Conformational Memory Effect Reverses Chirality of Vortex-InducedInsulin Amyloid SuperstructuresWojciech Dzwolak,*,† Weronika Surmacz-Chwedoruk,‡,§ and Viktoria Babenko†

†Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland‡Institute of Biotechnology and Antibiotics, Staroscinska 5, 02-516 Warsaw, Poland§Institute of High Pressure Physics, Polish Academy of Sciences, Sokolowska 29/37, 01-142 Warsaw, Poland

ABSTRACT: Formation of amyloid fibrils is often associatedwith intriguing far-from-equilibrium phenomena such asconformational memory effects or flow-driven self-assembly.Insulin is a model amyloidogenic polypeptide forming distinctstructural variants of fibrils, which self-propagate throughseeding. According to infrared absorption, fibrils from bovineinsulin ([BI]) and LysB31-ArgB32 human insulin analogue([KR]) cross-seed each other and imprint distinct structuralfeatures in daughter fibrils. In the absence of preformed [KR]amyloid seeds, bovine insulin agitated at 60 °C converts into chiral amyloid superstructures exhibiting negative extrinsic Cottoneffect in bound thioflavin T. However, when agitated bovine insulin is simultaneously cross-seeded with [KR] amyloid, daughterfibrils reveal a positive extrinsic Cotton effect. Our study indicates that dramatic changes in global properties of amyloidsuperstructures may emerge from subtle conformational-level variations in single fibrils (e.g., alignment and twist of β-strands)that are encoded by memory effects.

■ INTRODUCTION

Aggregation of misfolded protein molecules may lead toformation of linear β-sheet-rich aggregates (so-called amyloidfibrils). In vivo, amyloidogenesis is linked to severaldegenerative disorders such as Alzheimer’s disease or diabetesmellitus type II.1 Although amyloid fibrils from certain proteinsare biologically functional,2 and benign amyloid-like aggregatesobtained in vitro find applications in material science andnanotechnology,3,4 the clinical context remains relevant for themajority of studies in this field. Understanding the mechanismsof amyloidogenesis is crucial for finding successful therapies forrelated maladies, but this task is complicated by non-crystallizable character of fibrils, their polymorphism, andirreversibility of amyloidogenic self-assembly. The latter twoaspects are clear violations of the Anfinsen principle stating thatprotein folding is under thermodynamic control, and it isleading, for a given amino acid sequence, to a single native statecorresponding to the global free energy minimum.5,6 Whenmanifold of accessible aggregate structures is coupled to theirirreversible and autocatalytic growth, a conformational memoryeffect may emerge, which is observed upon seeding ofamyloidogenic proteins with preformed variants of fibrils.7−11

This effect, termed also “self-propagating polymorphism”,7

consists of copying structural features of the seed by daughterfibrils regardless of environmental biases favoring alternativeamyloid variants. Conformational memory effects are thoughtto underlie intriguing clinical problem of “prion strains”,12,13

but may also be observed in vitro upon aggregation ofnonpathogenic proteins and peptides such as insulin.8−10 Self-assembly of chiral amyloid superstructures induced by vortex

forces is a less common far-from-equilibrium phenomenonobserved upon misfolding and aggregation of insulin.14−17

Recently, much attention is being paid to the problems ofchirality of amyloid fibrils,18−21 and to superstructuresassembled upon influence of hydrodynamic forces.22−24 Wehave reported earlier that agitation-assisted aggregation ofinsulin results in the formation of ordered chiral superstructureswith very strong optical activity.14−17 On the other hand,without agitation and depending on insulin concentration andthe presence of cosolvents, insulin fibrils either do not mergeinto superstructures at all (and no enhancement of opticalactivity is detected through circular dichroism) or formdifferent mesoscopic architectures.25−27 Interestingly, whileaggregation of agitated bovine insulin at 60 °C results inamyloid superstructures with uniformly negative CD signals (inthe far-UV range, and, as induced circular dichroism, atapproximately 450 nm in ThT-stained fibrils28), so-called−ICD fibrils, lowering the temperature of aggregation to 35 °Cfavors superstructures with positive CD/ICD signals (+ICDfibrils). In the intermediate temperature range, the sign of ICDof bovine insulin chiral superstructures is stochasticallydetermined.15 In other words, a microscopic chiral fluctuationin aggregating insulin leads to conversion of a macroscopicamount of the hormone into superstructures with uniformlypositive or negative ICD characteristics. This describes thefundamental hallmark of the fascinating newly observed

Received: November 4, 2012Revised: December 10, 2012Published: December 12, 2012

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© 2012 American Chemical Society 365 dx.doi.org/10.1021/la304374q | Langmuir 2013, 29, 365−370

phenomenon in conformational transitions of aggregatingproteins: chiral bifurcation.14,15

This study reports an interesting behavior of aggregatinginsulin when two antagonistic amyloidogenic pathways, onecontrolled by a memory effect, another by hydrodynamic vortexforces, are coupled and forced to compete in recruiting nativeinsulin molecules. The results provide new insights into therelationship between conformation of single amyloid fibrils andchirality of their superstructural microarchitectures.

