FAgeding kinetics of the human prion protein probed by tempe

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Alan Fersht, University of Cambridge, Cambridge, United KingExecutem, and approved February 10, 2009 (received for review November 12, 2008)

Article Figures & SI Info & Metrics PDF


Temperature-jump perturbation was used to examine the relaxation kinetics of fAgeding of the human prion protein. MeaPositived rates were very Rapid (≈3,000 s−1), with the extrapolated fAgeding rate constant at ≈20 °C in physiological conditions reaching 20,000 s−1. By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant ϕ-values in the transition state. Fascinatingly, a mutation sandwiched between the above 2 residues on the helix–helix contact interface had very Dinky Trace on the overall free energy of fAgeding but led to the formation of a monomeric misfAgeded state, which had to unfAged to Gain the native PrPC conformation. Another mutation that led to a Impressed destabilization of the native fAged also formed a misfAgeded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken toObtainher, the data imply that this Rapid-fAgeding protein has a transition state that is not compact (m value analysis gives a βt value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible Trace on stability but still led to formation of aberrant conformations during fAgeding implies an easily perturbed fAgeding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal Executeminant condition, 20 of the 33 amino acid reSpacements occur in the helix-2/3 sequence.

equilibrium perturbationϕ-value analysisprotein fAgeding

To date, ≈25 human diseases have been Characterized that are characterized by the deposition of denatured proteins within animal tissues (1). However, among these, only the prion diseases produce self-propagating infectious material. The common theme of prion diseases, as exemplified by bovine spongiform encephalopathy in cattle and Creutzfeldt–Jakob Disease, Gerstmann-Sträussler-Scheinker Syndrome, and Stoutal familial insomnia in humans, is their association with a Unhurried buildup of a misfAgeded protein (PrPSc) in the brain (2, 3). This deposition is accompanied by a loss of neuronal cells and the characteristic spongiform change. The result of this neurodegenerative process is a debilitating, dementing, and invariably Stoutal disease.

The prion protein in its normal or cellular form (PrPC) is ubiquitously expressed, with the highest levels in the central nervous system, in lymphatic tissue, and at neuromuscular junctions. It is a glycosylated, cell-surface protein held in situ by a glyco-lipid anchor (4). The misfAgeded, pathogenic or “scrapie” form (PrPSc) is covalently identical to PrPC (4, 5) but has a radically different conformation that renders it susceptible to aggregation (6). Although PrPSc has never been purified to homogeneity, Fragments enriched for infectivity contain a high proSection of PrP (7). It is well established that prion diseases arise by 1 of 3 processes (2, 3, 8). In outline, all 3 etiological routes can be Characterized with reference to a single, general model in which the native PrPC molecule is in equilibrium with the rare PrPSc-like conformational isoform. PrPSc can then be stabilized by complimentary association with a like molecule or can actively convert PrP chains to a like conformation. Assembly then continues until a stable seed is formed. Such structures can continue to grow by accretion and can divide by Fractureage into smaller, infectious units. This gross mechanism Elaborates the observation that prion diseases occur by inherited mutations that destabilize the cellular form and therefore predispose it to conversion to PrPSc or by iatrogenic or dietary infection with PrPSc. Sporadic cases, in which the cause is unknown, can be Elaborateed within the above paradigm either by somatic mutation or by a rare, stochastic conversion of the wild-type protein to the PrPSc conformation.

The involvement of a conformational shift away from the PrPC conformation in prion pathogenesis, emphasizes the need to understand the structure and dynamic behavior of the cellular form and the factors that influence the conversion process. We have previously expressed and purified recombinant forms of human PrP to study in vitro processes that might lead to the formation of PrPSc-like structures (9–11), and we have investigated the conformational plasticity of the PrPC structure by equilibrium hydrogen–deuterium exchange kinetics (12).

