Both the cis-trans equilibrium and isomerizationdynamics of a single proline amide modulateβ2-microglobulin amyloid assemblyVladimir Yu. Torbeev and Donald Hilvert1

Laboratory of Organic Chemistry, ETH Zurich, CH-8093 Zurich, Switzerland

Edited by Ronald T. Raines, University of Wisconsin–Madison, Madison, WI, and accepted by the Editorial Board October 29, 2013 (received for reviewJune 1, 2013)

The human protein β2-microglobulin (β2m) aggregates as amyloidfibrils in patients undergoing long-term hemodialysis. Isomeriza-tion of Pro32 from its native cis to a nonnative trans conformationis thought to trigger β2m misfolding and subsequent amyloid as-sembly. To examine this hypothesis, we systematically varied thefree-energy profile of proline cis-trans isomerization by replacingPro32 with a series of 4-fluoroprolines via total chemical synthesis.We show that β2m’s stability, (un)folding, and aggregation prop-erties are all influenced by the rate and equilibrium of Pro32 cis-trans isomerization. As anticipated, the β2m monomer was eitherstabilized or destabilized by respective incorporation of (2S,4S)-fluoroproline, which favors the native cis amide bond, or the ste-reoisomeric (2S,4R)-fluoroproline, which disfavors this conforma-tion. However, substitution of Pro32 with 4,4-difluoroproline,which has nearly the same cis-trans preference as proline but anenhanced isomerization rate, caused pronounced destabilizationof the protein and increased oligomerization at neutral pH. Moreremarkably, these subtle alterations in chemical composition—incorporation of one or two fluorine atoms into a single prolineresidue in the 99 amino acid long protein—modulated the aggre-gation properties of β2m, inducing the formation of polymorphi-cally distinct amyloid fibrils. These results highlight the importanceof conformational dynamics for molecular assembly of an amyloidcross-β structure and provide insights into mechanistic aspects ofPro32 cis-trans isomerism in β2m aggregation.

protein conformation | polymorphism | amyloidogenesis |native chemical ligation

Human β2-microglobulin (β2m) is a component of the class Imajor histocompatibility complex (1). Although the WT

protein is generally stable at neutral pH, in kidney diseasepatients undergoing long-term hemodialysis, it forms insolubleamyloid deposits causing a condition known as dialysis-relatedamyloidosis (2). Collagen and glycosaminoglycans facilitate fibrilformation at neutral pH (3, 4), as do organic solvents (5), di-valent metal ions (Cu2+) (6, 7), and surfactants (8). Amyloids canalso be induced at low pH (<3) (9) or by “seeding” with stabilizedpreformed amyloids (10). Despite more than two decades of re-search, however, the molecular mechanism by which soluble β2m isconverted into insoluble fibrils remains poorly understood (11).Wild-type β2m (WT β2m) contains a highly conserved, cis-

configured proline at position 32 that is required for maintainingthe soluble native structure (Fig. 1) (12). Several lines of evi-dence, including detailed folding studies and mutagenesisexperiments on β2m, have suggested that cis-to-trans isomeriza-tion of Pro32 serves as a trigger to misfolding and subsequentaggregation (6, 13). In β2m amyloids, this residue adopts a transconformation (14, 15), and additives that increase the equilib-rium concentration of β2m conformers containing trans-Pro32have been shown to promote amyloid formation (11). A trans-proline amide is also observed in the X-ray structure of thehexameric species that is formed upon treatment of the H13Fvariant with Cu2+ ions (7). Although replacement of Pro32 with

glycine, which has a stronger propensity to adopt a trans amideconformation than proline, facilitates fibril elongation in seedingexperiments (13), population of a trans-amide at residue 32 is notsufficient in itself to induce fibrillation. Mutation of Pro32 toalanine and valine, which have much higher trans amide bondpropensities (>99.9%) than proline, does not result in enhancedamyloid formation (11). Other factors, such as protein confor-mational dynamics (11), are probably important for the initiationand assembly processes.In this paper, we report a robust chemical synthesis of the 99-

residue human β2m protein that allows us to probe the role ofPro32 cis-to-trans isomerization in amyloid formation with mech-anistically more informative substitutions than can be achievedusing conventional site-directed mutagenesis. Specifically, wegenerated three β2m variants in which Pro32 was replaced with(2S, 4S)-4-fluoroproline (4S-fpr), (2S, 4R)-4-fluoroproline (4R-Fpr),and 4,4-difluoroproline (F2Pro), respectively (16) (Fig. 1C).Fluorinated proline analogs have been used extensively to probethe structure and stability of many proteins (17–20). They arenearly isosteric to the natural amino acid and preserve the uniqueconformational properties of the five-membered pyrrolidine ringbut exhibit altered cis-trans amide isomer ratios. For example, theconfiguration at C4 in 4R-Fpr stabilizes the exo ring pucker viaa gauche effect, which, in turn, leads to higher stability of thetrans-prolyl conformer [6.7:1 versus 4.6:1 for proline in the modelcompound Ac-Xaa-OMe (20)] (Fig. 1D). The opposite is true for4S-fpr, which favors the endo ring pucker, reducing the prefer-ence for the trans-prolyl conformer to a factor of 2.5:1 (20). F2Prohas similar cis-trans peptide bond propensities as native proline