■ MATERIALS AND METHODSSamples. BI (insulin from bovine pancreas) was from Sigma-

Aldrich, U.S., and KR was manufactured by the Institute ofBiotechnology and Antibiotics (Warsaw, Poland) using recombinantDNA technology.29,30 D2O (“99.8 atom % D” grade) was fromARMAR Chemicals, Switzerland, and deuterium chloride (35 wt %DCl solution in D2O, 99 atom % D) was from Sigma-Aldrich, U.S.Mother amyloid fibrils were obtained through a quiescent

incubation of 1 wt % insulin solutions in 0.1 M NaCl in D2O, pD1.9 adjusted with diluted DCl (where pD is pH-meter readoutuncorrected for isotopic effects) at 60 °C for 48 h. Subsequently,insoluble aggregates of amyloid fibrils were subjected to FT-IR/AFM/ICD analysis, or, after sonication, used as seeds to induce daughteramyloid fibrils.15

For seeding experiments, sonicated mother fibrils were added tofreshly prepared 1 wt % insulin solutions in 0.1 M NaCl in D2O, pD1.9 at the native insulin:insulin fibrils mass ratio of 100:1 forexperiments carried out at 37 °C, or 20:1 for aggregation at 60 °C, oras specified in the Figure 4 caption. For the different temperature (37or 60 °C)/agitation (0 or 1400 rpm) regimes of aggregation, identical0.6 mL volumes of the fresh insulin solutions (with or without seeds)were incubated in an Eppendorf Thermomixer Comfort accessory, asdescribed in our previous works.15,16

FT-IR Spectroscopy. For FT-IR measurements, a CaF2 trans-mission cell equipped with a 0.05 mm Teflon spacer was used. All FT-IR spectra were collected on a Nicolet NEXUS FT-IR spectrometerequipped with a liquid nitrogen-cooled MCT detector. Typically, for asingle spectrum, 256 interferograms of 2 cm−1 resolution werecoadded. During measurements, the sample chamber was continuouslypurged with dry CO2-depleted air. All insulin spectra were correctedby subtracting the correct amount of buffer (D2O) and water vaporspectra prior to being baseline-corrected and then normalized by theintegrated intensity of the amide I′ band. Data processing (includingcalculations of self-deconvolved spectra: γ-factor set at 7.5/peak width15 cm−1, smoothing with Bessel function) was performed usingGRAMS software (ThermoNicolet, U.S.). Other details have beendescribed previously.31

ICD Spectroscopy. Insulin samples in acidified D2O-basedsolutions (as prepared for FT-IR measurements) were further diluted100 times with 0.1 M NaCl, pH 1.9 to the final fibril concentration of7.5 × 10−3 wt %, while the concentration of added ThT was 70 μM.All ICD spectra were collected at 25 °C and under quiescentconditions on a Jasco J-815 S spectropolarimeter using 10 mm quartzcuvettes. Other details were the same as in our previous works.15,16

AFM. Samples were diluted 60 times with deionized water. A smalldroplet (8 μL) of fibrils suspension was swiftly deposited onto freshlycleaved mica and left to dry overnight. AFM tapping-modemeasurements were carried out using a Nanoscope III atomic forcemicroscope from Veeco, U.S., and TAP300-Al sensors (res. frequency300 kHz) from BudgetSensors, Bulgaria. Other experimentalparameters were the same as in earlier studies.16

■ RESULTS AND DISCUSSION

At pH (pD) 1.9 and 60 °C, fibrillation of insulin proceedsaccording to a typical nucleation-and-growth scenario: after abrief lag period corresponding to the formation of aggregationnuclei, a rapid and autocatalytic elongation of fibrils follows.32

