Locating folds of the in-register parallel β-sheet of theSup35p prion domain infectious amyloidAnton Gorkovskiya, Kent R. Thurberb, Robert Tyckob, and Reed B. Wicknera,1

Laboratories of aBiochemistry and Genetics and bChemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes ofHealth, Bethesda, MD 20892

Contributed by Reed B. Wickner, September 19, 2014 (sent for review August 8, 2014; reviewed by Michael TerAvanesyan)

The [PSI+] prion is a self-propagating amyloid of the translationtermination factor, Sup35p, of Saccharomyces cerevisiae. TheN-terminal 253 residues (NM) of this 685-residue protein normallyfunction in regulating mRNA turnover but spontaneously form in-fectious amyloid in vitro. We converted the three Ile residues inSup35NM to Leu and then replaced 16 single residues with Ile, oneby one, and prepared Ile-1-13C amyloid of each mutant, seedingwith amyloid formed by the reference sequence Sup35NM. Usingsolid-state NMR, we showed that 10 of the residues examined,including six between residues 30 and 90, showed the ∼0.5-nmdistance between labels diagnostic of the in-register parallel am-yloid architecture. The five scattered N domain residues with widerspacing may be in turns or loops; one is a control at the C terminus ofM. All mutants, except Q56I, showed little or no [PSI+] transmissionbarrier from the reference sequence, suggesting that they couldassume a similar amyloid architecture in vitro when seeded withfilaments of reference sequence Sup35NM. Infection of yeast cellsexpressing the reference SUP35 gene sequence with amyloid of sev-eral mutants produced [PSI+] transfectants with similar efficiency asdid reference sequence Sup35NM amyloid. Our work provides a strin-gent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locationsof the folds. This architecture naturally suggests a mechanism of in-heritance of conformation, the central mystery of prions.

[PSI+] prion | solid-state NMR | dipolar recoupling | amyloid | Sup35

Prions are infectious proteins, mostly self-propagating amy-loids. Amyloid is a filamentous polymer, rich in β-sheet

structure, in which the β-strands run perpendicular to the longaxis of the filament and the hydrogen bonds joining β-strands tomake a sheet are along the long axis of the filament. In mam-mals, prions are uniformly lethal diseases, caused by amyloidformation of the PrP protein. In yeast and fungi, prions are notuniformly fatal and have widely varying effects (reviewed in ref. 1).Perhaps the most remarkable feature of prions is that they havestrains or variants, distinct self-propagating forms of the sameprotein, analogous to alleles of a gene, each relatively stably prop-agated. The existence of different self-propagating prion variantsimplies an array of self-propagating structures, each based on thesame protein sequence. Because each prion variant is self-propa-gating, and each variant represents a different amyloid structure/conformation, there must be some mechanism by which the proteincan template its conformation. This mechanism must operate foreach of the many conformations that are possible for a given prion.An amino acid residue in a β-sheet can have interactions in

three dimensions (Fig. 1A): (a) along the peptide chain; (b) withthe residues it faces within the β-sheet but perpendicular to thepeptide chain, including the residues to which its main chainN–H and >C = O are hydrogen bonded; and (c) with residueslocated in the direction perpendicular to the β-sheet, possiblyanother β-sheet, interacting with the distal part of the side chain.The distance between residues having the relation (b), betweenadjacent β-strands in the β-sheet, is generally 0.47–0.48 nm. Thedistance between adjacent sheets, as in (c), is about 1.0 nm.

Given that the filament is a regular/repeating structure, themolecules in a filament can have any of several architectures(Fig. 1B). Most β-sheets in soluble enzymes are antiparallel, withadjacent β-strands running in opposite directions (Fig. 1B). La-beling a single atom in each molecule and measuring the distancebetween nearest neighbors will usually produce a distance of>0.9 nm (the distance between strands in a β-sheet is ∼0.47 nm) foran antiparallel β-sheet. A parallel β-sheet can be in-register or out-of-register. In-register means that each residue in one molecule isaligned with the same residue in the neighboring molecule, pro-ducing lines of identical residues along the long axis of the filament.Labeling a single atom in each molecule will give a distance be-tween labels of ∼0.48 nm if the filament has the parallel in-registerβ-sheet architecture (Fig. 1B). For an out-of-register β-sheet, thedistance will be greater, depending on the register shift (Fig. 1B). Ina β-helix, the distance between single labels will be >0.9 nm if eachmolecule constitutes two turns of the helix, and even greater if eachmolecule comprises more than two turns (Fig. 1B).The first demonstration of an in-register parallel amyloid

structure was by Benzinger et al. studying the 10–35 fragment ofthe Aβ peptide, and established the approach for proving thisarchitecture (2). The peptide was synthesized with a single 13Catom as the carbonyl carbon of one residue. Amyloid was as-sembled from each such synthetic peptide, and the distance be-tween labels (necessarily in different molecules because there wasonly one label per molecule) was measured by solid-state NMRusing dipolar recoupling. The distance between labeled carbonylcarbons was ∼0.5 nm regardless of which residue was labeled,establishing the in-register parallel architecture for this peptide.The full-size Aβ is a peptide of 40–42 residues. Tycko’s group

used a similar approach to show that amyloid of Aβ40 has an in-register parallel β-sheet architecture, and long range intra-molecular cross-peaks showed that the sheet was folded alongthe long axis of the filaments (refs. 3 and 4, reviewed in ref. 5).