Significance

β2-Microglobulin is an abundant and normally soluble protein.In patients undergoing chronic dialysis, however, it forms in-soluble amyloid plaques, leading to medical complications. Ithas been suggested that the conformational transformation ofsoluble protein monomers into polymeric amyloids is mediatedby isomerization of a single amino acid, namely, proline 32. Inthis study, we probed the role of this amino acid by chemicallysynthesizing uniquely tailored protein analogs containingnoncanonical amino acids at position 32. Our results show thatboth the chemical equilibrium and rate of cis-trans isomeriza-tion of proline 32 are critical for the solubility of β2-micro-globulin and its self-assembly into morphologically distinctamyloid fibrils. These insights may aid ongoing efforts toprovide remedies against dialysis-related amyloidosis.

Author contributions: V.Y.T. and D.H. designed research; V.Y.T. performed research; V.Y.T.and D.H. analyzed data; and V.Y.T. and D.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. R.T.R. is a guest editor invited by the EditorialBoard.1To whom correspondence should be addressed. E-mail: hilvert@org.chem.ethz.ch.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1310414110/-/DCSupplemental.

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but a significantly lower activation barrier for isomerization (16).Biochemical characterization of WT β2m and the three analogsprovides unique insight into the mechanistic consequences of per-turbing the Pro32 peptide bond on protein (un)folding and sub-sequent aggregation to give oligomers and amyloid fibrils.

ResultsChemical Synthesis of Human β2m and Analogs by Native ChemicalLigation. WT β2m was assembled by “one pot” native chemicalligation of three unprotected peptide fragments (Fig. 2 and SIAppendix, Figs. S1 and S2) (21). The peptide segments weresynthesized using an “in situ neutralization” protocol for Boc-based solid phase peptide synthesis (22) and subsequently puri-fied to homogeneity by reverse-phase HPLC (RP-HPLC). Nativechemical ligation of the C terminal and middle fragments wasfollowed by thiazolidine (Thz) deprotection, ligation with theN-terminal peptide, and formyl-deprotection of tryptophans. AfterRP-HPLC purification, formation of the native disulfide bond byair-oxidation, and further RP-HPLC purification, the protein

was folded by dialysis against 25 mM sodium phosphate buffer(pH 7.5). High purity of synthetic β2m was verified by analyticalRP-HPLC and high-resolution mass spectrometry (Fig. 2B). Theprotein adopts a native fold as judged by CD spectroscopy (Fig.3A) and 1H-NOESY NMR spectroscopy (SI Appendix, Fig. S8).The three β2m analogs containing the noncanonical amino

acids 4R-Fpr, 4S-fpr, and F2Pro at position 32 were synthesizedanalogously using N-Boc-protected 4-fluoroprolines instead ofproline (see SI Appendix and Figs. S3–S6 for analytical data).Near-UV CD and 1H-NOESY spectra (SI Appendix, Figs. S7–S10) show that the variants adopt the same overall tertiarystructure as WT β2m. As expected for such close structuralanalogs, only small differences localized near residue 32 areobserved in the 1H-NOESY spectra (SI Appendix, Figs. S8–S10).

Fluoroprolines Modulate β2m Stability. Far-UV CD spectra andthermal denaturation curves for WT β2m and the three analogsare depicted in Fig. 3 A and B, respectively. Chemically synthe-sized β2m has a CD spectrum that is virtually identical to thepublished spectra of recombinantly expressed protein containingan additional N-terminal methionine (9, 23). Chief features area strong maximum at 203 nm and a weak minimum at 221 nm. TheCD spectra of [4S-fpr32]β2m and [4R-Fpr32]β2m are similar (Fig.3A). The relative stability of these three proteins was assessed bythermal denaturation. Their apparent melting temperatures (Tm)correlate with the cis-amide conformational preferences of therespective proline derivative at position 32 (Fig. 1D), decreasing inthe order [4S-fpr32]β2m > WT β2m > [4R-Fpr32]β2m (Table 1).The [F2Pro32]β2m protein deviates from these trends. Its far-

UV CD spectrum shows a diminished maximum at 203 nm and aslightly blue-shifted minimum at 218 nm (Fig. 3A). Interestingly,similar features were previously observed for β2m variants con-taining trans-configured Pro32, including the P5G point mutantand ΔN6, which lacks six N-terminal amino acids (23). Like theseproteins, [F2Pro32]β2m exhibits reduced thermal stability (Tm =56 °C) and less cooperative thermal denaturation compared withWT β2m (Fig. 3B).Chemical denaturation (Fig. 3C and SI Appendix, Fig. S13)

and folding–unfolding kinetics (SI Appendix, Fig. S14) of theFig. 1. Human β2-microglobulin (β2m). (A) Primary amino acid sequence ofthe 99-residue β2m with Pro32 highlighted in red and the two cysteine-containing ligation sites underlined. (B) Native structure of β2m showing theBC loop (cyan) containing cis-Pro32 (red) (based on PDB ID 2YXF) (C) Struc-ture of proline and the three fluorine-containing proline analogs used in thisstudy: (2S, 4S)-4-fluoroproline (4S-fpr), (2S, 4R)-4-fluoroproline (4R-Fpr), and4,4-difluoroproline (F2Pro). (D) Stereoelectronic effects associated with theC4 substituent influence both the proline ring conformation as well as thecis-trans amide bond equilibrium (17). An electron-withdrawing group atthe 4R position stabilizes the exo ring pucker via a gauche effect. This con-formation preorganizes main chain torsion angles, resulting in stabilizationof the trans peptide bond isomer via an n→π* orbital interaction. The equi-librium cis-trans propensities for proline and its fluorinated analogs followa different trend than the corresponding trans-cis isomerization rates (16).

