CHIRALITY 26:580–587 (2014)
Review Article Chirality and Chiroptical Properties of Amyloid Fibrils WOJCIECH DZWOLAK* Biological and Chemical Research Centre, Department of Chemistry, University of Warsaw, Warsaw, Poland
ABSTRACT Chirality of amyloid fibrils-linear beta-sheet-rich aggregates of misfolded protein chains-often manifests in morphological traits such as helical twist visible in atomic force microscopy and in chiroptical properties accessible to vibrational circular dichroism (VCD). According to recent studies the relationship between molecular chirality of polypeptide building blocks and superstructural chirality of amyloid fibrils may be more intricate and less deterministic than previously assumed. Several puzzling experimental findings have put into question earlier intuitive ideas on: 1) the bottom-up chirality transfer upon amyloidogenic self-assembly, and 2) the structural origins of chiroptical properties of protein aggregates. For example, removal of a single amino acid residue from an amyloidogenic all-L peptide was shown to reverse handedness of fibrils. On the other hand, certain types of amyloid aggregates revealed surprisingly strong VCD spectra with the sign and shape dependent on the conditions of fibrillation. Hence, microscopic and chiroptical studies have highlighted chirality as one more aspect of polymorphism of amyloid fibrils. This brief review is intended to outline the current state of research on amyloid-like fibrils from the perspective of their structural and superstructural chirality and chiroptical properties. Chirality 26:580–587, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: fibril symmetry; handedness; superstructural chirality; beta-sheet twist; filament; vibrational circular dichroism INTRODUCTION
A common generic route of self-assembly of polypeptide chains leads to formation of so-called amyloid fibrils.1 Occurrence of amyloid deposits in vivo is recognized as a histopathological hallmark in dozens of degenerative maladies, such as diabetes type II, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases.2,3 On the other hand, there are many examples of biologically functional amyloid fibrils. These include nonpathogenic yeast prions formed by Ure2p protein which are involved in regulation of nitrogen catabolism4 and Pmel17 amyloid-based scaffold of melanosomal ultrastructures in animals.5 A variety of unrelated proteins and peptides can be induced to form amyloid-like fibrils in vitro. Globular proteins,6,7 short synthetic peptides,8 polyα-amino acids,1 as well as poly-ε-amino acids9 are found among precursors competent to form fibrils which—regardless of the chemical composition—share many structural and physicochemical traits such as the characteristic cross-beta X-ray diffraction pattern.10 Because their noncrystallizable character makes fibrils often inaccessible to high-resolution methods (with several exceptions11–13), early concepts of amyloid structure were based entirely on the cross-beta fiber diffraction signature, leading to the suggestion that stacked β-sheets are the main conformational component of fibrils— corroborated by circular dichroism and infrared spectroscopy.10 The two reflections routinely captured for well-oriented amyloid samples appear at 4.8 Å (strong) and 10–11 Å (diffuse and dependent on the type of amino acid residue in the case of homopolypeptides1), and correspond to the hydrogen bonding distance between strands within a β-sheet and intersheet spacing, respectively.14 Implicating β-sheet as the main secondary structural components of amyloid fibrils (with interstrand © 2014 Wiley Periodicals, Inc.