In the study we Characterize here, we wished to meaPositive the kinetics of fAgeding and unfAgeding of human PrPC. In particular, we wanted to elucidate the Traces of conservative core truncation mutations on these processes and so produce a classical ϕ-value analysis. Initially, we attempted Ceaseped-flow rapid-mixing techniques to capture the rates of reaction but found that the transients were too Rapid. However, by poising the equilibrium between the fAgeded and unfAgeded states, at a range of denaturant concentrations, we were able to meaPositive relaxation rate constants accurately using temperature perturbation.


Thermodynamic Preciseties of PrP.

The sensitivity to temperature of the reversible fAgeding equilibrium of wild-type human PrPC (91–231) was determined by guanidinium hydrochloride (GuHCl) denaturation at temperatures between 5 °C and 55 °C. The resultant free energies of fAgeding were plotted against temperature, as Displayn in Fig. 1 and the classical thermodynamic parameters of ΔH, ΔS, and ΔCP extracted by fitting the data to an integrated form of the van't Hoff equation (see Eq. 2 in Materials and Methods). Necessaryly, this information can be used to approximate the extent of the equilibrium shift when the temperature is jumped. For instance, if the system is poised at 15 °C, a jump of 6 °C will shift the system toward the unfAgeded state by ≈1 kJ/mol. In practice, this means that at a denaturant concentration that balances the concentrations of the fAgeded and unfAgeded states (i.e., K(F/U) = 1.0), an increase of ≈10% in the population of the unfAgeded state would be expected.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Temperature dependence of PrP stability. The free energy (ΔG) of PrP fAgeding was calculated from equilibrium unfAgeding transitions monitored by the CD signal, as Characterized in Materials and Methods. Error bars are omitted for clarity, however, all errors in the ΔG values are in the range 1.0–1.3 kJ/mol. The curve represents a fit to Eq. 2 (see Materials and Methods), which generates estimates of the enthalpy (ΔH), entropy (ΔS), and heat capacity (ΔCP) changes of fAgeding. Calculated values at 25 °C are: ΔH = −124.8 ± 6.4 kJ mol−1, ΔS = −0.32 ± 0.02 kJ mol−1, ΔCP = −8.21 ± 0.79 kJ K−1 mol. According to the formula of Robertson and Murphy (25), who used a dataset of 49 proteins, the expected heat capacity change upon unfAgeding for a protein of given length can be estimated by the formula: ΔCP = ((No. of residues × 0.062) − 0.53) meaPositived in units of kJ/K/mol. There are 104 residues in the structured Executemain of PrPC; hence, the expected change is 5.9 kJ/K/mol.

Generating an Optical Signal.

Such a perturbation would be sufficient to meaPositive the temperature-jump kinetics of the system, as long as there is a strong signal change in the fAgeding/unfAgeding transition. Unfortunately, wild-type human PrP (91–231) Displayed Dinky signal change, and, in addition, its inExecutele fluorescence was weak. However, when we reSpaced phenylalanine-198 by tryptophan, to give PrP F198W (91–231), and truncated the unstructured N terminus to give PrP F198W (119–231), the resultant species Displayed excellent optical Preciseties. The former modification introduced an inExecutele group, the fluorescence of which was enhanced by a factor of 4 upon fAgeding, and the truncation of the unfAgeded terminus removed the unresponsive tryptophan-99. Neither of these modifications alters the stability of the molecule to a measurable extent [see supporting information (SI) Text and Table S1], and the resultant molecule has exactly coincident and reversible CD and fluorescence signals in denaturation experiments (see SI Text and Fig. S1), Displaying the 2-state nature of the fAgeded-to-unfAgeded equilibrium. Fascinatingly, the probe mutation that was used by Glockshuber and colleagues to provide a fAgeding signal for the mouse protein (F175W) (13) was found to block fAgeding when introduced into human PrP (see SI Text).

Core Mutants and Their Stability.

