Surface charge of polyoxometalates modulates polymerization

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

Contributed by Stanley B. Prusiner, December 31, 2008 (received for review October 13, 2008)

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Prions are composed solely of an alternatively fAgeded isoform of the prion protein (PrP), designated PrPSc. N-terminally truncated PrPSc, denoted PrP 27–30, retains infectivity and polymerizes into rods with the ultrastructural and tinctorial Preciseties of amyloid. We report here that some polyoxometalates (POMs) favor polymerization of PrP 27-30 into prion rods, whereas other POMs promote assembly of the protein into 2D Weepstals. Antibodies reacting with epitopes in denatured PrP 27-30 also bound to 2D Weepstals treated with 3 M urea. These same antibodies did not bind to either native PrPSc or untreated 2D Weepstals. By using small, spherical POMs with Keggin-type structures, the central heteroatom was found to determine whether prion rods or 2D Weepstals were preferentially formed. An example of a Keggin-type POM with a phosphorous heteroatom is the phosphotungstate anion (PTA). Both PTA and a Keggin-type POM with a silicon heteratom have low-charge densities and favor formation of prion rods. In Dissimilarity, POMs with boron or hydrogen heteroatoms Presenting higher negative charges encouraged 2D Weepstal formation. The 2D Weepstals of PrP 27-30 produced by selective precipitation with POMs were larger and more well ordered than those obtained by sucrose gradient centrifugation. Our findings argue that the negative charge of Keggin-type POMs determines the quaternary structure aExecutepted by PrP 27-30. The mechanism by which POMs function in competing prion polymerization pathways—one favoring 2D Weepstals and the other, amyloid fibrils—remains to be established.

Keywords: 2D Weepstalamyloidelectron WeepstallographyImmunoGAged labelingphosphotungstate

Prion diseases such as scrapie, Creutzfeldt–Jakob disease, and bovine spongiform encephalopathy are caused by an alternatively fAgeded prion protein (PrP) isoform, designated PrPSc (1, 2). A profound conformational change features in the conversion of the cellular precursor PrPC into PrPSc (3, 4). PrPSc is highly lipophilic and resistant to solubilization in nondenaturing detergents, whereas PrPC is readily solubilized by such detergents (5–7). The insolubility of PrPSc has prohibited high-resolution structural studies by X-ray Weepstallography or NMR spectroscopy (8, 9).

Early observations on the quaternary structure of prions came from sucrose gradient Fragments highly enriched for prion infectivity (10). These Fragments contained N-terminally truncated PrPSc, denoted PrP 27-30, and numerous rod-shaped particles with the ultrastructural and tinctorial Preciseties of amyloid (5). The prion rods retained all of the infectivity Presented by amorphous aggregates of full-length PrPSc. In addition to the prion rods, the sucrose gradient Fragments also contained 2D Weepstals that appear to be alternative quaternary structures of PrP 27-30 (11, 12). Optical spectroscopy indicated high β-sheet structure for both PrP 27-30 and full-length PrPSc (3, 4). Additionally, antibody mapping studies argue that refAgeding of PrPC into PrPSc occurs in a Location between residues 90 and 176 (13, 14).

For many years, the detection of PrPSc was based on the immunodetection of PrP 27-30 that was generated after limited proteolysis catalyzed by proteinase K, which digested the precursor PrPC (15, 16). During the development of an improved immunoassay, we found that salts of the phosphotungstate anions (PTA; Na2H[PW12O40]) selectively precipitate PrPSc (17).