hydrogen bonds running parallel, and main polypeptide chain perpendicular to the fibril axis) was a starting point to develop more detailed models of fibrils. In particular, it provided a simple conceptual link between the molecular chirality of amino acid residues and the higher-order chirality of amyloid fibrils that have been observed in electron micrographs (TEM and SEM) and atomic force microscopy (AFM) images: the helical twist. The vertical chiral transfer typically observed between protein primary and secondary structures is highly deterministic: a polypeptide chain built of L-amino acid residues is expected to fold either into a right-handed α-helix or a β-sheet with a left-handed twist.15,16 It should be stressed that while the sense of twist of β-sheets found in globular proteins is always the same, historically, there are two conflicting schools of categorizing it as either left- (according to the angle at which neighboring strands cross each other), or right-handed (twist of the peptide plane viewed alongside β-strand), as explained by Richardson.16 Although this would not necessarily hold true for fibrils built of β-helices,17 stacking of multiple β-sheets into superstructures should conserve the left-handedness on tertiary and quaternary levels of the amyloid architecture.14 This concept has been elegantly depicted by Aggeli et al. who looked into amyloidogenesis of synthetic peptides.18 The hierarchical self-assembly of chiral monomer units led to formation of tapes, ribbons, and fibrils—all of which were left-handed Contract grant sponsor: National Science Centre of Poland; Contract grant number: DEC-2011/03/B/ST4/03063. *Correspondence to: Wojciech Dzwolak; Department of Chemistry University of Warsaw, Pasteur 1, 02-093 Warsaw, Poland. E-mail: [email protected] Received for publication 06 January 2014; Accepted 28 March 2014 DOI: 10.1002/chir.22335 Published online 10 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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along the longer axis (Fig. 1). Another aspect of the compatibility between a precursor’s molecular chirality and higher-order chirality of amyloid fibril was captured by Wadai et al.: for an effective autocatalytic seeding of fibrils from β2-microglobulin K3 fragment, chiral types of amino acid residues within the amyloid template and in the seeded monomer must match.19 This contrasts with our earlier findings pointing to preferential self-assembly of racemic fibrils in mixed solutions of poly(L-lysine) and poly(D-lysine) compared to pure enantiomers.20,21 In the case of β2-microglobulin K3 amyloid, an L/D mismatch prevents proper docking interactions between monomer and seed and therefore precludes elongation of the latter one, while for polylysine an alternative stacking of L-only and D-only chains is preferred due to reduced thermodynamic frustration of peptide-solvating water.20,21 However, this mechanism cannot be generalized for homopolypeptides, as an ensuing study on mixed aggregates of poly(L-glutamic) and poly(D-glutamic) acids proved.22 Namely, α-helical samples of pure enantiomers of polyglutamic acid convert swiftly to very stable β2-fibrils (additionally stabilized by bifurcated hydrogen bonds), whereas co-aggregation of poly (L-glutamic) and poly(D-glutamic) acids leads to formation of metastable β1-sheet-rich aggregates which subsequently undergo slow spontaneous transition to the β2-aggregate.22 These studies have contributed to establishing the deterministic concepts of superstructural “chiral transfer” and “chiral matching” as the leading paradigms in research on amyloid handedness. HANDEDNESS OF AMYLOID FIBRILS
Periodic helical twist along fibril’s axis (i.e., handedness) is the most tangible chiral aspect of amyloid morphology and as such is expected to be intimately linked to molecular chirality of its building blocks. In their AFM investigation of fibrils from Aβ1-40 peptide—the main component of senile plaques hallmarking Alzheimer’s disease—Harper et al. found that
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all-L Aβ1-40 fibrils are left-handed while the opposite is true for fibrils from synthetic all-D Aβ1-40 peptide (Fig. 2).23 Other works on amyloid fibrils from naturally occurring and therefore all-L proteins such as SMA (fragment of immunoglobulin light-chain),24 or human amylin25 showed further evidence of morphological left-handedness. In an extensive electron microscopic study on amyloidogenesis of human calcitonin, Bauer et al. probed handedness of amyloid polymorphs appearing at different stages of the process.26 The lefthandedness was observed in early aggregates, protofibrilribbons and fibrils, and remained conserved when those merged, through distinct pathways, into higher-order structures: fibril-ribbons and cable-like entities.26 Rather unsurprisingly, all these works led to the notion that helical twist in amyloid fibrils composed of all-L proteins (or peptides) is always left-handed.27 Meanwhile, experimental evidence suggesting a more intricate nature of the relationship between molecular chirality and handedness in amyloid fibrils began to emerge. While examining the effects of growth conditions on morphology of α-synuclein amyloid, Hoyer et al. realized that certain combinations of pH and monomer concentration promotes formation of heterogeneous aggregates in which fibrils with two different helical periodicities, 45 nm and 95 nm, are prominent.28 According to those authors, the two types of fibrils had opposite handedness (fibrils with 45 nm periodicity being right-handed). It should be stressed, however, that this conclusion was primary based on conventional transmission electron microscopy (TEM)—a method ill-suited to assess the chirality of fibrils. Namely, in TEM images, unlike in scanning electron microscopy (SEM) or AFM, fibril’s back- and front sides are superimposed and the two images cannot be unambiguously told apart afterwards—the key structural information necessary to define handedness is irreversibly lost. That amyloidogenic aggregation of an all-L peptide may, indeed, produce right-handed fibrils was proven only several
Fig. 1. Conservation of left-handedness upon chiral transfer in amyloidogenic self-assembly. Model of hierarchical self-assembly of chiral rod-like units. Local arrangements (c–f) and the corresponding global equilibrium conformations (c’–f’) for the hierarchical self-assembling structures formed in solutions of chiral molecules. Adapted with permission from Ref. 18. Copyright 2001 National Academy of Sciences of the United States of America. Chirality DOI 10.1002/chir
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Fig. 2. AFM images of left-handed amyloid fibrils grown from all-L Aβ1-40 peptide (a) and right-handed fibrils from synthetic all-D Aβ1-40 peptide (b). Scale bar = 50 nm. Adapted with permission from Ref. 23. Copyright 1997 Elsevier.