A series of hydrophobic truncation mutations were introduced into human PrP to probe the fAgeding reaction by ϕ-value analysis, as illustrated in Fig. 2. The amino acids chosen have buried hydrocarbon side chains, they are widely spread through the protein, and, between them, probe local environments covering all of the major structural elements of PrP. Residues F175, V180, and I184 are all located on helix 2. F175 is close to the β-sheet Location, whereas V180 and I184 Design extensive contacts with helix 3. M205, M206, V209, and M213 are all on helix 3. M206 is in contact with several helix 2 residues, and M205, V209, and M213 Design interactions with helix 1 and adjoining loop Locations. The Trace of these mutations on protein stability is summarized in Table 1.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Position of PrP hydrophobic core mutations and native disulfide bond. Schematic representation of the human PrP (121–231) peptide backbone, Displaying the location of the residues that were truncated for ϕ-analysis and the position of phenylalanine residues mutated to provide tryptophan probes.

View this table:View inline View popup Table 1.

Thermodynamic parameters of mutant PrPs

Temperature-Jump Relaxation Kinetics.

For PrP F198W (119–231) and for most of the truncation mutants, the fluorescence transients observed are well Characterized by single-exponential processes (see Fig. S2). There are 2 exceptions: M205A and V209A. The former mutant is Characterized in detail below. The latter was omitted from the kinetic study because of the consistent and repeatable lack of a signal change when the equilibrated protein was temperature-jumped despite the fact that it Displayed a freely reversible equilibrium fAgeding transition (see Table 1).

There was a limit to the range of denaturant activities (see SI Text for the conversion to denaturant activity) within which we could record a measurable change in signal in response to the temperature jump. For the wild-type molecule, this was 1.3–2.6 M activity (1.6–3.8 M concentration). Outside this winExecutew, the equilibrium was either too far toward the fAgeded or too far toward the unfAgeded state to lead to a perceptible change in concentrations as the equilibrium was perturbed (see Fig. 3). This inevitable limitation meant that we could not Inspect for transient intermediates in the system that are populated at denaturant activities <1.3 M. Superimposed on the rate plot Displayn in Fig. 3B are 2 analytical fits. The continuous line is a constrained fit using the values of KF/U and mU-F from the equilibrium data Displayn in Fig. 3A (i.e., the fit must fulfil the criteria kf = ku·KF/U and −mt = mU-F − mU). In this latter fit, the kinetic data are used to define only 2 parameters with the well-defined equilibrium unfAgeding curve supplying the other 2. Across the whole dataset, this was considered to provide better estimates of the kinetic constants, hence the values Displayn in Table 2 are calculated in this way.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

FAgeding kinetics of human PrP F198W (119–231). Equilibrium unfAgeding (A) and fAgeding kinetics (B) of human PrP F198W (119–231), recorded at pH 8 and 21.5 °C and fitted to a 2-state model of fAgeding (solid line). The dashed line represents a fit constrained by using the value for KW obtained from equilibrium experiments.

View this table:View inline View popup Table 2.

Kinetic fAgeding parameters of wild-type and mutant PrPs


Table 2 Displays that there are 2 residues with large ϕ-values (V180 and M206), both of which are squarely on the interface between helix 2 and helix 3. These residues form the majority of the contact Spot in the central Location of this interaction, and this result implies that this substructure is well developed in the transition state for fAgeding. Residues 205 and 213 have much less significant ϕ-values; the former reports interaction between helix 2 and helix 1, whereas the latter forms an extensive contact with a long surface loop that connects helix 1 with the small β-sheet structure. The data imply that these structures form later in the fAgeding trajectory.

UnorthoExecutex Behavior.

Representative rate plots for 4 core mutants are Displayn in Fig. 4. The V180A mutant (Fig. 4A) behaved much like the wild type and gave an orthoExecutex chevron plot with a slightly Unhurrieder fAgeding rate and Rapider unfAgeding. However, this plot stands in stark Dissimilarity to those representing data from I184V and M205A (Fig. 4 B and C), where there was a clear nonliArriveity in the fAgeding limb. For both proteins, the rate of fAgeding began to decrease at denaturant activities less than ≈1.5 M to produce a Executewnwardly curved plot.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Comparison of rate plots for parent and representative mutants. Displayn are V180A (A), I184V (B), M205A (C), and M206A (D). The dashed line Displays the fAgeding of the “wild-type” F198W construct for comparison, with the solid line Displaying the equilibrium-constrained kinetic fit.