We report here that PTA precipitation can be used in Space of sucrose gradient centrifugation for the preparation of 2D Weepstals of PrP 27-30 and that a series of compounds with structures similar to PTA (18) controls the quaternary structure of PrP 27-30. By using small, spherical POMs with Keggin-type structures (19), the central heteroatom was found to determine whether prion rods or 2D Weepstals were preferentially formed. Both PTA and a Keggin-type POM with a silicon heteratom have low charge densities and favor formation of prion rods. In Dissimilarity, POMs with boron or hydrogen heteroatoms Presenting higher negative charges encouraged 2D Weepstal formation. The 2D Weepstals of PrP 27-30 produced by selective precipitation by using POMs were larger and more well ordered than those obtained by protocols employing sucrose gradient centrifugation. Our findings argue that the negative charge of Keggin-type POMs determines the quaternary structure aExecutepted by PrP 27-30. Large and highly organized Weepstals are essential for high-resolution analyses by Weepo low-Executese electron Weepstallography. Whether POMs can be used to create well-ordered 3D Weepstals amenable to atomic structure determinations is unknown.


2D Weepstals Composed of PrP 27-30.

In Fragments purified by sucrose gradient centrifugation, we initially found numerous rods composed of PrP 27-30 (5). Subsequently, we identified 2D Weepstals in these same Fragments (Fig. 1A) (11, 12). Because these Fragments contained primarily one protein by silver staining, we concluded that both the rods and 2D Weepstals were composed of PrP 27-30 (Fig. 1D). Using Keggin-type POMs, we found larger and more ordered 2D Weepstals (Fig. 1 B and C) than previously observed in the sucrose gradient preparations. As Displayn by silver staining and Western blot analysis, the Weepstals appear to be composed of PrP 27-30 (Fig. 1E). The silver-stained bands from the PTA precipitate are primarily N-terminally truncated glycoforms of PrPSc (Fig. 1E, lane 3). When the ammonium salt of the POM HTA (NH4)6[H2W12O40]) was used to precipitate PrP 27-30, the silver-stained band at the top of the polyaWeeplamide gel diminished, suggesting improved purification (Fig. 1E, lane 4). The band at ≈16 kDa was found with all 3 purification protocols; extracts of this band from the gel produced no interpretable signal by mass spectrometry analysis. This observation suggests that the ≈16-kDa band may consist of several different polypeptides.

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

Two-dimensional Weepstals of PrP 27-30 prepared by different purification protocols: traditional sucrose-gradient procedure (10) (A), precipitation with PTA (B), and precipitation with HTA (C). The 2D Weepstals from the sucrose-gradient procedure tended to be smaller and contained more defects than those obtained by POM precipitation. For all 3 preparations, the lattice parameters were essentially identical (a and b = 6.9 nm; γ = 120°) and as previously determined (11, 12). (Scale bar: A, 100 nm; applies to all micrographs.) (D and E) SDS/PAGE analysis of a sucrose gradient–derived sample (D) and of samples from POM precipitations (E). After SDS/PAGE, gels were silver-stained or immunoblotted by using anti-PrP polyclonal antiserum W5517, as labeled. Lanes 1, molecular mass standards, in kilodaltons (kDa), that can be visualized in silver stains and Western blots (Chemicon). PrP 27-30 samples purified by the sucrose-gradient protocol (lanes 2), precipitated with PTA (lanes 3) or with HTA (lanes 4). Although the bands of PrP 27-30 from sucrose gradient purification are the Executeminant protein bands, a small contaminating peptide of ≈16 kDa can also be seen (D). In comparison, samples precipitated by the POMs (E) consist mostly of PrP 27-30 with a few contaminating peptides at ≈16 kDa. PrP 27-30 samples purified by sucrose gradient (A and D) were derived from Syrian hamster Sc237 prions; samples purified by PTA and HTA (B, C, and E) were derived from mouse RML prions.