Fig. 2. Chemical synthesis of WT β2m and its analogs. (A) The syntheticstrategy involved one pot native chemical ligation of three polypeptidefragments. (B) Reverse-phase HPLC analysis and electrospray ionization (ESI)mass spectrum of chemically synthesized β2m (1–99) (SI Appendix, Fig. S2).Analogous analytical characterization of the fluoroproline-containing β2mvariants is presented in SI Appendix, Figs. S3–S6.

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Pro32 variants further illuminate the interplay between cis-transequilibrium propensities, the free-energy barrier for amide bondisomerization, and protein stability. As shown in Table 1, cis-transequilibrium preferences correlate with the chemical denaturationmidpoint ([D]50%]) and unfolding rate (ku), whereas the isomer-ization barrier is reflected in the cooperativity of denaturation (mvalue) and refolding kinetics. The Gibbs free energy for unfolding(ΔGu) is lowest for [F2Pro32]β2m and highest for [4S-fpr32]β2m.Because F2Pro and unmodified proline have comparable cis-trans

equilibrium ratios, the destabilizing effects due to incorporation of

residue F2Pro32 are counterintuitive. Although the 1H-NOESYNMR spectra (SI Appendix, Fig. S10) provide strong evidencethat [F2Pro32]β2m and WT β2m adopt similar folds, the far-UVCD spectra and folding data suggest that F2Pro32 is morestructurally disruptive than the monofluorinated prolines. Togain additional insight into the effects of this substitution, weprepared a derivative of [F2Pro32]β2m in which seven residues inthe hydrophobic core and an adjacent loop (Val9, Ile7, Val27,Gly29, Gly18, Ala15, Phe22) plus two residues flanking F2Pro32(Phe30 and Ile35) were 15N-labeled (SI Appendix, Fig. S11A).The 1H-15N resonances in the 1H-15N heteronuclear singlequantum coherence (HSQC) spectrum of this variant exhibitaverage chemical shifts that differ by less than 0.1 ppm from thecorresponding resonances in WT β2m (SI Appendix, Fig. S11). Inaddition, 19F NMR studies showed that the F2Pro32 amideadopts a cis conformation in the folded protein but a transconformation when denatured (SI Appendix, Fig. S12). Together,these observations strengthen the conclusion that the averagestructures of [F2Pro32]β2m and WT β2m are quite similar.The destabilization of [F2Pro32]β2m can be rationalized by

additionally considering the energy barrier for cis-trans isomeri-zation (16). Inserting fluorine atoms into the proline five-memberedring reduces the double-bond character of the prolyl amide andlowers the free-energy barrier for cis-trans isomerization. For themodel system Ac-Xaa-OMe, the energy barrier decreases in theorder Pro > 4R-Fpr ∼ 4S-fpr > F2Pro (Fig. 1D) (16). IntroducingF2Pro, the most conformationally labile residue in the series, atposition 32 of β2m may destabilize the protein by “melting” theBC loop that maintains Pro32 in the cis conformation (Fig. 1B).Consistent with a more dynamic structure and increased expo-sure of hydrophobic surfaces, [F2Pro32]β2m binds the environ-mentally sensitive dye 8-anilino-1-naphthalenesulfonic acid (ANS)to a greater extent than the other variants, giving rise to enhancedfluorescence and a characteristic blue shift of the emission maxi-mum (Fig. 3D). As for the previously published P32G variant (13),minor structural perturbations in the vicinity of residue 32, en-hanced by the rapid isomerization kinetics of the F2Pro32 amide,may be propagated to other parts of the molecule without alteringthe core structure of the protein. The lower stability of [F2Pro32]β2mcan then be attributed either to this slightly altered, less compactstructure and/or to changes in the denatured state.

Aggregation Properties of Chemically Synthesized β2m Analogs. Theoligomerization state of β2m variants was analyzed by size-exclusion chromatography. Upon incubation at pH 7.5 for a pe-riod of 2 wk, WT β2m and the monofluoro-proline analogs (40 μMeach) behaved as stable monomeric proteins, whereas [F2Pro32]β2mafforded a population of oligomers (Fig. 3E). Although previousstudies on amyloidogenic proteins have shown that partially fol-ded species serve as precursors to aggregates and, ultimately,amyloids (24–27), analysis of these samples by transmission electronmicroscopy (TEM) showed no evidence for amyloid fibrils even forthe least stable [F2Pro32]β2m protein.Oligomerization of β2m at neutral pH is known to be promoted