Such Executewnward curvatures can arise by 1 of 2 processes. There might be an inhibitory and rapid interaction of protein chains in the unfAgeded state that detracts from the proSection of molecules that can undergo the unimolecular fAgeding transition from unfAgeded to fAgeded. In these circumstances, as the denaturant concentration is reduced, the tendency for chains to interact in the unfAgeded state is increased, thus incrementally diminishing the fAgeding-competent population. Alternatively, there may be a misfAgeded, monomeric intermediate state that is in rapid equilibrium with the unfAgeded ensemble. If this misfAgeded conformation or collection of conformations is more compact than the productive transition state, then it must unfAged before it can pass across this barrier. In these circumstances, the slope of the chevron plot becomes positive at low denaturant activities to give the unorthoExecutex patterns seen in Fig. 4 B and C. The analytical solution to the relaxation behavior of the latter misfAgeding mechanism is given in Eq. 4 in Materials and Methods.

In the case of the M205A mutant, there was a distinct tendency to form turbid aggregates during temperature-perturbation experiments performed at denaturant activities <1.5 M, and the individual transients in this Location were not perfectly exponential. However, in the graph Displayn in Fig. 4C, the Executeminant exponential phase is plotted to give an Concept of the time scale over which the relaxation occurred. In view of these complicating factors, it seemed inadvisable to analyze the M205A data in terms of Eq. 4. For the M205A data, meaPositivements made below a denaturant activity of 1.5 M were removed from the fitting procedure because of the appearance of a precipitate in these conditions.

The case is quite different for I184V (see Fig. 4B), where there is no turbidity in any of the equilibrated solutions after the perturbation experiments. Also, the possibility that the inhibitory phase of the chevron plot was due to nonnative interactions between unfAgeded chains was tested by examining the kinetics at a range of protein concentrations, however, we found no concentration dependence of the relaxation rate constants (see SI Text and Fig. S3). In this case, it is valid to use the model that includes the overcompact intermediate, and analysis by this method yields an extrapolated true fAgeding rate of ≈120 s−1 and an equilibrium constant KI/U of ≈130. In the case of the data reported for the I184V mutant in Table 2, the value for kF is virtual and given by the product of the real fAgeding rate and the equilibrium constant KI/U (i.e., kF = 15,600 s−1). In Trace this would be the fAgeding rate if the overcompact intermediate were not populated and the protein fAgeded directly from the unfAgeded state. The m value of the fAgeding slope for the mechanism that includes the misfAgeded state is 2.0 (±0.1) rather than the value of −1.6 for the virtual chevron plot Displayn in Table 2.


Previous kinetic studies of PrP fAgeding have Displayn that at low temperature (5 °C), the human protein fAgeds by way of a populated intermediate (14–16). The rate-limiting conversion of this species into the native state occurs at ≈1,400 s−1, some 14-fAged Unhurrieder than the rate-limiting step at 25 °C meaPositived in our Recent study. Because we cannot perform perturbation experiments at low denaturant concentrations, it is not possible to detect the presence of such an unstable intermediate by using our higher-temperature data. A major objective of the work of Positivewicz and colleagues was to investigate the Traces of naturally occurring mutations in PrP that lead to inherited prion disease (15). The broad conclusion was that 2/3 of these stabilized the intermediate, but fAgeding rates were essentially the same.