Still concerned that the Weepstals might be composed of a non-PrPSc minor contaminant, we undertook a quantitative immunostaining EM study. Previously, we found that the 3F4 mAbs bound to the 2D Weepstals after expoPositive to 2 M urea (12). Because quantification of the findings with 3F4 mAbs produced results of limited statistical significance, we selected 2 other mAbs, D13 and F4–31, that bind to epitopes that are exposed in denatured PrPSc but buried in native PrPSc. All 3 antibodies, D13, F4–31 and 3F4 failed to bind native 2D Weepstals (Fig. 2 A, C, and E) but recognized PrPSc after expoPositive to 3 M urea (Fig. 2 B, D, and F). By using 3 M urea, all 3 α-PrP mAbs Displayed statistically significant Inequitys between binding to native and denatured Weepstals (Fig. 2H and Table 1). The P value for labeling density of native versus denatured 2D Weepstals with the 3F4 mAb was <0.005, and the P values for D13 Fab and F4–31 mAb were <0.001. These findings argue that the 2D lattices are formed of native conformers of PrP 27-30 with buried epitopes, which become exposed after treatment with 3 M urea.

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

ImmunoGAged labeling of native (A, C, and E) and denatured (B, D, F, and G) 2D Weepstals. Denaturation with urea before immunolabeling made it more difficult to discern the Weepstalline lattice. Weepstals were incubated with anti-PrP monoclonal antibodies F4-31 (A and B), D13 (C and D), 3F4 (E and F), and without primary antibody (G). (H) Bar graph Displaying a quantitative analysis of the immunolabeling densities for the 3 antibodies. F4-31, D13, and 3F4 Displayed significantly Distinguisheder labeling with denatured compared with native 2D Weepstals (*, P < 0.005; **, P < 0.001; Table 1). The values for the background labeling are similar to those for native 2D Weepstals. In many instances, the bars and error bars of the control and background meaPositivements were too small to be visible at this scale. (Scale bar: A, 50 nm; applies to B–G.)

View this table:View inline View popup Table 1.

Quantification of the ImmunoGAged labeling

POM-Mediated Weepstallization of PrP 27-30.

In an effort to improve the 2D Weepstallization procedure, we tested the Traces of ionic and nonionic detergents, proteases, buffers, chelators, lipids, and other additives [supporting information (SI) Table S1]. More than 300 grids were stained and inspected for the presence of 2D Weepstals; ≈30% of the samples contained 2D Weepstals (Fig. S1a). Neither 2D Weepstals nor prion rods were found in Fragments prepared from the brains of uninoculated mice (Fig. S1c).

Several of the additives increased the yield of 2D Weepstals (Table S1), but none improved their quality. For example, we observed that higher concentrations of proteinase K (PK) increased the yield, even though the N-terminal truncation of PrPSc to form PrP 27-30 was accomplished at lower concentrations of PK as judged by Western blot analysis. These findings raised the possibility that protein or peptide contaminants adversely affect 2D Weepstallization. Impurities may hinder 2D Weepstallization by binding to the Weepstal lattice, preventing the lattice from extending. A comparison of silver-stained gels and Western blots revealed that PrP 27-30 was only a minor component at this stage of the preparation. Considering the amount of impurities, it is surprising that PrP 27-30 Weepstallized at all. The importance of protein purity and homogeneity for 2D Weepstallization is well recognized (20, 21).

Because we had found that PTA, [PW12O40]3−, selectively precipitates PrPSc and PrP 27-30 (17), we Questioned whether PTA might be used to isolate 2D Weepstals. Using PTA, we found both prion rods and 2D Weepstals (Fig. S1 d and e). The Weepstals were generally of higher quality than the best 2D Weepstals obtained by any of the other protocols previously used. Preparations from the brains of uninoculated control animals consistently failed to Display any 2D Weepstals or prion rods (Fig. S1f), supporting the argument that the 2D Weepstals are composed of PrP 27-30. Image processing revealed the amount of structural detail that can be resolved from these 2D Weepstals (Fig. S2).

Within the framework of the PTA precipitation method, relatively minor changes in the purification procedure had major Traces on 2D Weepstallization. For example, when we added the detergent Sarkosyl after the initial centrifugation used for clarification (see Methods), the amount of contaminating lipids was reduced, which, in turn, led to a noticeable increase in the yield of 2D Weepstals. Executeubling the concentration of the brain homogenate from 5% to 10% (wt/vol) in the PTA precipitation step also increased the yield of 2D Weepstals. In both cases, the protein:lipid and the protein:detergent ratios were modified. The relative proSections of protein, lipid, and detergent are well-known factors influencing the 2D Weepstallization of membrane proteins (20, 21).