years later by Addadi and colleagues who employed SEM to show exclusively right-handed fibrils assembled from SAA1-12 peptide—an N-terminal fragment of serum amyloid A (SAA) protein.29 Sticking with the argument that a hierarchical organization of β-sheets would (for all-L proteins) always lead to left-handed twists, the authors suggested that β-helix—a fold more ambiguous in terms of superstructural chiral transfer—could be the actual secondary motif of SAA1-12 fibrils’ architecture. In their follow-up work, Addadi et al. compared the morphology of fibrils grown from several different peptides including SAA1-12—all of them obtained through different truncation scenarios of SAA protein.17 Interestingly, while the aforementioned SAA1-12 peptide (with the amino acid sequence: RSFFSFLGEAFD) produced uniformly righthanded fibrils, truncation of the single aspartic residue (D) from the C-terminus resulted in fibrils (formed by peptide now labeled as SAA1-11) adapting exclusively left-handed symmetry. Moreover, SAA2-9 octapeptide truncated at both termini formed heterogeneous mixtures of right-handed and left-handed (dominating) fibrils. These findings show that molecular all-L chirality of monomer does not warrant fibrils’ left-handedness, and the relationship between the two depends on very subtle effects controlled on the level of single amino acid substitutions (without L/D isomerization). Furthermore, the behavior of SAA2-9 peptide indicates that, for certain peptides, the superstructural chiral transfer can be under less strict deterministic control. This is likely to reflect coexistence of multiple competing aggregation pathways leading to fibrils with distinct handedness. The structural complexity of amyloid fibrils and scarcity of high-resolution data make the task of uncovering mechanisms of chirality transfer from the molecular to morphological levels a very difficult one. Recent works employing coarse-grained models are helpful in understanding different aspects of dynamics of chiral superstructural self-assembly. For example, Wales and colleagues have shown how lefthanded particles (dimers of Paramonov-Yaliraki ellipsoids) can self-assemble into right- and left-handed helices.30 Our own recent study pointed to low-barrier pathways to establishing dominant superstructural chirality in an aggregate of several chiral protofibrils.31 In spite of the insights provided by such models, the case of SAA1-12 / SAA1-11 peptides17 wherein truncation of a single amino acid residue changes the chiral outcome of the supermolecular self-assembly underscores the limitations of the coarse-grained approach. As this problem is becoming recognized, efforts are being made to elucidate links between chirality on different structural levels Chirality DOI 10.1002/chir
employing more realistic models. Using molecular dynamics, Ghattyvenkatakrishna et al. analyzed how internal organization of β-hairpins within an Aβ amyloid fibril affects its handedness.32 It was demonstrated that only negative staggers in the periodic alignment of β-hairpins would lead to the experimentally detected left-handedness of fibrils. A comprehensive model linking chirality on molecular and superstructural levels must address the polymorphism of amyloid fibrils, i.e., the existence of manifold of kinetically stable types of fibrils assembled from covalently (though not conformationally) identical monomers.33 From the energetic standpoint, fibril’s handedness is the outcome of competition between the intrinsic chiral bias of polypeptide chains and a mechanical strain suppressing twisting.18,34 Balance between these forces will depend not only on the amino acid sequence of the constituent peptide, but also on environmental factors such as pH and ionic strength that influence, for example, repulsive interactions within the fibril. This reasoning is best illustrated by the work of Adamcik and Mezzenga revealing that helical pitch of left-handed β-lactoglobulin fibrils grown in the absence of salts can be tuned by adding NaCl afterwards.35 In other words, the periodic twisting pitch reflects a balance between Coulombic interactions and torsional elastic energy of the fibril with NaCl affecting the former, and hence shifting the balance. Further