Significance

Infectious proteins (prions) are capable of encoding geneticinformation by templating their conformation, just as DNAtemplates its sequence. The mechanism of this templating hasnot been clear. We provide definitive proof that the architec-ture of amyloid of the prion domain of yeast prion proteinSup35p is a folded in-register parallel β-sheet, and our dataidentify some of the sites of folds in the sheet. This architecturenaturally suggests a templating mechanism based on favorableinteractions among aligned side chains of identical amino acids.This is the only mechanism suggested to date for such a con-formation templating.

Author contributions: A.G., K.R.T., R.T., and R.B.W. designed research, performed re-search, contributed new reagents/analytic tools, analyzed data, and wrote the paper.

Reviewers included: M.T., Institute of Experimental Cardiology.

The authors declare no conflict of interest.1To whom correspondence should be addressed. Email: wickner@helix.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1417974111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1417974111 PNAS | Published online October 13, 2014 | E4615–E4622

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Moreover, filament mass per length data implied that these foldedsheets were associated to form filaments with either two or threemolecules per layer (6, 7). Different filament formation conditionscan also produce minor differences in the conformation of themonomer (8–10), but seeding can fix the amyloid form (6). Notably,seeding recombinant Aβ peptide with brain material of differentAlzheimer’s disease patients produced different amyloid forms, withthe same form produced by seeds from different parts of the brainof a single patient (11). This suggests that there may be differentforms of Alzheimer’s disease based on different self-propagatingamyloid variants, a notion now supported by animal studies (12).Most other pathologic human amyloids have been found to have

the in-register parallel β-sheet architecture. Amylin/IAPP (type 2diabetes), alpha-synuclein (Parkinson’s disease), and β2-micro-globulin (dialysis-induced amyloidosis) all follow this pattern (13,14). An 11-residue peptide from transthyretin also forms an in-register parallel β-sheet structure (15), but full-length transthyretin(senile systemic amyloidosis) breaks the rule in that amyloid formedin vitro does not have this architecture (16, 17).The [Het-s] prion of Podospora anserina is responsible for

a heterokaryon incompatibility reaction, a phenomenon wide-spread in filamentous fungi that limits cell–cell fusion to theclosest relatives to block the spread of fungal viruses (18, 19).Infectious amyloid formed by the prion domain of the HET-sprion protein has a β-helix structure, with two helical turns permolecule and three β-sheets forming the sides of the coil (20, 21).There is only a single prion variant of [Het-s] and the amyloidforms an unique structure in vitro with very narrow peaks in 2Dsolid-state NMR experiments, enabling complete assignmentsand structural determination (20, 21).The yeast prions [URE3], [PSI+], and [PIN+] (22, 23) are

amyloids of Ure2p, Sup35p, and Rnq1p, respectively, with a re-stricted region (the prion domain) of each responsible for boththe prion properties and the amyloid structure (24–33). Amyloidof the Ure2p prion domain (residues 1–89) was shown by solid-state NMR to be in-register parallel using preparations with [13C]carbonyl labeling of either the two Leu or the four Val residues or[13C]methyl labeling of the single Ala residue (34). These residues

are scattered through the prion domain, and the result with thesingle Ala residue implies that the nearest neighbor distancemeasured was an intermolecular distance. This result was con-firmed by solid-state NMR studies of full-length Ure2p labeledwith Ile (three residues in the prion domain) (35) and by electronspin resonance (36).Sup35p consists of a Q/N-rich N-terminal domain (N, residues

1–123), a highly charged middle domain (M, residues 124–253),and the C-terminal domain essential for translation termination(C, residues 254–685). The Sup35p prion domain includes the Ndomain (residues 1–123) and an unknown part of the M domain,depending on how “prion domain” is defined. Residues 1–61fused to GFP are sufficient, in amyloid form, to transmit severaldifferent prion variants by protein transformation (31), but somevariants require up to residue 137 (37) and residues in the Mdomain are part of the barrier to [PSI+] prion transmission be-tween wild strains of Saccharomyces cerevisiae (38). H–D ex-change experiments indicate stable structures in the M domain inthe amyloid of some [PSI+] variants (39). Of the eight Leuresidues (seven of which are in M), solution NMR suggests thatonly four are fully unstructured (39). In agreement, solid-stateNMR experiments suggested that four of the eight Leu residuesare in parallel in-register structures (40).We previously showed that amyloid of Sup35NM having all

Tyr carbonyl carbons labeled with 13C showed signal decay ratesin dipolar recoupling experiments indicative of nearest neighbor13C at a distance of ∼0.5 nm (40, 41). Dilution of this Tyr-labeledsample with unlabeled Sup35NM and then formation of amyloidresulted in a dramatic increase in the nearest neighbor distance,exactly as predicted if the nearest neighbor were on anothermolecule. We inferred that the Sup35NM amyloid has an in-reg-ister parallel architecture, and data with Sup35NM amyloid labeledwith Phe-1-13C, Leu-1-13C or Ala-3-13C were consistent with thisconclusion. However, one could imagine that although the nearestneighbor of a given Tyr was on another molecule, it was not thesame Tyr. There are 20 Tyr residues in Sup35N (and none inSup35M), making this a real possibility. We therefore sought adefinitive proof of the in-register parallel architecture of Sup35NM.In addition, there continues to be a view that Sup35NM amyloidhas a β-helix architecture (42–45), and a more rigorous test wastherefore important to resolve this central issue.