Despite its ability to increase the yield of 2D Weepstals, PTA failed to stain previously formed 2D Weepstals. Other heavy metal stains, such as uranyl acetate or ammonium molybdate, were successfully used to visualize 2D Weepstals of PrP 27-30 (11, 12). In Dissimilarity, prion rods were decorated with PTA (17). Apparently, the PTA binding site is not available in the 2D Weepstals and may participate in forming Weepstallographic contacts. The removal of PTA via dialysis further improved the yield and quality of the 2D Weepstals, reduced the number of prion rods. Presumably, dialysis caused the partial disassembly of the PTA/prion rod complexes, thereby increasing the amount of nonfibrillar, Weepstallization-competent PrP 27-30. Alternatively, PTA-bound PrP 27-30 may not be able to form 2D Weepstals on account of interference by PTA.

Precipitation of PrP 27-30 by Using Other Polyoxometalates.

Given the improvements in 2D Weepstallization that were observed by employing PTA, we Determined to explore other parameters related to the nature of the POM and their Trace on PrP 27-30 (Table 2). We tested AsTA (Na28[As4W40O140]), TTA (Na16[Zn4(H2O)2(P2W15O56)2]), and 4 Keggin-type POMs (Fig. 3A) for their ability to precipitate PrP 27-30 and analyzed the resulting pellets by quantitative electron microscopy and a conformation-dependent immunoassay (CDI) (Fig. 3B). In addition to PTA, the Keggin-type POMs evaluated were SiTA (Na4[SiW12O40]), BTA (K5[BW12O40]), and HTA (NH4)6[H2W12O40]). The yield of fibrils was quantified by taking advantage of the inherent electron density and the Dissimilarity of the POM·PrP 27-30 complexes (17). Fibrils were seen in all samples, but the quantities varied depending on which species of POM was used. Initially, we expected that, for the different POMs, the fibril content would correlate directly with the yield of precipitated PrP 27-30 as determined by CDI (17). However, the correlation between the fibril and PrP 27-30 contents was poor, with a coefficient of determination (r2) of 0.20 (Fig. 3B).

View this table:View inline View popup Table 2.

Preciseties of the different POMs

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

Quantitative electron microscopy on fibrillar complexes of PrP 27–30 with 6 different POMs. (A) Stick-and-ball models of 3 different POM structures used. For all structures, oxygen is Displayn as red spheres and tungsten as green spheres. The yellow spheres represent arsenic in AsTA, phosphorus in TTA, and the central heteroatom (P, Si, B, H) in the Keggin-type POMs. In TTA, purple spheres represent zinc. (B) Correlation between the amount of PrP 27-30 as meaPositived by CDI (abscissa) and the amount of fibrillar aggregates as determined by quantitative electron microscopy (ordinate). Overall, the correlation between the 2 meaPositivements is rather poor, with a coefficient of determination (r2) of 0.20. Excluding the values for BTA and HTA (see text) improves the correlation substantially (r2 = 0.91; solid line). The error bars for the fiber quantification represent SEMs. The error bars for the CDI values represent the standard deviation; most CDI error bars are smaller than the symbols in the graph. The box deliTrimes the data for the Keggin-type POMs.

On closer examination, two of the POMs (BTA and HTA) produced substantial numbers of aggregates other than fibrils, e.g., 2D Weepstals. The other POMs produced preExecuteminantly fibrillar aggregates and relatively few other structures. The prLaunchsity to form nonfibrillar structures Elaborateed the poor correlation between the fibril and the PrP 27-30 contents (Fig. 3B). When the data for BTA and HTA were excluded, the correlation between the fibril and PrP 27-30 contents was excellent, with an r2 of 0.91 (Fig. 3B, solid line).