AFM studies on aggregation of β-lactoglobulin36,37 and lysozyme37,38 by Mezzenga and collaborators have depicted later stages of amyloidogenesis as dominated by lateral association of filaments into left-handed multistranded ribbons (with the helical pitch increasing with the number of filaments involved) followed by bending of the ribbons into a helical superstructure which would ultimately close into a smooth nanotube concealing its helical sense. More recently, while exploring fibrillation of bovine serum albumin using AFM-based methodology combined with statistical analysis, the same group came across a fascinating reversal of the fibril’s handedness occurring at higher levels of structural organization.39 Formation of early flexible left-handed twisted ribbons was identified as a critical stage of the fibrillation process followed by splitting of the aggregation pathway into two: one in which a twisted ribbon transforms into helical ribbons and ultimately nanotubes (similar to the case of β-lactoglobulin and lysozyme), another in which two lefthanded ribbons merge into a right-handed ribbon subsequently transforming into a rigid right-handed helical ribbon (Fig. 3). The right-handed helical ribbon in Fig. 3B does not close into a nanotube, making its surface handedness
CHIRALITY OF AMYLOID FIBRILS
Fig. 3. Schematic representations of the two main pathways in fibrillation of bovine serum albumin. (A) The single-fibril pathway occurs when the l flexible left-handed twisted ribbons F transform into the rigid nanotube-like 0 structure R . (B) The double-fibril pathway occurs when two left-handed l twisted ribbons 2F wind together forming the rigid right-handed helical r ribbon R . Adapted with permission from Ref. 39 (Usov et al., ACS Nano 2013;7:10465-10474). Copyright 2013 American Chemical Society.
accessible to AFM microscopy. Arguably, should the elastic energy of such fibril allow formation of a continuous nanotube, its surface twist would be no longer detectable, as is the case of the left-handed-helical-ribbon-turned-nanotube shown in panel A of Fig. 3. The inversion of amyloid handedness taking place on higher levels of superstructural selfassembly only stresses the complexity of these systems and the urgent need to develop more adequate mechanistic models to particular cases of superstructural chiral transfer. CHIROPTICAL PROPERTIES OF AMYLOID FIBRILS
While Raman optical activity (ROA) was employed to study amyloidogenic intermediate states of some proteins,40,41 its use in research on amyloid fibrils has been much more limited (e.g.,42). Meanwhile, applications of circular dichroism (CD) spectroscopy, especially in the infrared range (i.e., vibrational circular dichroism, VCD), has led to several fascinating findings on fibrils and their chirality. The conventional far-UV CD is routinely used to probe secondary structure of globular proteins in solution. However, when used to examine amyloid samples, the technique often turns out to be vulnerable to artifacts arising from optical anisotropy and light scattering on insoluble fibrils. Linear birefringence and linear dichroism of oriented samples overlap much weaker benign CD signals, which results in distortion of CD spectra43 (also see44 for a brief review). Similarly, a CD spectrum will be distorted when light scattering on chiral particles (resulting in the phenomenon of circular differential scattering45) takes place. Therefore, particular care must be taken when CD spectra of amyloid fibrils are collected: it often calls for advanced chiroptical solid state CD spectrometers.43,46,47 Navigating through the plethora of studies using conventional far-UV CD to simply assess the secondary structure of amyloid fibrils is beyond the scope of this review. It is noteworthy, however, that analysis of CD data (along with infrared spectra) allowed Andersen et al. to show that pronounced differences in the relative β-sheet/β-turn content characterize two morphologically distinct types of glucagon fibrils.48 Twisting of β-sheets has been predicted to affect the far-UV CD signature of proteins (e.g., by causing spectral shift and increasing CD intensity),49,50 but so far these findings have been only marginally exploited in the field of protein fibrils.51