ResultsTo test the in-register parallel model for amyloid of Sup35NM,it would be ideal to have singly 13C-labeled molecules so thatnearest neighbor distances would necessarily be intermolecular.At 253 residues, Sup35NM is too long to synthesize. Instead, weeliminated the only three Ile residues, all in the M domain(residues 152, 220, and 239) to do Ile scanning mutagenesis. Ile isa suitable choice for this role because adding labeled Ile to theculture completely suppresses endogenous synthesis, there is noincorporation into other amino acids, there are only three Ileresidues in Sup35NM to change to the nearly identical Leu, andIle is an amino acid compatible with prion formation (46).It would be ideal to have amyloid preparations with uniform

conformation for structural studies. Although efforts have beenmade using specific Sup35NM filament formation conditions (30)or seeding using Sup35p or Ure2p prion amyloid isolated fromcells (31, 35, 47), the relatively broad peaks found in 2D solid-stateNMR experiments (35, 47) and nonexponential H–D exchangecurves (39) indicate considerable heterogeneity of filaments madein vitro. Even single [PSI+] clones show segregation of prion var-iants and frequent mutation, indicating the existence of a cloud ofprion variants in vivo (48). Furthermore, single amino acid changescan, in many cases, block [PSI+] prion propagation (49, 50). Thus,we can only hope to approach this ideal.Single isoleucine residues were introduced throughout the N

domain, and one control at residue 239, presumed to be outside

Fig. 1. Amyloid structure. (A) An amino acid residue in amyloid has inter-actions in three directions: (a) along the peptide chain, (b) between alignedresidues in the β-sheet along the fibril axis but perpendicular to the peptidechain (∼0.48 nm distant), and (c) with residues in an adjacent β-sheet (∼1.0nm distant). Based on a model of Aβ amyloid (4). (B) Types of β-sheet. Thedots represent a single 13C-labeled atom in each protein molecule. Theexpected separation is ∼0.48 nm for in-register parallel, ∼0.94 nm for anti-parallel, and >0.9 nm for β-helix. (C) Amyloid fibers of Ile-1-13C–labeledSup35NM with mutations Y13I, I152L, I220L, and I239L.

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the amyloid-forming region. For each mutant, Ile-1-13C–labeledprotein was produced in Escherichia coli for NMR studies.Electron micrographs of amyloid preparations showed filamentssimilar to those previously described for Sup35NM (28) (Fig.1C). To ensure that the amyloid whose structure we are studyingis representative of prion amyloid in vivo, we took several steps.First, the mutant SUP35NM sequences were fused with thenormal SUP35C and used to replace the normal protein in vivo.Using cytoduction (cytoplasmic mixing), [PSI+] was transferredinto these cells from a strain with the wild-type Sup35p, and thetransmission efficiency was measured (Table 1). We found a hightransmission frequency for most of the mutants, with the cyto-ductants showing intensity of phenotype and stability of propa-gation similar to that seen with the wild-type recipient. Q56I,which resulted in a barrier to transmission, was presumed toblock formation of the structure and was not studied further. Asdescribed previously, Sup35 natural variants Δ19 and E9 largelyblocked transmission of [PSI+] (38). The second approach toensuring normal prion amyloid structures was to seed filamentformation by mutant protein with 1% of sonicated amyloid fil-aments composed of the wild-type Sup35NM sequence. Thethird method was to transfect filaments formed from wild-typeand mutant Sup35NM (seeded with wild-type Sup35NM fila-ments) into yeast expressing the wild-type Sup35p and assess theefficiency of transfection and the array of prion variants formed(Table 2). Filaments of wild-type Sup35NM produced mostlystrong, stable [PSI+] transfectants curable by growth on guani-dine. Filaments of mutant proteins produced [PSI+] transfectantsat efficiencies within twofold that of the wild-type sequence, andmost were strong variants, but many were unstable (Table 2).Although all mutants could form [PSI+] prions in vivo, the resultssuggest that the mutations often selected certain variants or even

altered the amyloid conformation, as could be expected fromprevious results (49–51).

One-Dimensional Solid-State NMR. Detecting the single 13C labelper molecule (Ile-1-13C) in a 253-residue protein is challenging;with 356 other carbonyl groups, 1.1% of which have the naturalabundance 13C, there is a total of 3.92 natural abundance 13Cresidues. In principle, the specific label may constitute only∼20% of the total carbonyl-13C. We used the aliphatic 13C signalas a measure of natural abundance in each amyloid sample, andwith the ratio of aliphatic to carbonyl 13C signal from an unlabeledamyloid sample determined the amount of natural abundancecarbonyl 13C to subtract to give the signal derived from the Ile-1-13Clabel. This correction was applied to adjust the dipolar recouplingexperiments for natural abundance signal (Methods). This methodwas also applied directly to 1D spectra, subtracting the Fourier-transformed spectrum of unlabeled Sup35NM fibrils from spectraof labeled fibrils to cancel the aliphatic region, with the remainingcarbonyl peak(s) attributable to the Ile-1-13C label (Fig. 2). Inall samples the carbonyl peak was narrowed after subtracting thenatural abundance signal and showed a chemical shift indicativeof β-sheet structure (Table 3). Rehydration of several samplesshowed significant narrowing of the carbonyl peaks and againchemical shifts typical of β-sheet structure (Table 3 and Fig. S1).