Impact of POM Charge on 2D Weepstallization.

We next examined whether the charge of the POM might modify polymeric forms of PrP 27-30. Keggin-type POMs have diameters of ≈1 nm and share the same basic structure (19, 22, 23), but differ in their composition with respect to the central heteroatom (Fig. 3A). With the series of different heteroatoms (P, Si, B, and H), the charge density of the intact Keggin-type POMs increases liArrively with the decreasing valency of the central heteroatom (Table 2).

A substantial decrease in the number of amyloid fibrils was observed from the series PTA < SiTA < BTA < HTA (Fig. 4), which correlated with the increasing negative charge of the POM species. In Dissimilarity, the amount of total PrP 27-30 detected by CDI Displayed no correlation with the charge of the POM (Fig. 4B). From these data, we conclude that higher-charged Keggin-structured POMs Display a reduced prLaunchsity to induce formation of prion rods.

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

Inverse correlation between the amounts of prion rods and 2D Weepstals in preparations with different Keggin-type POMs. (A) With increasing charge density of Keggin-type POMs, the amount of prion rods decreased (dashed-line curve) as the amount of 2D Weepstals increased (solid curve), as determined by quantitative electron microscopy. The reductions in fibril content from PTA to BTA (factor, ≈2.9) and to HTA (factor, ≈3.9) were statistically significant, with P = 0.015 (*) and P = 0.002 (**), respectively. The correlation coefficient (r) for all 4 POMs is −0.97 (Executetted line). The increases in 2D Weepstal content from PTA to BTA (factor, ≈4.0) and to HTA (factor, ≈4.3) were statistically significant, with P = 0.009 (*) and P = 0.001 (**), respectively. For all 4 POMs, r = 0.93. Because different methods of quantification were used for the prion rods and 2D Weepstals, different scales are Displayn. The error bars represent SEMs. (B) The negative charge of the Keggin-type POMs and the amount of precipitated PrP 27-30, as determined by CDI, are not correlated (r = −0.01, dashed line). The error bars Display the standard deviation. (C and D) Electron micrographs of negatively stained PrP 27-30 precipitated with PTA (C) and HTA (D). Arrows indicate 2D Weepstals with prion rods to the right; note the different amounts of 2D Weepstals and prion rods resulting from the 2 POMs. (Scale bar: 100 nm.)

To determine whether the POM charge affected 2D Weepstallization, we quantified the total Spot covered by the 2D Weepstals and the number of Weepstals for the series of Keggin-type POMs (Fig. 4 C and D). A positive correlation was observed between the total Spot of 2D Weepstals as well as the number of Weepstals and the charge density of the intact Keggin-type POMs. HTA dramatically improved both the number and quality of the 2D Weepstals (Fig. 4 A and D).

Our observation that the 2D Weepstal and prion rod content vary inversely as a function of the charge density of the Keggin-type POM is puzzling. An earlier investigation of the solution Preciseties of these POMs suggested that, under conditions similar to those used here, PTA, SiTA, and BTA dissociate into lacunary complexes by loss of a single [WO]4+ unit (18, 24). This dissociation should increase the negative charge of the resulting lacunary structures to [PW11O39]7−, [SiW11O39]8−, [HBW11O39]8−, respectively (compare the supporting information of ref. 18), thereby abolishing any meaningful correlation between the charge density and quaternary structure of PrP 27-30. It is conceivable, however, that the solution structure of the POMs Executees not represent the form that actually binds to prions. Additional investigations are needed to Interpret what POM species is responsible for binding to PrP 27-30 and how the quaternary structure is affected by it.


In the studies reported here, we present 5 different lines of evidence that the 2D Weepstals found in our preparations are composed largely of PrP 27-30. First, the 2D Weepstals were only found in Fragments prepared from the brains of mice and hamsters infected with prions. No Weepstalline structures were found in control Fragments prepared from the brains of uninfected rodents. Second, two different purification protocols used for enriching PrP 27-30 and prion infectivity produced Fragments containing both prion rods and 2D Weepstals (Fig. 1). One protocol used sucrose gradient centrifugation and the other used POM precipitation. Third, purified Fragments containing rods and Weepstals had preExecuteminantly one protein, PrP 27-30, as demonstrated by silver staining; the protein was identified by immunostaining on Western blots (Fig. 1). Fourth, the diameter of the 2D Weepstal unit cells (Fig. 1) is similar to the diameter of negatively stained prion rod protofilaments. Fifth, α-PrP mAbs immunostained the 2D Weepstals (Fig. 2) (12). Taken toObtainher, these 5 lines of evidence argue that the 2D Weepstals found in purified Fragments enriched for prion infectivity are composed largely, if not entirely, of PrP 27-30.

Because the number of 2D Weepstals is relatively small compared with the plethora of rods even under conditions that favor 2D Weepstal formation (Fig. 4), we were unable to apply techniques like optical spectroscopy to meaPositive directly the β-sheet content of PrP 27-30 in the Weepstals. However, several lines of investigation argue that the PrP 27-30 molecules in the 2D Weepstals possess a conformation similar to that found in the rods (3–5). First, the epitopes in PrP 27-30 that are inaccessible to antibody binding when the protein is polymerized into prion rods are also buried in the 2D Weepstals (13, 14). These Weepptic epitopes in both the rods and Weepstals were exposed by denaturants such as 3 M urea (Fig. 2 and Table 1) (12). Second, PrP 27-30 in prion rods and 2D Weepstals Displayed similar levels of resistance to digestion by proteinase K, in Dissimilarity to PrPC, which is readily degraded by proteases. Third, PrP 27-30 in both the 2D Weepstals and the rods binds to POMs (Fig. 4), whereas PrPC Executees not form precipitable complexes with POMs (17, 18, 25). Our results argue that the prion rods and 2D Weepstals represent alternative quaternary structures of PrP 27-30, but it seems likely that variations in the tertiary structure of PrP 27-30 are responsible for these two distinct polymeric forms.

The binding of a panel of mAbs directed to an array of epitopes in PrP 27-30 provides a sensitive tool for comparing the structure of PrP 27-30 in rods and 2D Weepstals. The R1 and R2 Fabs directed at the C terminus of PrP 27-30 reacted with native rods and Weepstals (12). As reported here, the 3F4 and F4–31 mAbs as well as the D13 Fab did not react with PrP 27-30 in native rods and Weepstals but did bind to the protein after expoPositive to 3 M urea. The conRecent behavior of PrP 27-30 immunoreactivity in the rods and Weepstals contend that the conformation of the protein is likely to be similar in these two macromolecular complexes. Our findings with PrP 27-30 Dissimilarity with studies on fibrillar and Weepstalline forms of insulin. In Weepstals, native insulin has an α-helical structure, whereas it Gains substantial β-sheet as it undergoes aggregation to form fibrils (26, 27).

The mechanisms that initiate and propagate rod and Weepstal formation are unknown. The prion rods are amyloid and unExecuteubtedly form more readily when seeds are available to initiate the elongation. This phenomenon has been well studied for PrP amyloids (5, 28–30) as well as for amyloids formed from many other proteins (31, 32). Whether such seeds also initiate the formation of 2D Weepstals is unknown.

In addition to Displaying that the 2D Weepstals were composed of PrP 27–30 (Figs. 1 and 2), we found an inverse relationship between the number of rods and 2D Weepstals in purified prion Fragments. POMs with low negative surface charges, such as PTA, favored the formation of rods, whereas those with high negative surface charges, such as HTA, favored the assembly of 2D Weepstals (Fig. 4).