When insulin fibrillation takes place at 37 °C, the population ofaggregation-prone intermediate states is much smaller, and thespontaneous nucleation becomes less likely. This is responsiblefor concomitant dramatic extension of the lag phase withdecreasing temperature. However, even at 37 °C, aggregationmay be very rapid if preformed sonicated fibrils are added asseeds to native insulin solution. Because our objective was tostudy effects of cross-seeding on agitation-assisted insulinaggregation under conditions promoting fast self-assembly ofonly one type of chiral superstructure (−ICD), that is, at 60 °Cwith agitation at 1400 rpm, the problem of competitive de novoaggregation had to be addressed. Thus, for initial seedingexperiments at 60 °C, high (5 wt % of the total native insulin)concentrations of sonicated amyloid seeds were used (Materialsand Methods). Fourier self-deconvolved infrared spectra ofamyloid fibrils formed upon quiescent incubation of acidifiedD2O-based solutions of KR and BI at 60 °C are shown inFigure 1A. While the positions and bandshapes of the amide I′vibrational band are in either case indicative of parallel β-sheetstructure, there are pronounced spectral differences betweenthe two types of fibrils. Spectra of [KR] and [BI] amyloid are

Figure 1. A conformational memory effect upon cross-seeding of [KR]and [BI] amyloid fibrils captured in FT-IR spectra. (A) Deconvolvedamide I′ vibrational band of daughter bovine insulin fibrils induced at37 °C by [KR] template ([BI]KR − thin red line) resembles thespectrum of mother [KR] fibrils (thick black line), rather than thespectrum of spontaneously formed bovine insulin amyloid ([BI] −thick red line). (B) Positions of the amide I′ band of amyloid fibrilsfrom BI or KR formed under different aggregation conditions(according to original absorption spectra). The blue and green arrowshighlight absorption maxima of the mother [KR] and daughter[BI]VKR fibrils, respectively. In all seeding experiments, the seed-to-native-insulin weight ratio was 1:20 for aggregation at 60 °C, and1:100 when aggregation was induced at 37 °C.

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dissimilar in terms of the relative intensities of components at1620 and ca. 1632/1628 cm−1, which can be attributed todifferences in local twists of stacked β-sheets and strength ofinter-β-strand hydrogen bonding.33,34 These fingerprint fea-tures are characteristic for spontaneously formed “mother”[KR] and [BI] fibrils enabling a straightforward distinctionbetween them. When [KR] mother amyloid is used as acatalytic template for inducing fibrils in a native KR solution at37 °C (i.e., when competitive de novo nucleation andformation of mother fibrils is unlikely), the resulting daughter[KR]37KR fibrils reveal an amide I′ band very similar to that ofthe mother seed (Figure 1A).30 Interestingly, when the sametemplate is used for seeding bovine insulin, the mother seeds’infrared features are again passed to daughter [BI]37KR fibrils,even though a spontaneous fibrillation of BI leads to aggregates(mother [BI]) with different spectral characteristics (bottomspectrum in Figure 1A). Hence, [BI] fibrils may adopt at leasttwo different packing modes of aggregated β-sheets: one that isfavored upon spontaneous de novo aggregation of the dissolvedprotein at 60 °C with the amide I′ band centered at 1628 cm−1,the other one mimicking the packing mode of spontaneouslyformed [KR] amyloid, with the absorption maximum shifted toca. 1620 cm−1.30 Upon cross-seeding and formation of [BI]KRfibrils, the latter assembly mode corresponds to the kineticallypreferred structure reached through the shortcut of the faster,that is, seeded aggregation pathway.35 Figure 1B summarizesseveral cross-seeding experiments between KR and BI fibrils(and vice versa) carried out at 37 or 60 °C, and under quiescentconditions or agitation through vortexing at 1400 rpm. Mother[BI]/[KR] seeds used for all seeding experiments had beenprepared under the conditions of quiescent spontaneousaggregation at 60 °C. The memory effect becomes somehowweakened for KR seeded with [BI] fibrils at 60 °C, when the denovo aggregation starts to compete with fibril extension.36

Influence of agitation on the memory effect is more equivocal,as it can, on the one hand, promote de novo aggregation bytriggering insulin denaturation at turbulent air/water inter-face,32 and, on the other hand, favor fibril extension by breakingseeds into shorter pieces and multiplying the total number ofcatalytic ends. Moreover, the memory effect appears to beasymmetric; that is, the amide I′ band frequency of [KR] fibrilsis imprinted on daughter [BI]KR amyloid with a higher fidelitythan when [BI] fibrils pass their infrared features to [KR]BIaggregate. This nonequivalence is likely to stem from either (i)lower catalytic efficiency of [BI] fibrils as compared to [KR], or(ii) differences in conformational dynamics of aggregation-prone intermediate states of BI and KR. However, these issuesare of minor importance for this study.ICD spectra of ThT-stained mother insulin fibrils prepared at