by Cu2+ ions (6, 7). Although this process is characterized bya several hour lag phase in the case of WT β2m, the P32A mutantproduces protein oligomers instantaneously in the presence ofmetal ions (6). The trans amide conformation adopted by Ala32apparently preorganizes the protein for tighter Cu2+ binding,triggering formation of oligomers. The synthetic proteins alsoundergo Cu2+-induced oligomerization at rates that correlate withtheir copper affinity and preference for the trans amide confor-mation (Fig. 3F). Thus, the [4R-Fpr32]β2m analog, which favorsthe trans-amide and has the highest affinity for Cu2+ (Kd = 7.31 ±0.53 μM), shows the most pronounced fluorescence response ina ThT-binding assay, followed by WT β2m (Kd = 10.3 ± 0.49 μM)and [4S-fpr32]β2m (Kd = 20.2 ± 0.84 μM). Interestingly, no olig-omerization was detected with [F2Pro32]β2m, which has the low-est affinity for Cu2+ (Kd = 70.8 ± 4.59 μM) (SI Appendix, Fig. S15).Enhanced flexibility of the BC loop in this variant, as discussedabove, may hinder formation of an appropriately preorganized

Fig. 3. Properties of chemically synthesized WT β2m and three chemical ana-logs. (A) Far-UV CD spectra at 37 °C. (B) Thermal denaturation monitored by CD.(C) Chemical denaturation with GdmCl. A two-state folding mechanism wasassumed to compare the data for different analogs. The data are summarized inTable 1. (D) Fluorescence of ANS dye [concentration (c) 10 μM] upon addition ofWT β2m and three analogs (c protein 10 μM). (E) Size-exclusion HPLC of samples(c 40 μM) incubated for 2 wk at pH 7.5, 37 °C. (F) Kinetics of oligomerization ofthe variants (c 40 μM) in the presence of Cu2+ (c 80 μM) at pH 6.8. (G) The pH-dependent solubility of the synthetic proteins upon incubation for 2 wk (initial c40 μM in 0.2 M Na.phosphate, 0.1 M Na.citrate buffers). For each plot, data forWT β2m are shown in black, data for [4S-fpr32]β2m are shown in green, data for[4R-Fpr32]β2m are shown in blue, and data for [F2Pro32]β2m are shown in red.

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environment for Cu2+ coordination even though F2Pro andunsubstituted proline have similar conformational preferences.Protein solubility was further examined as a function of pH.

The synthetic proteins (40 μM) were incubated at 37 °C withoutstirring for a period of 2 wk. The concentration of soluble proteinin filtered (0.2 μm) aliquots was quantified by absorbance at 280nm. The resultant solubility curves are shown in Fig. 3G. Themost stable analog, [4S-fpr32]β2m, only precipitated below pH3.5, whereas the other proteins began to form insoluble aggre-gates at higher pH values. The least stable protein, [F2Pro32]β2m,displayed the least cooperative behavior. However, in no samplewere amyloid fibrils detected above pH 4, either by ThT fluores-cence or TEM.

Polymorphism of β2m Amyloids Regulated by cis-trans Pro32Isomerization. Incubation at acidic pH (<3) is the most reliableway to produce β2m amyloids (9). Indeed, all synthetic variantsspontaneously form amyloid fibrils at pH 2.5 within a few days toa week (Fig. 4A). Monitoring amyloid growth at this low pHvalue showed that WT β2m, [4S-fpr32]β2m, and [4R-Fpr32]β2mform fibrils at qualitatively similar rates, whereas [F2Pro32]β2maggregates more slowly. Nevertheless, intrinsic tryptophan fluo-rescence suggests that the different Pro32 analogs give rise todistinct fibril polymorphs (SI Appendix, Fig. S16). For example,WT and [4S-fpr32]β2m amyloids afford similar fluorescencespectra, but the fluorescence emission of [4R-Fpr32]β2m amy-loids is red-shifted and strongly diminished; amyloids derivedfrom [F2Pro32]β2m show intermediate values. These resultssuggest that Trp60, which is buried in WT amyloids (28), islocated in very different environments in the other polymorphsas a consequence of altered self-assembly of the respective cross-β structures.We performed seeding experiments to characterize the ki-

netics of amyloid growth and structural differences in amyloidsderived from the different protein variants. The preformed am-yloid seeds serve as growth templates, effectively eliminating thenucleation requirement and hence the kinetic lag phase (29).This templating effect is structure-dependent: amyloid seeds of aparticular protein polymorph are generally most efficient at in-ducing fibrils of the same protein polymorph (30, 31). Fibrilformation was monitored by a standard ThT fluorescence assayusing 40 μM protein in the presence of seeds derived from pre-formed amyloids of all four variants (16 combinations in total)(Fig. 4B).The results obtained using cognate seeds, which likely re-

semble the nucleating species that arise spontaneously duringunseeded aggregation, show the same qualitative trend for ag-gregation rates observed in the absence of seeds, namely WTβ2m ∼ [4S-fpr32]β2m ∼ [4R-Fpr32]β2m > [F2Pro32]β2m. Be-cause [4R-Fpr32]β2m does not aggregate faster than other var-iants, this trend does not support the simple hypothesis thataggregation is driven by the cis-trans amide bond ratio of residue32. Other factors, including isomerization kinetics and the al-tered conformational preferences of the proline ring itself, pro-bably play a role.In cross-seeding experiments, both growth onset and assembly