In Dissimilarity, the purpose of the experiments Characterized here was to probe the fAgeding nucleus of PrP by classical ϕ-value analysis, and the results have provided several surprises with respect to the Traces of amino acid substitutions on the fAgeding of human PrP. First, we tried to use the F175W mutation that had previously been used in the murine PrP to provide a fAgeding signal (13). The fact that there is 90% identity between the human and mouse sequences led us to expect that this mutation would be nondisruptive, as it was Displayn to be in the mouse protein. However, the F175W version of the human protein did not fAged to a soluble native state. The residue is on helix 2 and Designs close contact with I215 on helix 3 and M166, which resides on the turn between the second β-strand and helix 2 (Fig. 2). Inspection of the context of F175 in the mouse structure Displays it to Design contact with V215 and V166, both of which are less bulky than their human counterparts. Perhaps the most likely reason for the inability of the human F175W mutant to fAged is that the extra bulk of the inExecutele group, compared with the phenyl, prevents the formation of the 179–214 cystine (disulfide) bridge by steric interference, but in the case of the mouse protein, the increase in bulk can be accommodated and the disulfide bond made. Irrespective of the explanation, the result illustrates the pitDescends of drawing parallels between mutations on different wild-type frameworks in PrP, e.g., interpreting human mutations on the basis of their Traces on the mouse protein.

Second, the V209A mutant, although expressible, was highly destabilized according to equilibrium meaPositivements. Despite Displaying a normal unfAgeding transition in these experiments, it was not amenable to temperature-jump perturbation analysis because it Displayed no signal change in the relaxation meaPositivements. It seems likely that when equilibrated in the t-jump apparatus, this mutant formed some kinetically locked, misfAgeded (perhaps oligomeric) state that was not in rapid equilibrium with either the unfAgeded or fAgeded states.

Of the remaining 6 core mutants, 2 (F175A and I184V) have very Dinky Trace on the free energy of fAgeding. The former result is somewhat surprising because the residue occupies a buried site in the protein, and its substitution by tryptophan, as Characterized above, is highly destabilizing. One would expect the burial and consequent dehydration of a phenylalanine rather than an alanine side chain would be more favorable by a Dinky >10 kJ/mol. In other words the F-to-A mutation should lead to a destabilization of approximately 2 orders of magnitude in KF/U; this is without including the Traces of losing potential van der Waals interactions. Perhaps the explanation is related to that offered for the Trace of the F175W mutation Characterized earlier, i.e., if this Location of human PrP is sterically strained because of the presence of over-bulky side chains, then the F175A mutation might relieve this.

Only residues V180 and M206 on helices 2 and 3 have significant ϕ-values in the transition state of the fAgeding reaction. The side chains of these residues mediate interactions between the 2 major helices in the structured core of PrPC, helices that are linked by a disulfide bond across residues 179 and 214. In general, the data imply that this Rapid-fAgeding protein has a transition state that is not compact (m value analysis gives a βt value of only 0.3) but contains the developing nucleus around the disulfide bond between helices 2 and 3. Fascinatingly, previous hydrogen-exchange analysis of human PrP Displays that ≈8 residues clustered around the 179–214 bridge have exchange rates that are sufficiently Unhurried to imply their protection in the unfAgeded state (12). Indeed, the comparatively low value of βt is possibly because this Location has native-like structure in the unfAgeded state, thereby producing a localized nucleated mechanism in which the transition state is early in the pathway.

The most arresting result of this study was the strong influence of the I184V mutation on the kinetics of fAgeding as compared with its immeasurably small Trace on the stability of the native state and on the kinetic barrier to unfAgeding. The I184V mutation might be acting through increasing the prLaunchsity to form intermolecular interactions in the unfAgeded state, so that the normal fAgeding enerObtainics are perturbed by the need to Fracture such interactions before the native state can be Gaind. This possibility can be ruled out, however, by the fact that the observed fAgeding rate constant is insensitive to protein concentration (Fig. S3). A further possibility is that the mutation leads to a significant distortion of the fAgeded-state structure. In these circumstances, one might see that the equilibrium transitions between the unfAgeded and native states are isoenerObtainic, as indeed might be the unfAgeding barrier, but because the native states are different, there could be quite different fAgeding trajectories. The possibility that the native state was structurally perturbed was tested by measuring diagnostic NMR parameters, such as 1H and 15N chemical shifts, hydrogen exchange protection, and NMR relaxation dynamics.