60 °C either under quiescent conditions ([BI], [KR]) orvortexing at 1400 rpm ([BI]V, [KR]V) are presented in Figure2A. We have shown earlier that intensive agitation ofaggregating bovine insulin results in the formation of orderedsuperstructures of amyloid fibrils with powerful chiropticalproperties.16 ThT is an achiral molecular-rotor-type fluoro-phore, which binds to surfaces of amyloid fibrils.28,37 Dockingto fibril surface enforces a twisted and therefore chiralconformation of ThT, which is the base for the extrinsicCotton effect observed as induced circular dichroism in thewavelength range corresponding to an electronic transition inThT at around 450 nm. Thus, a nonzero sign of ICD of ThT-stained insulin aggregates is a consequence of the net chiral biasof ThT-binding surface moieties of insulin amyloid. It was

demonstrated in our previous works that bovine insulinamyloid superstructures formed at 60 °C give rise to uniformlynegative ICD signals and that agitation of aggregating insulinsamples is a necessary condition for self-assembly of thesesuperstructures.14−16 The ICD spectra in Figure 2A confirm theprevious observations but, more importantly, also reveal thatvortexed KR insulin forms aggregates with a flat ICD signal,suggesting thereby that the subtle differences in amino acidsequence between BI and KR (Table 1) cause not only differentpacking modes of aggregated β-sheets that affect infraredspectra (Figure 1A), but also predispose flow-induced fibrillarsuperstructures to a different chiral bias of surface ThT-bindingmoieties, as was captured through ICD. In the followingexperiments, agitation-assisted aggregation of BI or KR insulinwas coupled to cross-seeding with alternative mother fibrils

Figure 2. ICD spectra of different types of mother (A) and daughter(B) insulin amyloid fibrils obtained after staining with ThT. The signof the extrinsic Cotton effect reflects the net chiral bias of ThT-bindingsurface moieties, which are imprinted in twisted conformations of thedye.

Table 1. Amino Acid Sequences of the A and B Chains of BIand KR Insulinsa

type ofinsulin symbol sequence

bovineinsulin

BI A-chain: GIVEQCCASVCSLYQLENYCNB-chain:FVNQHLCGSHLVEALYLVCGERGFFYTPKA

LysB31-ArgB32

KR A-chain: GIVEQCCTSICSLYQLENYCN

humaninsulinanalogue

B-chain:FVNQHLCGSHLVEALYLVCGERGFFYTPKTKR

aAmino acid substitutions relative to human insulin are marked withbold letters. Underlined are insulin’s two amyloidogenic regions(according to Eisenberg et al.39,40).

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(Figure 2B). Mother seeds used for the cross-seeding were“ZERO-ICD” fibrils grown through spontaneous aggregation at60 °C and under quiescent conditions; that is, the seedsprovided effective templates for conformational memory effectwithout contaminating daughter aggregates with their ownresidual ICD signals. Moreover, seeding with vortex-inducedaggregates is problematic, as such entities are stronglyagglomerated and poorly dispersed in solution. This makesthem rather ineffective templates allowing side de novoaggregation instead (the problem cannot be circumventedthrough sonication of seeds, as such treatment destroys the+ICD/−ICD superstructures15). The most striking result ofthis study is the ICD spectrum of fibrils formed upon agitation-assisted cross-seeding of BI with ICD-silent [KR] fibrilsdisplayed in Figure 2B. The ICD spectrum of [BI]VKR fibrilsreverts its sign to +ICD, implying a dramatic change of chiralityof amyloid surface moieties. This is an unexpected resultshowing that the memory effect thought to template fibrilsecondary and tertiary structures can affect the chiropticalproperties determined on superstructural levels.14−16 Cross-seeding with [KR] is capable of overcoming the inherenttendency of BI to form −ICD fibrils at 60 °C, which was earliershown to hold under the conditions of homologous seedingwith ZERO ICD/−ICD/+ICD [BI] fibrils.15 On the otherhand, seeding of agitated KR with [BI] fibrils does not lead tothe formation of superstructures with strong chiropticalproperties. This implies that the covalent structure of BI (butnot of KR) must be equipped with elements necessary for theself-assembly of stable ICD superstructures. KR insulin has twoadditional positively charged (in the acidic environment inwhich peptide fibrillates) amino acid residues introduced to theC-terminus of the B-chain (Table 1). Strong repulsive forcesbetween positively charged [KR] fibrils could prevent a properalignment and lateral association needed for the formation ofchiral superstructures. In fact, the tendency to agglomerate ismore pronounced for [BI] than for [KR] fibrils, as ensuingAFM data suggest.Amplitude AFM images shown in Figure 3 were collected for

ICD-silent mother fibrils formed spontaneously underquiescent conditions, fibrils formed upon agitation of BI andKR, and daughter fibrils obtained through cross-seeding ofagitated insulin samples ([BI]VKR and [KR]VBI). Comparison ofmorphologies with the corresponding ICD signals reported inFigure 2 enables linking of the strong ICD signals tosuperstructural arrangements of individual fibrils into twistedbraids, as has been reported in our earlier studies.16 It has beenproposed that hierarchically ordered amyloid microarchitec-tures may favor coupling of transition moments of individualchromophores resulting in the powerful enhancement ofcircular dichroism.38 Figure 4A summarizes the followingexperiments focused on the dependence of decreasing ratio of[KR] seeds on the ICD signs of [BI]VKR amyloid. The dataindicate that the reversal of ICD sign takes place only at thehighest [KR] seeds concentration, 5 wt % of total protein. At60 °C and intensive agitation, the de novo aggregation of BIproceeds quickly enough even in the absence of templates,leading to the formation of [BI]V superstructures of the −ICDphenotype. Clearly, with less [KR] templates, the spontaneousaggregation outcompetes the seed-extension pathway con-trolled by the memory effect. In agreement with thisexplanation, the effect of the decreasing concentration of[KR] seeds on the amide I′ band’s position of daughter fibrils isgradually diminishing (Figure 4B).

In conclusion, we have shown that two far-from-equilibriumprocesses, an autocatalytic fibril extension (spreading “memo-rized” pattern of packing of aggregated β-sheets) and flow-driven self-assembly, can be induced to compete in thethermodynamically downhill process of conversion of nativeinsulin into amyloid fibrils. When the concentration of amyloidseeds is sufficient to hijack the aggregation pathway, theresulting aggregates have their superstructural chirality reversedin comparison to amyloid formed in the absence of seeds. Ourdata indicate that tiny secondary/tertiary structure perturba-tions in amyloid template may have powerful implications forchiroptical characteristics of daughter amyloid assemblies. As

Figure 3. AFM amplitude images of mother amyloid fibrils anddaughter fibrils formed through cross-seeding of vortexed insulinsolutions.

Figure 4. ICD intensities of ThT−amyloid complexes for fibrilsformed upon agitation-assisted aggregation of BI cross-seeded withgradually decreasing ratio of [KR] at 60 °C and 1400 rpm (A).Corresponding amide I′ band positions of daughter [BI]VKR fibrils (B).The relative (to the total polypeptide weight) concentrations of seedswere 5 (a), 1 (b), 0.5 (c), 0.2 (d), 0.1 (e), and 0 (f) wt %. ICD signalsand amide I′ band positions were calculated as the average of fourparallel experiments.

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properties of amyloid-like fibrils are proving advantageous fromthe nanotechnological perspective, the possibility of tuningthem through interplay of memory effects and hydrodynamicforces may open new synthetic routes to amyloid-basedadvanced materials.

■ AUTHOR INFORMATIONCorresponding Author*Phone: +48 22 8220211 ext. 528. Fax: +48 22 822 5996. E-mail: wdzwolak@chem.uw.edu.pl.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was supported by the Polish Ministry of Educationand Science (grant NN 301 101236 to W.D.). Preparation ofLysB31-ArgB32 analogue of human recombinant insulin wasfunded by POIG Key Research Project (contract no. POIG01.01.02-00-007/08-00), and insulin samples were kindlyprovided by Dr. Piotr Borowicz from the Institute ofBiotechnology and Antibiotics, Warsaw.

■ ABBREVIATIONSAFM, atomic force microscopy; BI, bovine insulin; FT-IR,Fourier transform infrared; ICD, induced circular dichroism;KR, recombinant LysB31-ArgB32 human insulin analogue; ThT,thioflavin T; [X], mother fibrils of protein X formedspontaneously at 60 °C and under quiescent conditions;[X]V, fibrils of protein X formed through vortexing at 1400 rpmat 60 °C; [X]37Y, daughter fibrils formed upon seeding proteinX with preformed fibrils of protein Y at 37 °C and underquiescent conditions; [X]VY, daughter fibrils formed uponseeding protein X with preformed fibrils of protein Y at 60 °Cand vortexing at 1400 rpm

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