Dipolar Recoupling Experiments. The distance of a single labeledatom, the carbonyl 13C of the lone isoleucine in each molecule, tothe nearest neighbor 13C is measured using the PITHIRDS-CTmethod of dipolar recoupling (52). The rate of signal decay in thisexperiment is proportional to 1/r3, where r is the distance beingmeasured. Because of this relation, the measurement is only usefulwithin the range of 0.2–0.8 nm, but this is exactly the range to testfor the expected ∼0.5-nm spacing between identical atoms indifferent molecules of an in-register parallel β-sheet.One control was the natural abundance 13C in amyloid of

unlabeled Sup35NM. Because only 1.1% of 13C atoms are la-beled, the nearest neighbor distance should be >>1.0 nm. In-deed, there is only a very gradual decay of the signal in thePITHIRDS-CT experiment for this sample. As another control,labeling residue I239, which is naturally isoleucine, and is beyondthe prion domain by all measures, shows a decay indicative of>0.8 nm. We carried out PITHIRDS-CT experiments with 15other samples, each with a single site labeled with Ile-1-13C (Fig.3 A–C). Many residues showed the rapid decay indicative of in-register parallel β-sheet structure. Y13I, Q22I, Y35I, Y46I, N48I,Q71I, and N109I are clearly in this group. Q62I, Y82I, Y101I,and Q90I show slightly slower signal decay, suggestive of in-reg-ister parallel structure and far too rapid for a β-helix. The slightlyslower decay for these four samples may be due to errors in thelarge correction for natural abundance 13C, the presence ofsome protein not in amyloid form, or a mixture of prion variants.The Y29I sample shows rapid initial decay followed by a highplateau, consistent with this residue being parallel in-register insome fibrils, but not others. I239, G51I, G58I, and Y73I are clearlynot parallel in-register. The error bars in these experiments arenecessarily quite wide because, as described above and in Methods,one is subtracting a large natural abundance signal and setting thedifference to 100 at the 0 recoupling time.To confirm the validity of our correction for natural abun-

dance 13C, we prepared one mutant, Y13I, with Ile-1-13C, usingeither the usual dextrose containing 1.1% natural abundanceisotope, or 13C-depleted dextrose, so that the natural abundance13C would be a smaller proportion of the total. The 1D spectrumshows the expected lower proportion of natural abundance 13Cin the aliphatic region (Fig. 2). From these data, we calculate thatthe fibers made with 13C-depleted dextrose had only 0.24% 13C, or∼0.85 residues per Sup35NM molecule. The correspondingraw PITHIRDS-CT data show a more robust decay, and when

Table 1. Cytoduction test of mutant protein compatibility

Cytoductants

Donor Recipient Sup35 allele [PSI+], % Total

779-6A [PSI+] ρ+SUP35reference

4830 ρo Reference 95 57

Δ19 1 114E9 5 80

3I→3L, Y13I 80 153I→3L, Q22I 100 733I→3L, Y29I 74 503I→3L, Y35I 84 1353I→3L, Y46I 91 1053I→3L, N48I 99 1973I→3L, G51I 80 603I→3L, Y55I 84 683I→3L, Q56I 15 393I→3L, G58I 92 263I→3L, Q62I 98 623I→3L, Q71I 83 1233I→3L, Q73I 100 353I→3L, Y82I 95 873I→3L, Q90I 82 1523I→3L,Y101I 89 183I→3L, N109I 96 963I→3L, L126I 93 423I→3L, H225I 94 333I→3L, 239I 97 88

Cytoplasmic mixing (cytoduction) was used to introduce [PSI+] from thedonor strain (779-6A) expressing the reference Sup35p into the recipientstrain 4830 carrying Sup35p with the indicated sequence. Cytoductants werescored for transmission of the [PSI+] prion.

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corrected for this natural abundance 13C, a result similar to thatof the Y13I sample made with the usual dextrose, but witha smaller variance (Fig. 3D).

DiscussionOur previous claim that Sup35NM amyloid has the in-registerparallel β-sheet architecture relied on our demonstration thatthe distance from a 13C carbonyl label on a Tyr residue to an-other 13C carbonyl Tyr in a different molecule was about 0.5 nm.However, because each molecule had 20 labeled Tyrs, that datadid not prove it was the same Tyr in the neighboring moleculethat was nearby. Y35 in one molecule might have been near Y13in another molecule. Here, using singly labeled molecules, weshow that this cannot be the case at least for five Tyr residuesand five other residues scattered through the N domain. Thesedata prove the in-register parallel model for the N domain.If the N domain, which is largely in β-sheet form, were a single

flat sheet, it would appear as fibers more than 0.35 nm/residue ×∼100 residues = 35 nm wide, but the diameter of filaments ofSup35NM amyloid is only 11.5 ± 1.5 nm (28) or 9 ± 1 nm (41)wide. We infer that the β-sheet must be multiply folded. Wepropose that residues not showing the ∼0.5-nm spacing are in theturns or loops present at each fold (Fig. 4), because residues inturns or loops are not as tightly constrained by main chainH-bonds to the neighboring molecules to be close to the corre-sponding residues. Transfection experiments (Table 2) and solid-state NMR lineshapes (Fig. S1) indicate that our mutantSup35NM fiber samples are not structurally homogeneous, so wecannot determine a unique secondary structure for Sup35NM inthese fibers. It is possible that residues showing decay ratesslightly above the 0.5-nm level are parallel in-register in somefibers, but in a turn or loop in others. Thus, our data confirm thepresence of turns of the β-strands (i.e., folds in the β-sheet).Paradoxically, alteration of Q56, which seems to be in a loop

or turn based on the results with Q51 and Q58, shows a barrier totransmission of [PSI+], but similar alteration of other residueslocated in in-register regions do not produce such a barrier.Resolution of this paradox will require more detailed examina-tion of the structure in this region, and it remains possible thatthis residue is not actually in a loop.Krishnan and Lindquist proposed that Sup35NM amyloid was

a β-helix including residues 30–90, based on chemical modifica-tion of cysteine-substituted mutants with pyrene maleimide, alarge fluorescent probe (43), and single fiber stretching experi-

ments (44). Our data are incompatible with the claim that resi-dues 30–90 are in a β-helix form. Six of the 10 residues examinedin this region show the ∼0.5-nm spacing incompatible with thisarchitecture. We suggest that the other four are in loops or turns.In addition, the mass per length of a β-helical fiber is less than 0.5molecules per 0.47 nm, but that of amyloid fibers of Sup35NM orfull-length Sup35p is consistently ∼1.0 (7, 53), ruling out theβ-helix model.Hydrogen–deuterium exchange experiments showed protection

from exchange varying with amyloid formation conditions andwith location in the sequence, with most N residues examinedmore protected than most M residues (39). Luckgei et al. andSchutz et al. have been able to make NMR assignments for res-idues 4–27 of amyloid of Sup35NM (54) and residues 2–30 of full-length Sup35p (55) and find that most residues in this domain arein β-sheet. Another study examined the chemical shifts and dy-namic features of different amino acid types, finding that the Ndomain is more rigid than the M domain (47). None of thesestudies deals with the issue of which type of β-sheet forms theamyloid core of Sup35NM.The folded in-register parallel β-sheet architecture naturally

suggests an explanation for the heritability of protein conformationand the existence of multiple heritable prion variants (1, 56). Thefavorable interactions between identical side chains—hydrogenbonds between Q, N, S, or T side chains or hydrophobic inter-actions between F, Y, W, I, L, or V side chains—can occur only ifthe peptides are in-register. Therefore, these interactions keep thestructure in-register. Charge repulsion between identical K, R, E,or D residues would impair this structure, but such residues are fewin the Ure2p, Sup35p, and Rnq1p prion domains shown to havethis architecture. The same interactions force the prion domain ofa molecule newly joining the end of the filament to have the sameconformation as the last molecule on the end of the filament, andthus have its folds/loops in the same locations as the other mole-cules in that filament. We suggest that different prion variants havefolds in different locations, but once formed the conformation israther faithfully propagated by this mechanism (1, 56).The yeast prions [URE3], [PSI+], and [PIN+] each have mul-

tiple prion variants in vivo and form heterogeneous/polymorphicamyloid in vitro. Seeding with filaments isolated from cells canreduce the variability of filaments to some extent (30–32, 47), andspecific filament formation conditions may favor one group ofvariants over another (39). However, this approach may be limitedby the known existence of a “cloud” of prion variants in a single

Table 2. Transfection of yeast with mutant Sup35NM amyloid filaments

Amyloid Weak stable [PSI+] Strong unstable [PSI+] Strong stable [PSI+] [PSI+]/μg amyloid

Wild type 0 2 89 14H2O 0 0 0 0Y35I 0 1 93 34N48I 0 69 20 12Q71I 0 23 60 36Wild type 0 0 50 13Y46I 8 44 27 26Wild type 4Q62I 0 59 31 2Y101I 0 39 54 4Wild type 5Y13I 0 74 20 5N48I 2 68 23 6Q90I 0 84 6 5N109I 2 34 58 17H2O 0 0 0 0

Ade+ clones were checked for guanidine-curability, for strength of the prion (Ade+) phenotype on 1/2 YPDand for prion stability in subclones.

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strain (48). Further progress in obtaining more detailed structuralinformation on yeast prion amyloid filaments will depend on de-velopment of new methods to obtain homogeneous preparations.

MethodsStrains and Media. Media are as described (57). Strain 779–6A [MATα kar1-1ade2-1 SUQ5 his3 leu2 trp1 ura3 [PSI+] (58)] was from Dan Masison, NationalInstitutes of Health, and strain 4830 [MATa kar1-1 ade2-1 SUQ5 leu2 trp1ura3 lys2 sup35::kanMX p1215 (CEN URA3 PSUP35SUP35C)] has been de-scribed (38).