rate depend on the origin of the seeds. In Fig. 4B, the kinetics offibril growth of the four studied proteins is quantitatively similarin the case of WT and [F2Pro32]β2m seeds, whereas [4S-fpr32]β2mand [4R-Fpr32]β2m seeds produced very different growth patterns.Furthermore, analysis of the samples upon completion of the

reaction by TEM and intrinsic fluorescence indicates that seed-ing with WT or [F2Pro32]β2m amyloid seeds resulted in fibrilswith well-defined morphology in all cases (SI Appendix, Fig. S17);they also exhibit a strong intrinsic tryptophan emission signal (SIAppendix, Fig. S18). In contrast, experiments with [4S-fpr32]β2mand [4R-Fpr32]β2m seeds yielded well-defined fibrils for [4S-fpr32]β2m and [4R-Fpr32]β2m, respectively, but gave mixtures oflong, straight amyloids and large amounts of amorphous pre-cipitate for the WT β2m and [F2Pro32]β2m proteins (SI Appendix,Fig. S17).Although [4S-fpr32]β2m and [4R-Fpr32]β2m seeds have, in-

dividually, distinctive templating properties, hybrid seeds derivedfrom amyloid fibrils grown from a mixture of [4S-fpr32]β2m and[4R-Fpr32]β2m proteins recapitulate the properties of WT β2mseeds (SI Appendix, Figs. S19 and S20). Thus, growth kinetics forthe four β2m variants seeded with WT and hybrid seeds wasequivalent (compare Fig. 4B, Top Left, and SI Appendix, Fig.S20A). Intrinsic tryptophan fluorescence of hybrid amyloidsgenerated from a 1:1 mixture of [4S-fpr32]β2m and [4R-Fpr32]β2mproteins (SI Appendix, Fig. S20B) also suggests that their struc-tural properties approximate those of WT fibrils. At pH 2.5, β2mexists as a mixture of partially structured and extensively un-folded conformers (32). Because the BC loop that maintainsPro32 in the cis conformation will be (partially) denatured underthese conditions, the average trans-cis amide bond ratio for anequimolar mixture of 4R-Fpr (6.7:1) and 4S-fpr (2.5:1) effec-tively mimics that of unmodified proline (4.6:1) (20). Becausehybrid fibrils formed by two β2m analogs containing isomeric 4-fluoroprolines behave like WT β2m amyloids, it is unlikely thatreplacement of hydrogen with a larger fluorine atom modulates thestructure and stability of the amyloid fibrils for steric reasons. In-stead, polymorphism would appear to have a conformational origin.

DiscussionFluorinated proline derivatives are useful probes of structure–function relationships in proteins (16–20). In addition to alteringthe equilibrium population of cis and trans isomers via stereo-electronic effects, fluorine substitution lowers the barrier forisomerization by reducing the double-bond character of theprolyl amide (16). Fig. 1D shows schematically the relative orderof the two effects. The values for trans-cis equilibrium ratios forproline and its three 4-fluoroproline variants follow a differenttrend than the corresponding trans-cis isomerization rates. Thesecounterbalanced properties are useful for studying folding, mis-folding, and amyloid formation in β2m. A robust chemical syn-thesis of β2m allows site-specific replacement of one out of thefive prolines with unnatural 4-fluoroproline analogs to dissecthow the conformational properties of Pro32 influence foldingand misfolding mechanisms.Previous studies with WT β2m showed that trans→cis prolyl

bond isomerization of Pro32 is the rate-limiting step in the pro-tein-folding mechanism (33, 34). As a consequence, the apparentfolding rate of WT β2m is independent of denaturant concentra-tion (SI Appendix, Fig. S14A). In the case of [4S-fpr32]β2m and[4R-Fpr32]β2m, a weak dependence on denaturant concentrationis evident on their folding kinetics (SI Appendix, Fig. S14 B and C),in agreement with lower cis-trans isomerization barriers for both4S-fpr and 4R-Fpr residues. In contrast, folding of [F2Pro32]β2mshowed a strong dependence on denaturant concentration (SIAppendix, Fig. S14D) and proceeded much faster than folding ofWT β2m at low denaturant concentrations. In essence, the

Table 1. Thermodynamic stability and unfolding kinetics

Variant Tm, °C mΔG, kcal/mol/M;mean ± SD [D]50%, M; mean ± SD ΔGH2Ou , kcal/mol ΔΔGH2O

u , kcal/mol 10−4 × kH2Ou , s−1

[4S-fpr32]β2m 64 2.33 ± 0.17 2.79 ± 0.02 6.5 −0.27 0.92WT β2m 61.5 2.72 ± 0.21 2.29 ± 0.02 6.23 0 2.2[4R-Fpr32]β2m 58.5 2.68 ± 0.25 2.08 ± 0.02 5.57 0.66 5.14[F2Pro32]β2m 56 2.15 ± 0.21 2.29 ± 0.04 4.92 1.31 7.32

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trans→cis Pro32 isomerization step is no longer rate-limitingwhen Pro32 is replaced by F2Pro, the residue with the lowestbarrier for cis-trans isomerization.Thermal and chemical denaturation of the folded β2m variants

shows that their stability decreases in the order [4S-fpr32]β2m >WT β2m > [4R-Fpr32]β2m > [F2Pro32]β2m (Table 1). Thus,monomeric β2m is stabilized relative to WT when Pro32 isreplaced with 4S-fpr, which favors the native-like cis-Pro32conformer, and destabilized when proline is replaced with4R-Fpr, which favors the nonnative trans-Pro32 conformer. Inenergetic terms, these effects are relatively modest, however. The

0.66 kcal/mol destabilization observed for [4R-Fpr32]β2m wasnot sufficient to induce protein oligomerization at neutral pH.Unexpectedly, [F2Pro32]β2m proved to be the least stable vari-ant. Even though F2Pro and proline have similar cis-trans-amideratios in model compounds, the Pro32 to F2Pro substitutiondestabilized β2m by 1.3 kcal/mol. This seemingly innocuoussubstitution also increased the dynamic properties of the protein,judging from enhanced ANS binding and facile oligomerization,probably by introducing structural flexibility in the BC loopwhere residue 32 is located. Although the NMR evidence showsthat the [F2Pro32]β2m tertiary structures are, on average, verysimilar to that of WT β2m (SI Appendix, Figs. S10 and S11),enhanced flexibility of the F2Pro32 amide bond likely increasesthe solvent exposure of normally buried hydrophobic groups.Much experimental evidence suggests that fibril formation

occurs via metastable, partially unfolded protein conformers (26,27). In the case of β2m, such species are obtained when Pro32,normally in a cis conformation, slowly isomerizes to trans (13, 33,35). Cis-to-trans isomerization of this residue exerts a destabilizingeffect on protein structure (13, 23), and ensuing conformationalchanges lead to exposure of hydrophobic residues and intermo-lecular aggregation via the D strand of the β-sandwich structure(6). The destabilized [F2Pro32]β2m variant possesses the char-acteristics of a partially unfolded conformer, leading to proteinaggregation over a wide pH range. However, above pH 4, theproducts of [F2Pro32]β2m aggregation are either soluble oligom-ers or insoluble amorphous aggregates. Well-ordered amyloidfibrils were not observed for [F2Pro32]β2m, even after incubatingthe samples at neutral pH for a period of several weeks. Longerincubation may be necessary to convert such amorphous aggre-gates into “crystalline” amyloid fibrils.At pH 2.5, all four synthetic proteins spontaneously formed

typical amyloid structures, albeit with unique morphologies andspectroscopic signatures. Amyloid polymorphism is well known:a single sequence, under different conditions, can generate arange of amyloid structures (36). For example, WT β2m formsamyloids of different morphology and stability from solvent mix-tures containing varying amounts of trifluoroethanol (37). Copre-cipitation of WT β2m and the truncated ΔN6 variant was alsoshown to produce a distinct polymorph (38). Here, substitution ofone or two hydrogens in Pro32 by fluorine elicits a similar effect.The polymorphic nature of the fibrils is best illustrated in Fig.

4B. The four β2m variants show distinct amyloid growth kinetics,which vary depending on what kind of “seeds”were used to initiategrowth. Amyloids of two proteins, WT β2m and [F2Pro32]β2m,served as efficient seeds for all four proteins, whereas seeds derivedfrom [4S-fpr32]β2m and [4R-Fpr32]β2m selectively initiated am-yloid growth of the cognate proteins only. Thus, the onset andkinetics of amyloid growth essentially depend on i) structural

Fig. 4. Different polymorphic forms of the amyloids formed by WT β2m andits variants. (A) Substitution of Pro32 by 4S-fpr, 4R-Fpr, or F2Pro led to for-mation of fibrils of different morphology as seen in the TEM images. Growthconditions: 50 μM protein in 50 mM citrate buffer, 100 mM NaCl, pH 2.5 for2 wk at 37 °C with shaking (250 rpm). Relative growth rates: WT β2m ∼ [4S-fpr32]β2m ∼ [4R-Fpr32]β2m > [F2Pro32]β2m. (Scale bar, 200 nm.) (B) Kineticsof fibril growth of WT β2m and the chemical analogs upon seeding withpreformed WT, [4S-fpr32]β2m, [4R-Fpr32]β2m, or [F2Pro32]β2m amyloidseeds monitored by the increase in ThT fluorescence at pH 2.5. Differences inmaximum ThT fluorescence levels for the different protein analogs arepresumably due to structurally distinctive fibril surfaces that bind ThT dye todifferent extents. Control absorption measurements (at 280 nm) of proteinremaining in solution confirmed that most of the sample (>90%) had pre-cipitated at the end of each experiment.

Fig. 5. Schematic representation of how cis-trans Pro32 isomerizationmight influence the mechanism of β2m amyloid assembly. The trans-Pro32conformer dictates self-assembly into a polymorph that places Trp60 in anenvironment where fluorescence emission is quenched, whereas coassocia-tion of cis-Pro32 and trans-Pro32 conformers leads to some of the Trp60shielded in the core of cross-β structure and hence stronger fluorescence.Subsequent Pro32 cis→trans isomerization leads to rearrangement of theβ-strands to give the distinctive cross-β structure of WT β2m amyloids.

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information encoded in amyloid seeds and ii) the competence ofa particular β2m analog to perpetuate this structural information.Although favoring aggregation, population of a trans-Pro32

amide conformation does not fully explain amyloid formation inWT β2m. The [4R-Fpr32]β2m variant, which favors trans-Pro32to a greater extent than native β2m, formed amyloids that arespectroscopically distinct fromWT fibrils (SI Appendix, Fig. S16).The structural and seeding properties of WT amyloids werebetter mimicked by fibrils grown from mixtures of this variantand [4S-fpr32]β2m, which should favor cis-Pro32 to a greaterextent than native β2m. These observations suggest that cis-Pro32 conformers may be important for productive assembly ofWT-like fibrils, presumably via transient hybrid oligomers (Fig.5). Subsequent isomerization of the prolyl bond during fibrilmaturation would then exclusively afford the trans isomer ob-served by solid-state NMR (14, 15). Furthermore, growth kineticsfor [F2Pro32]β2m amyloids shows that the activation energy forisomerization of amide32 also influences fibril assembly. The[F2Pro32]β2m protein, which has the most flexible His31-Xaa32peptide bond and forms oligomers and amorphous precipitatemore readily than the other β2m variants, showed the slowest ki-netics of amyloid growth, even with “homologous” seeds (Fig. 4B).

In conclusion, this study links conformational isomerization ofa single residue in the β2m sequence (Pro32) with global char-acteristics of the resultant amyloids such as aggregation andpropagation properties. Extending this type of conformationalanalysis to multiple protein sites can be expected to delineatesubtle molecular details and functional aspects of the poly-morphic states of these and many other amyloids. Infectiousprions in which conformational polymorphism gives rise tomultiple distinct heritable strains are a pertinent example (39).

Materials and MethodsDetails concerning the synthesis and characterization of the protein samplescan be found in SI Appendix, Materials and Methods and SI Appendix, Figs.S1–S6. Biophysical characterization of the proteins is described in SI Ap-pendix, Biophysical Characterization and SI Appendix, Figs. S7–S15. Proteinoligomerization studies are detailed in SI Appendix, Protein Aggregationand SI Appendix, Figs. S16–S20.

ACKNOWLEDGMENTS. We are grateful to Dr. Marc-Olivier Ebert for helpwith NMR measurements and Dr. Cindy Schulenburg for advice on proteinfolding. Electron microscopy measurements were performed at the ElectronMicroscopy Center of the ETH Zurich (EMEZ). This work was generouslysupported by the ETH Zurich and an ETH Fellowship to V.Y.T.

1. Adams EJ, Luoma AM (2013) The adaptable major histocompatibility complex (MHC)fold: Structure and function of nonclassical and MHC class I-like molecules. Annu RevImmunol 31:529–561.

2. Smith DP, Ashcroft AE, Radford SE (2010) Hemodialysis-related amyloidosis. ProteinMisfolding Diseases: Current and Emerging Principles and Therapies, eds Ramirez-Alvarado M, Kelly JW, Dobson CM (John Wiley and Sons, Hoboken, NJ).

3. Myers SL, et al. (2006) A systematic study of the effect of physiological factors on β2-microglobulin amyloid formation at neutral pH. Biochemistry 45(7):2311–2321.

4. Yamamoto S, et al. (2004) Glycosaminoglycans enhance the trifluoroethanol-inducedextension of β 2-microglobulin-related amyloid fibrils at a neutral pH. J Am SocNephrol 15(1):126–133.

5. Rennella E, et al. (2010) Folding and fibrillogenesis: Clues from β2-microglobulin.J Mol Biol 401(2):286–297.

6. Eakin CM, Berman AJ, Miranker AD (2006) A native to amyloidogenic transitionregulated by a backbone trigger. Nat Struct Mol Biol 13(3):202–208.

7. Calabrese MF, Eakin CM, Wang JM, Miranker AD (2008) A regulatable switch medi-ates self-association in an immunoglobulin fold. Nat Struct Mol Biol 15(9):965–971.

8. Yamamoto S, et al. (2004) Low concentrations of sodium dodecyl sulfate induce theextension of β 2-microglobulin-related amyloid fibrils at a neutral pH. Biochemistry43(34):11075–11082.

9. McParland VJ, et al. (2000) Partially unfolded states of β(2)-microglobulin and amyloidformation in vitro. Biochemistry 39(30):8735–8746.

10. Kihara M, et al. (2005) Seeding-dependent maturation of β2-microglobulin amyloidfibrils at neutral pH. J Biol Chem 280(12):12012–12018.

11. Eichner T, Radford SE (2011) Understanding the complex mechanisms of β2-micro-globulin amyloid assembly. FEBS J 278(20):3868–3883.

12. Trinh CH, Smith DP, Kalverda AP, Phillips SEV, Radford SE (2002) Crystal structure ofmonomeric human β-2-microglobulin reveals clues to its amyloidogenic properties.Proc Natl Acad Sci USA 99(15):9771–9776.

13. Jahn TR, Parker MJ, Homans SW, Radford SE (2006) Amyloid formation under phys-iological conditions proceeds via a native-like folding intermediate. Nat Struct MolBiol 13(3):195–201.

14. Barbet-Massin E, et al. (2010) Fibrillar vs crystalline full-length β-2-microglobulinstudied by high-resolution solid-state NMR spectroscopy. J Am Chem Soc 132(16):5556–5557.

15. Debelouchina GT, Platt GW, Bayro MJ, Radford SE, Griffin RG (2010) Magic anglespinning NMR analysis of β2-microglobulin amyloid fibrils in two distinct morpholo-gies. J Am Chem Soc 132(30):10414–10423.

16. Renner C el al. (2001) Fluoroprolines as tools for protein design and engineering.Angew Chem Int Ed 40(5):923–925.

17. Shoulders MD, Raines RT (2009) Collagen structure and stability. Annu Rev Biochem78:929–958.

18. Shoulders MD, Kamer KJ, Raines RT (2009) Origin of the stability conferred uponcollagen by fluorination. Bioorg Med Chem Lett 19(14):3859–3862.

19. Merkel L, Budisa N (2012) Organic fluorine as a polypeptide building element: In vivoexpression of fluorinated peptides, proteins and proteomes.Org Biomol Chem 10(36):7241–7261.

20. Bretscher LE, Jenkins CL, Taylor KM, DeRider ML, Raines RT (2001) Conformationalstability of collagen relies on a stereoelectronic effect. J Am Chem Soc 123(4):777–778.

21. Bang D, Kent SBH (2004) A one-pot total synthesis of crambin. Angew Chem Int EdEngl 43(19):2534–2538.

22. Schnölzer M, Alewood P, Jones A, Alewood D, Kent SBH (2007) In situ neutralizationin Boc-chemistry solid-phase peptide synthesis – rapid, high yield assembly of difficultsequences. Int J Pept Res Ther 13(1–2):31–44.

23. Eichner T, Radford SE (2009) A generic mechanism of β2-microglobulin amyloid as-sembly at neutral pH involving a specific proline switch. J Mol Biol 386(5):1312–1326.

24. Colon W, Kelly JW (1992) Partial denaturation of transthyretin is sufficient for amy-loid fibril formation in vitro. Biochemistry 31(36):8654–8660.

25. Pan KM, et al. (1993) Conversion of α-helices into β-sheets features in the formation ofthe scrapie prion proteins. Proc Natl Acad Sci USA 90(23):10962–10966.

26. Kelly JW (1996) Alternative conformations of amyloidogenic proteins govern theirbehavior. Curr Opin Struct Biol 6(1):11–17.

27. Uversky VN, Fink AL (2004) Conformational constraints for amyloid fibrillation: Theimportance of being unfolded. Biochim Biophys Acta 1698(2):131–153.

28. Chatani E, et al. (2010) Pre-steady-state kinetic analysis of the elongation of amyloidfibrils of β(2)-microglobulin with tryptophan mutagenesis. J Mol Biol 400(5):1057–1066.

29. Harper JD, Lansbury PT, Jr. (1997) Models of amyloid seeding in Alzheimer’s diseaseand scrapie: Mechanistic truths and physiological consequences of the time-dependentsolubility of amyloid proteins. Annu Rev Biochem 66:385–407.

30. Chien P, Weissman JS, DePace AH (2004) Emerging principles of conformation-basedprion inheritance. Annu Rev Biochem 73:617–656.

31. Petkova AT, et al. (2005) Self-propagating, molecular-level polymorphism in Alz-heimer’s β-amyloid fibrils. Science 307(5707):262–265.

32. Yanagi K, et al. (2012) The monomer-seed interaction mechanism in the formation ofthe β2-microglobulin amyloid fibril clarified by solution NMR techniques. J Mol Biol422(3):390–402.

33. Kameda A, et al. (2005) Nuclear magnetic resonance characterization of the refoldingintermediate of β2-microglobulin trapped by non-native prolyl peptide bond. J MolBiol 348(2):383–397.

34. Sakata M, et al. (2008) Kinetic coupling of folding and prolyl isomerization of β2-microglobulin studied by mutational analysis. J Mol Biol 382(5):1242–1255.

35. Chiti F, et al. (2001) A partially structured species of β 2-microglobulin is significantlypopulated under physiological conditions and involved in fibrillogenesis. J Biol Chem276(50):46714–46721.

36. Tycko R, Wickner RB (2013) Molecular structures of amyloid and prion fibrils: Con-sensus versus controversy. Acc Chem Res 46(7):1487–1496.

37. Chatani E, Yagi H, Naiki H, Goto Y (2012) Polymorphism of β2-microglobulin amyloidfibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol.J Biol Chem 287(27):22827–22837.

38. Sarell CJ, et al. (2013) Expanding the repertoire of amyloid polymorphs by co-poly-merization of related protein precursors. J Biol Chem 288(10):7327–7337.

39. Halfmann R, Lindquist S (2010) Epigenetics in the extreme: prions and the inheritanceof environmentally acquired traits. Science 330(6004):629–632.

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