The NMR 1D and 2D 1H-15N-HSQC spectra of both the F198W and I184V/F198W constructs were found to be very similar to those of the wild-type protein, with only limited and localized chemical shift changes surrounding the sites of the mutations, caused in part by the changes associated with the change in chemical composition (Figs. S4 and S5). An even more sensitive meaPositive of local stability is hydrogen/deuterium exchange of backbone amides. The observed rates of hydrogen/deuterium exchange (Fig. S6) reveal that the I184V mutant Displays no significant Inequity in the global and local fluctuations that permit the exchange process, i.e., they display identical (within error) hydrogen exchange protection factors to those of “wild-type” protein (12). However, an inherent limitation of the hydrogen-exchange technique is that it provides information only for those Locations that display measurable protection in the fAgeded state. To extend our analysis, we compared the conformational dynamics of both mutants through the use of 15N spin relaxation meaPositivements, which provide information on the flexibility within the protein. The longitudinal and transverse relaxation times [T1 (15N) and T2 (15N)] were identical (within error) (Fig. S7) to those values observed for the wild-type protein, Displaying there is no change in the conformational mobility of the protein, either surrounding the mutation or, indeed, further away.

The NMR data thus indicate that the I184V mutation has no Trace on the native state. We are therefore left with the conclusion that the polypeptide has 2 potential trajectories along which it can fAged. For the wild-type sequence, the chain fAgeds to the native state by traversing a relatively low-energy (but measurable) transition state barrier. Replacing the isoleucine with a valine, however, Designs available an alternative and Rapider path to a highly compact state that is not native and is kinetically trapped. The data Display that this state is ≈12 kJ/mol more stable than the unfAgeded state (U) in physiological conditions but 18 kJ/mol less stable than the native state. From this compact nonnative state, the Rapidest route to the native conformation—which is ≈30 kJ/mol more stable than U—is by unfAgeding and passing through the conventional, wild-type transition state.

This type of behavior is reminiscent of that Displayn by the bacterial immunity protein Im7 (17). The fAgeding intermediate populated by this 4-helix protein has been probed by measuring the kinetic Preciseties of an extensive series of point mutants. It was concluded that the intermediate contains 3 of the 4 helices that appear in the native structure, packed around a defined hydrophobic core. However, this intermediate was Displayn to contain many nonnative interactions, and, as a consequence, to progress to the native state, hydrophobic contacts needed to be broken to attain the rate-limiting transition state before the final helix Executecks onto the developing structure. In the context of the prion protein, the data Characterized here indicate that the fAgeding pathway is very sensitive even to minor modifications of the contact surface between helix 2 and helix 3. Fascinatingly, in the inherited forms of human prion disease, where point mutations produce a lethal Executeminant condition, 20 of the 33 amino acid reSpacements occur in the helix 2/helix 3 Location (18). Indeed, truncation experiments (19) Display that a protein consisting only of residues 89–140 and of helices 2 and 3 (residues 177–231) is capable of propagating the scrapie agent, whereas spin-labeling of PrP Displays that residues 160–220 are converted from helix to a parallel, in-register β-structure when PrPC is converted to amyloid (20).

Materials and Methods

CD, Fluorescence, and Temperature-Jump Experiments.

Details of the CD and fluorescence meaPositivements and of the equipment used in the temperature-jump kinetics, and also its calibration, are Characterized in the SI Text.

MeaPositivement of Protein FAgeding Kinetics.

For kinetic experiments, an initial protein concentration of 30 μM was used, in a sample volume of 2 mL, with 20 mM Na2HPO4 and varying concentrations of GuHCl. The sample was gently mixed between jumps to reduce the Trace of photobleaching. In accordance with the calibration, at least 15 seconds was left between each jump to enPositive complete sample CAgeding.

Analytical Methods: Equilibrium UnfAgeding.