Plasmid Constructs. Plasmid p1399 is pET21a(+) (EMD Millipore) into whichSUP35NM with a His6 tag was inserted with codons optimized for E. coli(Gene Art, Life Technologies). Using the QuikChange Lightning Multi-Site-Directed Mutagenesis Kit (210515; Agilent Technologies), we simultaneouslymade I152L, I220L, and I239L, converting all of the Ile residues in Sup35NMto Leu making p1426. Single residues were then changed to Ile in individualplasmid constructs using the QuikChange Lightning Site-Directed Muta-genesis Kit (210519; Agilent Technologies). These plasmids were used forprotein production in E. coli. For testing the biological properties of mutantproteins in yeast, we used the InFusion kit (639649; Clontech) to transfer theSUP35NM mutants, without the His6 tag, into p1422 (LEU2 CEN PSUP35 BamHI

NdeI AUG SUP35C), producing an in-frame fusion with SUP35C under controlof the SUP35 promoter on a single-copy plasmid.

Cytoduction. Transfer of cytoplasm from cell to cell without altering nuclei orplasmids uses the kar1-1 mutation, which interferes with nuclear fusion (59).The [PSI+] ρ+ donor strain 779–6A (carrying a strong stable [PSI+]) was mixedin twofold excess over the [psi-] ρo strain 4830 with a derivative of p1422 asthe only source of Sup35p. After 7 h of mating on a rich plate, matingmixtures were streaked for single colonies on plates selecting against thedonor strain. Colonies that were respiration-competent and were not dip-loids were tested for growth on –Ade plates to determine if [PSI+] had beensuccessfully transmitted. Recipients carrying either the reference (S288C)Sup35, the deletion of residues 59–77 in the oligopeptide repeat region ofSup35N (Δ19), or the “E9” allele (N109S with several M domain differences)(38) were used as positive and negative controls.

Expression, Labeling, and Purification of Sup35NM. Proteins were expressed inE. coli strain BL21-CodonPlus (DE3) RIPL (Agilent Technologies) growing insynthetic complete medium containing 100 μg/mL of ampicillin (41). AtOD550 at 0.5, cells were collected and resuspended in the same medium butwith isoleucine replaced with 100 mg/L isoleucine-1-13C (Cambridge IsotopeLaboratories), incubated with shaking at 37 °C for 15 min, then made 1 mMin isopropyl β-D-1-thiogalactopyranoside. After 4–6 h further growth, cellswere harvested, lysed by addition of 8 M guanidine containing 100 mMTris·Cl (pH 8.0) and 150 mM NaCl with one “complete, EDTA-free” proteaseinhibitor tablet (Roche). After overnight incubation at 4 °C, the extract wasspun at 30,000 rpm in a 45Ti Beckman rotor for 1 h. The supernatant wasapplied to a 4-mL (packed volume) NiNTA column (Qiagen), washed with200 mL of 8 M urea 0.1 M Tris·Cl (pH 8.0) and 150 mM NaCl, then with 10 mLof the same buffer with 10 mM imidazole and eluted with the same buffercontaining 200 mM imidazole. Protein-containing fractions were immedi-ately applied to a PD-10 desalting column equilibrated with 5 mM K PO4

(pH 7.2) and 150 mM NaCl. The eluate was immediately seeded with 1%wt/wt of wild-type Sup35NM filaments and incubated at room temperaturewithout agitation to allow filament formation. Filaments were washed twicewith water, dried by lyophilization, and packed in 3.2-mm-thick-walled zirconium

Y13C-depleted dextrose

ile-1- C

Y13minus unlabeled signal

ile-1- C

Y13 ile-1- C

G58 ile-1- Cminus unlabeled signalunlabeled

150 100 50 0 ppm 150 100 50 0 ppm

13

1313

13

13

13

13

13

Q62I ile-1- C

Y46I ile-1- C

239 ile-1- Cminus unlabeled signal

Fig. 2. Solid-state NMR spectra of Sup35NM amyloid samples. Spectra ofSup35NM labeled with Ile-1-13C at single sites (or unlabeled to measure thenatural abundance signal) were recorded at 9.39 T at 9 kHz MAS. Eachprotein has the I152L, I220L, and I239L mutations (except the I239 sample,which has only the first two changes) as well as a single other residuechanged to Ile. All (except the “unlabeled” sample, which is Sup35NM Y29I)were fully labeled at the indicated single site with Ile-1-13C. The Fourier-transformed signal from the unlabeled samples was subtracted from theindicated labeled sample signals to eliminate the aliphatic signal and,therefore, the natural abundance part of the carbonyl signal. The carbonylchemical shift determined from these corrected spectra was consistentlywithin 0.2 ppm of that determined from the raw spectra. The peak between100 and 150 ppm is background signal from plastic in the probe.