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Congo Red (CR) is a diazo dye with a high affinity to amyloid fibrils. The CR molecule is flat and therefore achiral, yet upon binding to fibrils—either obtained from diseased tissues52 or in-vitro-grown insulin and SMA amyloids53—an extrinsic Cotton effect in the stain (induced circular dichroism, ICD) was observed. Molecular mechanisms of such chiral transfer may involve twisting of CR conformation upon docking at amyloid surface moieties, arranging flat CR molecules into a helical staircase, or a combination of both. Thioflavin T (ThT) is another amyloid-specific organic stain with the characteristics of molecular-rotor-type fluorophore.54 Quantum yield of fluorescence of ThT increases dramatically when its intramolecular rotation is hindered—for example, in highly viscous media or through intercalation in amyloid’s stacked β-sheets. Because all rotamers of ThT are chiral (except those twisted at the right angle or perfectly planar), we sought to use ICD coupled to ThT to probe the surface chirality of amyloid fibrils.55 Namely, surface ThT-binding moieties of amyloid fibrils would imprint their local chirality in flexible molecules of the stain. Eventually, the net bias of amyloid surfaces, as probed by docking interactions with ThT, should manifest in nonzero-induced CD at the wavelength corresponding to the electronic transition in ThT. Hence, ICD of amyloid-bound ThT is a spectroscopic tool complementary to the microscopic means of studying amyloid fibril chirality. Certainly, as for the aforementioned case of CR, the observed spectral effects could correspond to twisting of single dye molecules, arranging them into helical staircases of coupled chromophores, or both. This approach allowed us to discover that agitation of acidified insulin solutions in the presence of NaCl triggers rapid formation of superstructures of insulin amyloid fibrils with strong chiroptical properties manifesting through ICD a sign of ThT-stained fibrils around 450 nm, as well as in far-UV CD spectra.56–58 Moreover, within a certain temperature range of fibrillation, the ICD sign of insulin amyloid superstructures cannot be predicted, i.e., a microscopic fluctuation results in a macroscopic excess of chiral aggregates with either positive (+ICD fibrils) or negative (–ICD fibrils) chiroptical properties (Fig. 4). This fascinating phenomenon bearing all the hallmarks of chiral bifurcation draws a few analogies to symmetry-breaking observed upon agitation-assisted formation of chiral crystals in achiral solutions.59,60 We have shown recently that formation of the + ICD/–ICD type of amyloid superstructures may be rather rare among amyloidogenic proteins.61 A covalent modification of insulin which decreases its capacity to form dimers through C-termini of B-chains prevents self-assembly of these superstructures.61 We have also shown that out of two component chains of insulin, A and B themselves amyloidogenic, only the latter one reveals a tendency to form chiral superstructures.62 While the current understanding of structural prerequisites for selfassembly of such amyloid superstructures remains limited, the picture emerging from these two articles accentuates the role of tight and orderly lateral alignment of fibrils, which is the case of insulin amyloid provided by the C-terminal part of B-chains acting as molecular velcros.61,62 More efforts must be made to explain the origins of the observed strong chiroptical properties of insulin amyloid superstructures. In our very recent study, +ICD and –ICD fibrils were used as catalytic molecular hosts for photocyclodimerization of 2-anthracenecarboxylate, a photochemical reaction transforming the achiral substrate into up to four different types Chirality DOI 10.1002/chir
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Fig. 4. Chiral bifurcation: stochastic formation of chiral variants of insulin fibrils of the + ICD and –ICD phenotypes reflected in sign of induced circular dichroism after staining with ThT. The multiple spectra correspond to 10 amyloid samples obtained through parallel agitation of insulin samples 48 h at 1400 rpm and at different temperatures, according to Ref. 57.
of covalent dimers, two of which are chiral.63 Approximately opposite enantiomeric excesses of one of the chiral products (anti-head-to-head dimer) were obtained when + ICD and –ICD insulin fibrils were used as hosts. This result constitutes strong evidence that the opposite supermolecular chiralities of these fibrils are represented at the molecular level. Certainly, more efforts must be made before a satisfactory mechanistic explanation of the chiroptical properties of +/–ICD insulin fibrils is obtained. Some of the most interesting results on chiroptical properties of amyloid fibrils have been obtained using VCD spectroscopy. While ICD of amyloid-bound stains reports on the surface chiral bias, VCD is a more adequate probe of the within aspects of chirality of fibrils—such as twisting of stacked β-sheets. Nafie and colleagues first reported unusually strong VCD spectra of lysozyme and insulin fibrils formed—unlike the previously discussed “+/–ICD fibrils”— under quiescent conditions.64 The amide I vibrational region revealed a characteristic signature consisting of five component peaks with the sign pattern “+ + – + +”: the middle negative peak being the most intensive one (placed at 1627 cm-1 for insulin fibrils; Fig. 5). Even more interestingly, a followup study from the same group showed that when insulin fibrillation takes place at lower pH the precipitating fibrils have a distinct VCD spectrum: less intensive, but most importantly with a quasi-reversed sign pattern.65 Hence, the two types of fibrils were termed ”normal“ (NF) and ”reversed“ (RF). The similarity of deep-UV resonance Raman spectra collected for both the types of aggregates indicated that the NF/RF-type polymorphism is established on the quaternary structural level—i.e., through association of individual protofilaments which are identical in terms of the secondary structure.65 Eventually, ”reverse“ fibrils turned Chirality DOI 10.1002/chir
Fig. 5. VCD (top) and corresponding infrared absorption spectra (bottom) of native lysozyme, centrifuged lysozyme supernatant, and centrifuged lysozyme fibril gel at pH 2. VCD spectrum of fibrils reveals the characteristic “+ + – + +” pattern. The supernatant and the gel were obtained after heating at 60°C for 2 days. Adapted with permission from Ref. 64 (Ma et al., J Am Chem Soc 2007;129:12364-12365). Copyright 2007 American Chemical Society.
out to be metastable—a readjustment of pH of grown RF triggers an irreversible RF/NF transition.66 Intensity differences apart, VCD spectra of RF and NF are nearly mirror reflections. Yet although NF are left-handed in AFM, RF are flat.66,67 The authors have put forward a hypothesis that aggregating insulin first forms protofilaments which are lefthanded (stable, formed at pH 2.4–3.2), or right-handed (unstable, formed at pH 1.1–2.1). The handedness of protofilaments may manifest only in VCD; however, it is inaccessible to AFM.67 It is only the subsequent hierarchical lateral association of left-handed protofilaments that would produce thicker left-handed proto-fibrils and fibrils recognizable in AFM. Importantly, due to chiral conflict, a righthanded protofilament could only merge into flat tape-like assemblies retaining the RF signature in VCD spectra.67 A very recent study from the same group has indicated that the correlation between AFM/SEM-accessible morphological patterns and VCD signatures may in fact be quite universal for amyloidogenic proteins.68 These studies on insulin amyloid64–68 not only proved that VCD spectroscopy may be a very insightful approach to protein fibrils, but they have also made a strong argument that at least certain types of polymorphism of fibrils are defined on quaternary structural levels. Finally, these works provide evidence that higher-order chirality of protein aggregates can escape the deterministic control of the molecular chirality of building blocks—in agreement with the thesis of our earlier studies based on the ICD of ThT-stained fibrils.56,57
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VCD has also been useful in distinguishing between parallel and anti-parallel β-sheets in fibrils of Aβ16-22 peptide,69 monitoring handedness of fibrils from peptidomimetic compounds,70 and detection of supramolecular assembly of polyglutamine peptides.71 The technique will become an even more powerful tool for amyloid research when the link between spectral features and the amyloid structure is better understood. This concerns the sign pattern but also the remarkable enhancement of VCD absolute intensity described not only for fibrils from insulin,64,65 lysozyme,64 polyglutamine,71 but also poly-L-glutamic acid,72,73 and short synthetic octapeptide (termed AKY8) whose fibrils yielded a particularly strong VCD signal.74 Measey and SchweitzerStenner, in their theoretical work based on an excitonic coupling model, proposed that intrasheet vibration coupling and helical twisting of stacked β-sheets are primarily responsible for the VCD enhancement.75 More recently, Density Functional Theory (DFT) has been used to model VCD spectra of amyloid fibrils.76,77 Welch et al. suggested that a mutual rotation of stacked β-sheets could be another structural feature conducive to magnified VCD signals.76
CONCLUSIONS
Recent years have witnessed several exciting and often entirely unexpected discoveries regarding chirality and chiroptical properties of amyloid fibrils. The microscopic studies have demonstrated that fibrils formed from all-L peptides could—depending on the primary structure and aggregation conditions—be left- or right-handed. Hierarchical assembly of amyloid fibrils does not have to preserve the left-handedness of protofilaments. These two morphological aspects of the “loosened” vertical chiral transfer in fibrils are supported by chiroptical evidence of amyloid polymorphs with quasi-opposite VCD/ICD spectra. The intrinsic structural complexity and polymorphism of fibrils, and the fact that the different research groups do not work on the same samples, leave many fundamental questions unanswered: 1. Does the chiroptical dimorphism of insulin fibrils contradict the structural models based on stacked βsheets, since β-sheets, unlike β-helices, are expected to conserve the left-handedness upon hierarchical selfassembly? 2. Could anomalously tilted (and therefore metastable) stacked β-sheets underlie the reversed VCD spectra detected for some amyloid species? 3. How does the reversal of fibril’s chirality to a microscopically detectable right-handedness caused by amino acid truncation or supermolecular intertwining affect the corresponding VCD/ICD spectra? 4. What are the origins of ICD signals of insulin fibrils and why agitation of aggregating, but not already aggregated, insulin samples favors so strongly the formation of the chiral superstructures? These are just few out of many questions facing scientists in this field. Addressing them will not only deepen current understanding of the biological role of amyloid chirality, but will also enable exploration of possible nanotechnological and optical applications of chiral amyloid fibrils.78,79
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25. Goldsbury CS, Cooper GJS, Goldie KN, Müller SA, Saafi EL, Gruijters WTM, Misur MP, Engel A, Aebi U, Kistler J. Polymorphic fibrillar assembly of human amylin. J Struct Biol 1997;119:17–27. 26. Bauer HH, Aebi U, Häner M, Hermann R, Müller M, Arvinte T, Merkle HP. Architecture and polymorphism of fibrillar supramolecular assemblies produced by in vitro aggregation of human calcitonin. J Struct Biol 1995;115:1–15. 27. Jiménez JL, Nettleton EJ, Bouchard M, Robinson CV, Dobson CM, Saibil HR. The protofilament structure of insulin amyloid fibrils. Proc Natl Acad Sci U S A 2002;99:9196–9201. 28. Hoyer W, Antony T, Cherny D, Heim G, Jovin TM, Subramaniam V. Dependence of α-synuclein aggregate morphology on solution conditions. J Mol Biol 2002;322:383–393. 29. Rubin N, Perugia E, Goldschmidt M, Fridkin M, Addadi L. Chirality of amyloid suprastructures. J Am Chem Soc 2008;130:4602–4603. 30. Olesen SW, Fejer SN, Chakrabarti D, Wales DJ. A left-handed building block self-assembles into right- and left-handed helices. RSC Adv 2013;3:12905–12908. 31. Gruziel M, Dzwolak W, Szymczak P. Chirality inversions in self-assembly of fibrillar superstructures: A computational study. Soft Matter 2013;9:8005–8013. 32. Ghattyvenkatakrishna PK, Uberbacher EC, Cheng X. Effect of the amyloid β hairpin’s structure on the handedness of helices formed by its aggregates. FEBS Lett 2013;587: 2649–2655. 33. Kodali R, Wetzel R. Polymorphism in the intermediates and products of amyloid assembly. Curr Opin Struct Biol 2007;17:48–57. 34. Volpatti LR, Vendruscolo M, Dobson CM, Knowles TPJ. A clear view of polymorphism, twist, and chirality in amyloid fibril formation. ACS Nano 2013;7:10443–10448. 35. Adamcik J, Mezzenga R. Adjustable twisting periodic pitch of amyloid fibrils. Soft Matter 2011;7:5437–5443. 36. Adamcik J, Jung JM, Flakowski J, De Los Rios P, Dietler G, Mezzenga R. Understanding amyloid aggregation by statistical analysis of atomic force microscopy images. Nat Nanotechnol 2010;5:423–428. 37. Lara C, Adamcik J, Jordens S, Mezzenga R. General self-assembly mechanism converting hydrolyzed globular proteins into giant multistranded amyloid ribbons. Biomacromolecules 2011;12:1868–1875. 38. Lara C, Handschin S, Mezzenga R. Towards lysozyme nanotube and 3D hybrid self-assembly. Nanoscale 2013;5:7197–7201. 39. Usov I, Adamcik J, