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.
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ABSTRACT |
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INTRODUCTION |
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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 -helical conformation and susceptibility to degradation by proteolytic enzymes such as proteinase K. By contrast, PrPSc forms large insoluble oligomers that are rich in
-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
-helices and two short
-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
-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
-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
-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.
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EXPERIMENTAL PROCEDURES |
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Circular Dichroism SpectroscopyThe 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 UreaThe 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 ExperimentsThermal 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 50250 mM NaCl or Na2SO4. These experiments were performed on a Jasco J-810 spectropolarimeter equipped with a Peltier temperature control system.
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RESULTS |
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To obtain a thermodynamic description of salt effect on huPrP90231 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, 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
GUH2O values indicates that sodium chloride induces a significant decrease in the thermodynamic stability of huPrP90231. However, the drawback of this type of analysis is that the
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
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
GU[D]50% values were calculated according to the formula
GU[D]50% =
m
[D]50%, where
[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
GU[D]50% values provide a more reliable measure of relatively small differences in protein stability than the absolute
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
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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Fig. 2 shows the effect of different salts (NaF, Na2SO4, CH3COONa, NaCl) on the thermodynamic stability of huPrP90231 at pH 4 as assessed by the GU[D]50% values. At low concentrations, all salts studied induced a marked protein destabilization (decrease in
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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The observed decrease in the thermodynamic stability of huPrP90231 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 (23 kJ/mol) at pH 4 and 7. However, in relative terms, the effect was significantly stronger at acidic pH because GUH2O for huPrP90231 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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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 (huPrP23231). The glycine-rich N-terminal region (missing in huPrP90231) 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 huPrP90231 (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 122231. 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 huPrP122231 was similar to that observed for huPrP90231 and huPrP23231.
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Thermal Denaturation ExperimentsAs 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.
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DISCUSSION |
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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 huPrP90231 and huPrP122231. 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
> 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 PrP121231, 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 PrP23231 (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.
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FOOTNOTES |
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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; huPrP90231, recombinant human prion protein fragment 90231; huPrP122231, recombinant human prion protein fragment 122231.
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 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).
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ACKNOWLEDGMENTS |
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REFERENCES |
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