Table 3. Analysis of 13C solid-state NMR spectra

Chemical shift, ppm FWHM, ppm

Sup35NM sample Raw Net Net

Y13I 172.99 172.83 3.4Q22I 172.96 172.98 2.2Y29I 172.28 172.19 3.8Y35I 172.52 172.16 3.7Y46I 173.12 173.19 3.5Y46I wet 172.7 2.2N48I 172.60 172.25 4.1N48I wet 172.2 2.6G51I 172.97 172.98 4.6G51I wet 173.0 3.4G58I 172.75 172.93 4.3Q62I 172.35 172.10 4.6Q71I 172.96 172.94 4.0Y73I 173.38Y82I 172.75 172.75 3.7Y82I wet 173.0 2.8Q90I 173.28 173.45 3.0Y101I 173.15 173.19 3.5N109I 172.92 172.95 5.8I239 172.16 172.46 4.1Unlabeled 172.98 6.7

Each sample was labeled with Ile-1-13C (except for the ‟unlabeled” sample)and a 1D solid-state NMR spectrum was obtained on the dry sample and, inseveral cases, after rehydration (wet). Chemical shifts determined from theuncorrected 1D spectra are in the ‟raw” column, whereas those after sub-traction of the natural abundance signal are shown as ‟net.” Only net peakwidths are shown, except for the unlabeled sample. The Ile carbonyl 13Cchemical shift for random coil conformation is 174.7, and a significantly lowercarbonyl chemical shift is characteristic of β-sheet structure (64).

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oxide rotors for solid-state NMR. Filaments stained with uranyl acetate wereexamined using an FEI Morgagni transmission electron microscope operat-ing at 80 kV.

Solid-State NMR. Solid-state NMR experiments were carried out at 100.4 MHz13C frequency (9.4 T) on a Varian InfinityPlus spectrometer and 100.8 MHz 13Cfrequency on a Bruker Avance spectrometer with lyophilized amyloid samplespacked in thick-walled 3.2-mm zirconium oxide rotors using a magic anglespinning (MAS) NMR probe (Varian). Sample temperatures were maintainedat ∼24 °C during PITHIRDS-CT measurements by cooling with cold nitrogengas. One-dimensional 13C NMR spectra used 9-kHz MAS with 1.5-ms 1H-13Ccross-polarization (60) and 100-MHz two-pulse phase-modulated 1H decou-pling (61). Dipolar recoupling experiments were carried out at an MAS fre-quency of 20 kHz using the PITHIRDS-CT method (52) and spin-lock detection(62). T2 values were measured for each sample by varying the dipolarrecoupling period but with no recoupling, and in all cases >20% of the initialspin-locked 13C NMR signal remained after 76.8 ms, implying T2 relaxationtimes greater than 48 ms for carbonyl sites under PITHIRDS-CT conditions.

One-dimensional NMR spectra were recorded of amyloid of each mutantSup35NM labeled with Ile-1-13C and one that had only natural abundance 13C.The unlabeled sample and several labeled samples were rehydrated by ad-dition of 4 μL of water, and 1D spectra were again obtained. Signal in thealiphatic region could come only from natural abundance 13C, whereas thatin the carbonyl region was a combination of natural abundance and thesingle residue of fully labeled Ile-1-13C. The ratio of aliphatic to carbonylsignal in the unlabeled sample compared with the same ratio for the Ile-1-13Clabeled samples allowed us to measure the fraction of the carbonyl signal dueto the Ile-1-13C label. The slow rate of decay of magnetization of naturalabundance carbonyl 13C in the PITHIRDS-CT experiment was measured usingamyloid of Sup35NM Y29I lacking any added label. This was used to correctthe PITHIRDS-CT results for the labeled samples as follows.

P3lab(t) = PITHIRDS-CT signal from a labeled sample.

P3NA(t) = PITHIRDS-CT signal from natural abundance sample (unlabeled).

Starting with integrals of the carbonyl (CO, 182–167 ppm) and aliphatic(AL, 73–4 ppm) regions of the 1D spectra of labeled (lab) and unlabeled

Fig. 3. Measurements of 13C-13C dipole–dipole couplings of Sup35NM samples singly labeled with Ile-1-13C. The PITHIRDS-CT method (52) was used with20-kHz MAS spinning in a 9.4 T magnet and sample cooling to maintain room temperature. Simulated curves for PITHIRDS-CT results with linear chains of 13Catoms separated by 0.4 (—), 0.5 (– –·), 0.6 (···), or 0.7 (gray—) nm are shown. (A) Amyloid filaments of Sup35NM Y13I, Y46I, Q62I, Q71I, Y73I, Y82I, and I239(each having the I152L, I220L, and I239L, except for the I239 sample) singly labeled with Ile-1-13C are compared with unlabeled filaments. Error bars are notshown in A or B for clarity but are generally 5–12%. (B) Amyloid filaments of Sup35NM Y35I, Q22I, N109I, Y101I, G58I, and G51I were analyzed. (C) Results forN48I, Q90I, and Y29I are shown. (D) Sup35NM Y13I was singly labeled with Ile-1-13C either with glucose containing the 1.1% natural abundance 13C (Y13I rawand Y13I corrected) or with ∼99.93% 13C-depleted glucose (Y13Id raw and Y13I depleted corrected). Curves labeled “raw” are not corrected for naturalabundance 13C, whereas the natural abundance 13C signal has been subtracted from those labeled “corrected.”

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(NA for natural abundance) samples, potentially from different numbers ofscans and slightly different sample sizes, we first rescale the labeled samplevalues to the NA scale by assuming the aliphatic signal in both cases arisessolely from NA 13C.

Let 1DCONA = raw integral of carbonyl region of 1D spectrum of theunlabeled sample.

1DALNA = raw integral of aliphatic region of 1D spectrum of theunlabeled sample.

1DCOlab = raw integral of carbonyl region of 1D spectrum of thelabeled sample.

1DALlab = raw integral of aliphatic region of 1D spectrum of thelabeled sample.

Multiply both 1DCOlab and 1DALlab by [1DALNA/1DALlab] to rescale.

From the rescaled CO signal of the labeled sample, subtract 1DCONA toget the part that is due to the label = [1DALNA/1DALlab]*1DCOlab –

1DCONA = 1DCOlab2.

The fraction of 1DCOlab that is from the label is flabCO = 1DCOlab2/1DCOlab.

The fraction of 1DCOlab that is from NA 13C is 1− flabCO = flabNA.

The part of the PITHIRDS-CT signal of the labeled amyloid that was due tothe label (and not the natural abundance), scaled to 100 at t = 0 is

P3labcorðtÞ =  �P3labðtÞ− P3NAðtÞ× flabNA

�=�flabCO

�:

The noise estimates for carbonyl peak integrals were made by integratingsimilar frequency intervals above and below the carbonyl frequencyand calculating the SD (σ). The SD of differences between labeled (lab)

and unlabeled (NA) samples was calculated by σdiff =ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiσ2lab + fNACOσ2NA

q .flabCO

,

where flabCO is the fraction of the raw carbonyl signal due to the label(see above).

Transfection of Amyloid. Recipient cells (779-6A [psi-]) were grown overnightat 30 °C in yeast extract-peptone-dextrose-adenine medium (YPAD). Onemilliliter of culture was used to inoculate 50 mL YPAD and cells were grownfor two to three doublings (5 h) at 30 °C. Cells were washed once with 20 mLwater, twice with 25 mL ST buffer [1 M sorbitol and 10 mM Tris·HCl (pH 7.5)]and resuspended in 5 mL ST buffer. Cells were protoplasted for 40 min at30 °C by addition of 20 μL lyticase (5 U/μL Sigma L5263 in 50% glycerol).Protoplasts were collected by centrifugation at 216 × g (1,000 rpm in a Sor-vall Legend T centrifuge equipped with a Sorvall Heraeus 75006434 rotor)for 3 min, washed two times with 10 mL STC buffer [1 M sorbitol, 10 mMTris·HCl (pH 7.5), and 10 mM CaCL2)] and resuspended in 1 mL STC buffer. Toreduce shearing protoplasts were resuspended by gentle rocking of the tube(50-mL polypropylene tubes; Corning).

Amyloid fibril suspensions were sonicated on ice three times for 45 s (dutycycle 40%, output 4) using a Branson 250 sonifier equipped with a microtip.When previously lyophilized amyloid fibrils were used, 45-s sonications weredone up to 10 times and the duty cycle was raised to 90%. Between soni-cations samples were kept on ice. Amyloid filaments were used at a con-centration of ∼6 μg/μL. To 100 μL of protoplasts was added 1 μL of single-strand DNA (calf sperm DNA, 10 μg/μL), 4 μL of pRS316 plasmid (URA3 CEN,0.9 μg/μL) (63),and 7 μL of amyloid filament suspension. The protoplast/DNA/protein mixture was incubated for 10 min at room temperature. Next, 900 μLPTC buffer was added and the mixture was incubated for 20 min at roomtemperature. Protoplasts were then collected by centrifugation for 3 min ina microcentrifuge at 250 × g (1,600 rpm, Eppendorf 5415R with F-45-24-11rotor). To the pellet was added 200 μL SOS buffer and the protoplasts wereleft to recover for 30 min at 30 °C. Recovered protoplasts were pipetted into10 mL of CS+A.1-U or CS+A5-U medium kept at 50 °C. To reduce shearingthe ends of pipette tips were cut off. Fourteen-milliliter round-bottompolypropylene tubes (BD Falcon) were used. The solution was mixed byinverting the tubes and poured directly into Petri dishes containing 20 mL ofthe same solidified medium. Plates were incubated for 6 d at 30 °C.

PTC buffer: 20% (wt/vol) PEG8000, 10 mM Tris·HCl (pH 7.5), and 10 mM CaCL2

SOS buffer: 1 M sorbitol, 7 mM CaCL2, 1/3 YPD (per 1 L: 3.3 g yeast extract,6.7 g peptone, and 6.7 g glucose)

CS+A.1-U: 1 M sorbitol, 0.67% yeast nitrogen base, 2% glucose, 1× com-plete amino acid mix, 0.1 mg/L adenine sulfate, and 20 g/L agar

CS+A5-U: 1 M sorbitol, 0.67% yeast nitrogen base, 2% glucose, 1× com-plete amino acid mix, 5 mg/L adenine sulfate, and 20 g/L agar

10× complete amino acid mix (per 1 L): 200 mg Met, 500 mg Tyr, 500 mgIle, 500 mg Leu, 500 mg Phe, 1,000 mg Glu acid, 2,000 mg Thr, 1,000 mgAsp, 1,500 mg Val, 4,000 mg Ser, 200 mg Arg, 200 mg His, 300 mg Lys, and300 mg Trp

The 10× complete amino acid mix was filter-sterilized before adding toautoclaved CS media containing the other components.

ACKNOWLEDGMENTS. This work was supported by the Intramural Programof the National Institute of Diabetes and Digestive and Kidney Diseases.

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