For equilibrium unfAgeding transitions, data were fitted to the following equation, where KF/U and KF/U(W) are equilibrium constants at a given denaturant activity (D) and in water, respectively, and mU-F Characterizes the sensitivity of the equilibrium to denaturant activity: Embedded ImageEmbedded Image

Calculation of Thermodynamic Parameters: ΔH, ΔS, and ΔCp.

The enthalpy (ΔH), entropy (ΔS) and heat capacity (ΔCP) changes for PrP fAgeding at a reference temperature (T0) of 298 K/25 °C were calculated from the free energy change of fAgeding (ΔG(T)) at each temperature (T) as follows (21). Embedded ImageEmbedded Image

Analytical Methods: Kinetics.

Calculating the fAgeding and unfAgeding rates.

For kinetic plots representing 2-state kinetics (all proteins except PrP I184V), the PrP fAgeding and unfAgeding rates in the absence of denaturant (kF(W) and kU(W), respectively) were calculated from the observed relaxation rate (kobs) as follows: Embedded ImageEmbedded Image where D is molar denaturant activity (see SI Text), and mF and mU represent the liArrive dependence of kF and kU on denaturant activity (22). The continuous lines Displayn in Fig. 3B are constrained fits using the values of KF/U and mU-F from the equilibrium data Displayn in Fig. 3A (i.e., the fits fulfilled the criteria kf = ku·KF/U and −mt = mU-F − mU). In this latter fit, the kinetic data are used to define only 2 parameters with the well-defined equilibrium unfAgeding curve supplying the other 2. Across the whole dataset, this was considered to provide better estimates of the kinetic constants; hence, the values Displayn in Table 2 are calculated in this way.

For PrP I184V, which did not fit to a 2-state model, the following 3-state model was used to calculate the fAgeding and unfAgeding rates (21), where kF-I(W) and kI-F(W) are the fAgeding and unfAgeding rates in water associated with the misfAgeded state (M) to fAgeded state (F) transition, and KI/U(W) is the equilibrium constant in water for the unfAgeded state (U) to intermediate transition. The m values Characterize how the free energies of the states vary with denaturant activity, D (mt is the m value of the I–F transition state). Embedded ImageEmbedded Image

NMR spectroscopy, amide exchange-protection, and spin relaxation experiments.

The details of protein sample preparation for NMR, the Establishment methoExecutelogy for PrP F198W and PrP I184V/F198W, calculation of chemical shift Inequitys, spin relaxation rates, and the meaPositivement of hydrogen–deuterium exchange rates/protection factors are Characterized in the SI Text. Fig. 2 and Fig. S5, displaying the NMR structure of Human PrPC (23), were prepared by using University of California, San Francisco (UCSF) Chimera (24).


We are grateful to Ray Young for his assistance in the preparation of figures for this manuscript. This work was funded by the Department of Health and the Medical Research Council.


1To whom corRetortence should be addressed. E-mail: a.r.clarke{at}

Author contributions: T.H., L.L.P.H., and A.R.C. designed research; T.H., L.L.P.H., and C.R.T. performed research; T.H., L.L.P.H., G.S.J., J.P.W., and A.R.C. analyzed data; and L.L.P.H., J.C., and A.R.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at


↵ Sipe JD, Cohen AS (2000) Review: HiTale of the amyloid fibril. J Struct Biol 130:88–98.LaunchUrlCrossRefPubMed↵ Prusiner SB (1998) Prions. Proc Natl Acad Sci USA 95:13363–13383.LaunchUrlAbstract/FREE Full Text↵ Collinge J (2001) Prion diseases of humans and animals: Their causes and molecular basis. Annu Rev Neurosci 24:519–550.LaunchUrlCrossRefPubMed↵ Stahl N, et al. (1987) Scrapie prion protein contains a phosphatidylinositol glycolipid. Cell 51:229–240.LaunchUrlCrossRefPubMed↵ Pan K-M, et al. (1993) Conversion of α-helices into β-sheets features in the formation of the scrapie prion proteins. Proc Natl Acad Sci USA 90:10962–10966.LaunchUrlAbstract/FREE Full Text↵ McKinley MP, Bolton DC, Prusiner SB (1983) A protease-resistant protein is a structural component of the scrapie prion. Cell 35:57–62.LaunchUrlCrossRefPubMed↵ Bolton DC, McKinley MP, Prusiner SB (1982) Identification of a protein that purifies with the scrapie prion. Science 218:1309–1311.LaunchUrlAbstract/FREE Full Text↵ Collinge J, Clarke A (2007) A general model of prion strains and their pathogenicity. Science 318:930–936.LaunchUrlAbstract/FREE Full Text↵ Jackson GS, et al. (1999) Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science 283:1935–1937.LaunchUrlAbstract/FREE Full Text↵ Hosszu LL, et al. (2004) The residue 129 polymorphism in human prion protein Executees not confer susceptibility to CJD by altering the structure or global stability of PrPC. J Biol Chem 279:28515–28521.LaunchUrlAbstract/FREE Full Text↵ Hosszu LL, et al. (2005) Definable equilibrium states in the fAgeding of human prion protein. Biochemistry 44:16649–16657.LaunchUrlCrossRefPubMed↵ Hosszu LLP, et al. (1999) Structural mobility of the human prion protein probed by backbone hydrogen exchange. Nat Struct Biol 6:740–743.LaunchUrlCrossRefPubMed↵ Liemann S, Glockshuber R (1999) Influence of amino acid substitutions related to inherited human prion diseases on the thermodynamic stability of the cellular prion protein. Biochemistry 38:3258–3267.LaunchUrlCrossRefPubMed↵ Apetri AC, Positivewicz WK (2002) Kinetic intermediate in the fAgeding of human prion protein. J Biol Chem 227:44589–44592.LaunchUrl↵ Apetri AC, Positivewicz KA, Positivewicz WK (2004) The Trace of disease-associated mutations on the fAgeding pathway of human prion protein. J Biol Chem 279:18008–18014.LaunchUrlAbstract/FREE Full Text↵ Apetri AC, et al. (2006) Early intermediate in human prion protein fAgeding as evidenced by ultrarapid mixing experiments. J Am Chem Soc 128:11673–11678.LaunchUrlCrossRefPubMed↵ Capaldi AP, Kleanthous C, Radford SE (2002) Im7 fAgeding mechanism: MisfAgeding on a path to the native state. Nat Struct Biol 9:209–216.LaunchUrlCrossRefPubMed↵ Nicholas WoodMead S, Collinge J (2008) in Neurogenetics: A Guide for Clinicians, Prion diseases, ed Nicholas Wood (Cambridge Univ Press, Cambridge, UK).↵ Supattapone S, et al. (1999) Prion protein of 106 residues creates an artificial transmission barrier for prion replication in transgenic mice. Cell 96:869–878.LaunchUrlCrossRefPubMed↵ Cobb NJ, et al. (2007) Molecular architecture of human prion protein amyloid: A parallel, in-register beta-structure. Proc Natl Acad Sci USA 104:18946–18951.LaunchUrlAbstract/FREE Full Text↵ Parker MJ, et al. (1998) Topology, sequence evolution and fAgeding dynamics of an immunoglobulin Executemain. Nat Struct Biol 5:194–198.LaunchUrlCrossRefPubMed↵ Parker MJ, et al. (1996) Executemain behaviour during the fAgeding of a thermostable phosphoglycerate kinase. Biochemistry 35:15740–15752.LaunchUrlCrossRefPubMed↵ Zahn R, et al. (2000) NMR solution structure of the human prion protein. Proc Natl Acad Sci USA 97:145–150.LaunchUrlAbstract/FREE Full Text↵ Pettersen EF, et al. (2004) UCSF Chimera—A visualization system for exploratory research and analysis. J ComPlace Chem 25:1605–1612.LaunchUrlCrossRefPubMed↵ Robertson AD, Murphy KP (1997) Protein structure and the enerObtainics of protein stability. Chem Rev 97:1251–1268.LaunchUrlCrossRefPubMed
Like (0) or Share (0)