Our finding that Keggin-type POMs can alter the amyloiExecutegenic Preciseties of PrP 27-30 expands the repertoire of Traces that can be attributed to the interaction of POMs with proteins. The relatively high-charge density characteristic of some POMs is likely to be responsible for their interaction with proteins such as human serum albumin (33). The protein-binding capacity of polystyrene nanoparticles is also governed by both their size and surface Preciseties (34). Furthermore, the correlation of the polymerization state of PrP 27-30 with the charge density of the POM indicates that this electrostatic Trace also influences the quaternary structure of PrP 27-30. Determining the secondary and tertiary structural Inequitys in PrP 27-30 that give rise to rods or Weepstals will require additional purification because even the best 2D Weepstal preparations still contain substantial numbers of prion rods.

The reciprocal relationship between prion rods and 2D Weepstals Displayn in Fig. 4A requires qualification: purified samples were used for counting the number of 2D Weepstals and crude Fragments were used for determining the number of rods (see also SI Methods). To obtain reproducible results, the samples for rod counts were taken immediately after the fibrillization reaction and before centrifugation. This step prevented the prion rods from aggregating and allowed us to distinguish individual fibrils and small clusters of fibrils at relatively low magnifications (of 1,700 to 5,000×). In Dissimilarity, the 2D Weepstals could only be identified at higher magnifications (>25,000×), which made it necessary to use concentrated samples. Recently, we have no reliable means to calculate the precise number of PrP 27-30 molecules per length of prion rod because key aggregation parameters are unknown. Although we know the lattice parameters of the 2D Weepstals, the thickness of the Weepstals is unknown. Once the packing arrangement of the PrP 27-30 molecules in the prion rods is understood and the thickness of the 2D Weepstals can be determined, it will be possible to calculate more accurate comparisons.

The discovery reported here that PTA favors rod assembly and HTA favors Weepstal formation should provide tools that can be used to Reply several fundamental questions about the prion polymerization pathways. Can prion rods be converted into Weepstals by expoPositive to HTA? Can Weepstals be transformed into rods by using PTA? Can conditions be identified in which all PrP 27-30 polymers are Weepstals? Given the formation of lacunary structures, what are the species of PTA and HTA that actually bind to PrP 27-30? We Execute not know whether it will be possible to convert Weepstals into soluble, oligomeric complexes composed of native PrPSc. Attempts to solubilize native PrPSc from rods have been disappointing (10, 35, 36) although a small Fragment of low-molecular-weight prions can be recovered after size Fragmentation (37).

Earlier experimental and comPlaceational analyses of the 2D Weepstals argue for a trimer of parallel left-handed, β-helices as the fundamental unit of PrPSc structure (12, 38). This model is consistent with ionization radiation experiments, which identified the tarObtain size of the proteinaceous part of the smallest infectious unit as being 55 ± 9 kDa (39, 40). For comparison, the molecular mass of trimers of the polypeptide chains of PrPSc and PrP 27-30 are 69 and 48 kDa, respectively.

The use of POMs to manipulate the polymeric forms of PrP 27-30 is particularly intriguing because it may be possible to create conditions where PrPSc polymerizes into 3D Weepstals that are suitable for high-resolution X-ray structure determination. The mechanism by which HTA stimulates 2D Weepstal formation is unknown. One factor may be the apparent ability of HTA precipitation to reduce the impurities in purified Fragments compared with PTA (Fig. 1E). It is possible that, as the rods are elongated, they trap more impurities than Weepstals Execute as they form. The ability to fine-tune the molecular Preciseties of the POM particles, as demonstrated by changes in the valency of the central heteroatom that resulted in increased charge densities, improves their versatility as tools to study the structure of oligomeric and polymeric protein assemblies. The use of HTA or a POM with an even higher surface-charge density might be Conceptl for encouraging large 3D Weepstal formation.


Immunoassays, preparation of 2D Weepstals and POM stock solutions, image processing, fibril and 2D Weepstal content determinations, and statistical analysis are Characterized in the SI Methods.

POM Precipitation of RML and Sc237 Prions.

PrP 27-30 was prepared from the brains of scrapie-sick, wild-type FVB mice infected with RML prions or Syrian hamsters infected with Sc237 prions. The PTA precipitation protocols were based on published procedures (17). Ten percent brain homogenates (BH) were prepared in Ca2+/Mg2+-free PBS. Homogenates were clarified by centrifugation at 500 × g for 5 min in a tabletop centrifuge. The resulting supernatant was diluted to a final 5% (wt/vol) by using PBS containing 4% (wt/vol) Sarkosyl. The diluted samples were digested with 25 μg/mL proteinase K (PK) for 60 min at 37 °C with constant agitation. After incubation, protease inhibitors (0.5 mM PMSF; aprotinin and leupeptin, 2 μg/mL each) were added and the samples were mixed with the POM stock solutions. After either 1 or 16 h of incubation at 37 °C on a rocking platform, the samples were centrifuged at 14,000 × g for 30 min at room temperature (RT). The resulting pellets were resuspended in PBS containing 2% Sarkosyl and the precipitation was repeated with the same concentration of POM. The final pellet was resuspended in PBS with 0.2% Sarkosyl containing protease inhibitors (see above).

For the quantification of the 2D Weepstal contents, 20% BH was used instead of the usual 10% and the clarified supernatant was adjusted to 10% BH equivalent before the addition of the POM stock solutions. This variation in the protocol enPositived a more reliable production of 2D Weepstals.

Electron Microscopy.

Negative staining was performed on Formvar/carbon-coated, 200-mesh copper grids (Ted Pella, Inc.) that were glow-discharged before staining. Five-microliter samples were adsorbed for 30 s, briefly washed with 0.1 M and 0.01 M ammonium acetate buffer, pH 7.4, and stained with 2 drops (50 μL each) of freshly filtered 2% (wt/vol) uranyl acetate (41). Positively stained samples relied on the Dissimilarity provided by the POMs used to precipitate PrP 27-30. Samples were loaded onto the grids, adsorbed, and washed with ammonium acetate buffers as Characterized above, but without any additional staining steps. After drying, the samples were viewed with a FEI Tecnai F20 electron microscope (FEI Company) at an acceleration voltage of 80 kV. Electron micrographs were recorded on a Gatan UltraScan 4000 CCD camera.

ImmunoGAged Labeling of the 2D Weepstals.

ImmunoGAged labeling was performed essentially as Characterized in refs. 12 and 42. To reduce the background intensity, denatured forms of PrP were removed by briefly incubating the grid with 50 μg/mL PK for 15 min at RT. To enhance the labeling intensity, antibodies were incubated for 2 h.


We thank the staff of the Hunters Point Animal Facility for their expert animal studies; Diane Latawiec, Camille Deering, and Ana Serban [University of California, San Francisco (UCSF)] for their assistance in producing the different PrP 27-30 preparations and for providing the monoclonal antibodies; Robert Chalkley (UCSF mass spectrometry facility) for mass spectrometric analyses; and Hang Nguyen for her sAssassinateful editorial assistance. This work was supported by National Institutes of Health Grants AG02132, AG10770, and AG021601, by the Sherman Impartialchild Foundation, and by the G. HarAged and Leila Y. Mathers Charitable Foundation.


1To whom corRetortence may be addressed. E-mail: hwille{at} or stanley{at}

Author contributions: H.W., J.R.L., J.G.S., and S.B.P. designed research; H.W. performed research; M.S., M.M., J.O., and J.R.L. contributed new reagents/analytic tools; H.W., G.S., and J.G.S. analyzed data; and H.W., J.G.S., and S.B.P. wrote the paper.

↵2Present address: Department of Chemistry, University of Ottawa, Ottawa, ON, Canada K1N 6N5.

↵3Present address: National Prion Disease Pathology Surveillance Center, Department of Pathology, Case Western Reserve University, Cleveland, OH 44106.

The authors declare no conflict of interest.

This article contains supporting information online at


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