series (chaotropes) act as destabilizing agents (38). To the best of our knowledge, no other protein has been reported to be destabilized by kosmotropes such as fluoride, sulfate, or acetate. Therefore, the present finding points to rather unique properties of the prion protein.

In a recent theoretical study, Morrissey and Shakhnovich (25) have pointed out an unusual characteristic of helix 1 in PrPC; this helix (DYEDRYYREN) is uniquely polar and has a remarkably low capacity to form tertiary hydrophobic interactions. Therefore, it must be stabilized almost exclusively by electrostatic interactions. The most important of the latter appear to be two internal salt bridges, one linking Asp-144 with Arg-148 and one between Asp-147 and Arg-151 (or Arg-151 and Glu-152). Our data are fully consistent with this theoretical model, providing direct experimental support to the notion that electrostatic interactions play an unusually important role in the stability of PrPC. Regardless of their ranking in the Hofmeister series, ionic compounds would be expected to weaken intrahelical salt bridges. In the absence of stabilizing hydrophobic tertiary interactions, this would greatly compromise the stability of helix 1. Given the cooperative nature of intramolecular interactions in folded proteins, such a local effect could result in global destabilization of the native PrPC conformer.

It should be noted that the effect of salts on the stability of prion protein is biphasic. For anions ranking high in the Hofmeister series, the destabilizing effect observed at low salt concentrations is gradually reversed when the concentration of ions is increased above ~50 mM. This biphasic response suggests that the destructive effect on the structure-stabilizing salt bridges in PrP saturates at relatively low ion concentrations. At higher salt concentrations, the classical "kosmotropic" effects become dominant, resulting in gradual protein stabilization as predicted by the Hofmeister series. Consistent with this interpretation, no reversal of the destabilizing effect was observed for the chloride ions that rank in the middle of the Hofmeister series. Although the molecular basis of the Hofmeister series is not fully understood, the ranking of anions in the series is related to their effect on the surface tension of water (33, 38).

It was recently reported that, at neutral pH, salts such as sodium chloride, sodium sulfate, and sodium fluoride decrease the apparent temperature of the thermal denaturation of mouse and sheep prion protein (37). This effect was especially pronounced at high salt concentrations. Based on the comparison of thermal denaturation curves for the full-length PrP and the N-truncated variant PrP121–231, the authors have attributed this effect to specific interactions of anions with glycine residues present in the unstructured N-terminal part of PrP. It was proposed that the preferential interaction of anions with the flexible N-terminal part somehow "signals conformational changes in the structured region," resulting in a global destabilization of the protein molecule. The results of the present study are at odds with the above interpretation. In contrast, the present data demonstrate that the unusual effect of salts on prion protein thermodynamic stability is essentially independent of the presence of the glycine-rich N-terminal region. This effect can be best rationalized by unusual properties of helix 1 and, especially, the role of intrahelical salt bridges in prion protein stability. To explain this apparent discrepancy, we have reexamined the thermal denaturation of the prion protein under experimental conditions similar to those used previously (37). The ellipticity versus temperature curves obtained in our experiments with the full-length human prion protein were essentially identical to those previously reported for murine PrP23–231 (37). Consistent with the latter report, we have also found that salts induce a significant shift of these curves to lower temperatures (data not shown). However, we have noticed that, under the experimental conditions used (0.1 M Tris buffer, pH 7.5), thermal denaturation of prion protein is not fully reversible and results in protein aggregation. This effect was especially significant in the presence of salts. For example, in Tris-HCl buffer containing 250 mM Na2SO4, only ~35% of the original ellipticity was recovered upon cooling of the sample to room temperature. Under these conditions, the majority of the protein formed large aggregates and precipitated from solution. Given the irreversibility of the reaction and protein aggregate formation, no reliable conclusions can be drawn from thermal denaturation experiments regarding the effect of salts on the thermodynamic properties of PrP. The findings reported in the previous study could be largely because of the salt-mediated effect of the glycine-rich region on prion protein aggregation, not its thermodynamic stability. The latter possibility is especially plausible because the reported decrease in the apparent midpoint denaturation temperature is particularly large in the presence of high concentrations (e.g. 0.25 M) of sodium sulfate, i.e. under the conditions that are especially conducive to prion protein aggregation at elevated temperatures. The present urea unfolding experiments clearly demonstrate that the decrease in the thermodynamic stability of the PrP monomer occurs only at low concentrations of Na2SO4. At higher salt concentration, this destabilization is gradually counteracted by the increasing strength of the Hofmeister-type effects.

The interpretation of the present data in terms of salt-induced destabilization of ionic interactions in helix 1 is generally consistent with the results of a recently published study (39) in which Asp-144 and/or Asp-147 in hamster PrP were replaced with Asn. These substitutions, designed to abolish one or two salt bridges in helix 1, resulted in a decreased thermodynamic stability of the protein. The magnitude of this effect was relatively small. However, it should be noted that the unfolding experiments in the latter study were performed using an ionic denaturant guanidine HCl. The present observation that any ionic species destabilizes the structure of the wild-type PrP could complicate the interpretation of these recently published data.

It has also been reported that mutants designed to destroy salt bridges in helix 1 convert more efficiently to a PrP-res form (39). This, in combination with the present finding, may explain the intriguing observation that sodium chloride acts as a strong promoter of prion protein transition to a scrapie-like form (23). Destabilization or removal of salt bridges in helix 1, either by the action of salt or mutations, could promote formation of partially unfolded intermediates that may be necessary for the PrPC -> PrPSc conversion. The role of such intermediates in prion protein folding is strongly supported by recent kinetic stopped-flow and NMR data (29, 40, 41). Alternatively, as proposed recently (39), the removal of salt bridges in helix 1 could facilitate the conversion reaction by eliminating the population of non-convertible conformers on the PrPC -> PrPSc conversion pathway. Regardless of the specific mechanism, the present data demonstrate a highly atypical effect of salts on prion protein properties, suggesting that ionic species present in the cellular environment may control the PrPC -> PrPSc conversion by modulating the thermodynamic stability of the native PrPC isoform.


    FOOTNOTES
 
* This work was supported by National Institutes of Health Grant NS38604 (to W. K. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-0139; Fax: 216-368-1693; E-mail: wks3{at}po.cwru.edu.

1 The abbreviations used are: PrPSc, scrapie PrP isoform; PrP, prion protein; PrPC, cellular PrP isoform; PrP-res, proteinase K resistant form of PrP; huPrP90–231, recombinant human prion protein fragment 90–231; huPrP122–231, recombinant human prion protein fragment 122–231. Back

2 It should be noted that the apparently good mathematical fit of experimental data to a two-state model may be fortuitous because equilibrium unfolding data alone are insufficient to prove the applicability of this model. Recent kinetic stopped-flow experiments point to the involvement in prion protein folding of a transient intermediate (29). However, under equilibrium conditions this intermediate is populated at a very low level. Therefore, the {Delta}{Delta}GU[D]50% values obtained from the analysis of equilibrium unfolding data provide a reliable measure of the total free energy difference between the native and fully unfolded states of the prion protein (29). Back


    ACKNOWLEDGMENTS
 
We thank Dr. Krystyna Surewicz for her contribution to the molecular biology (construct preparation) part of this work.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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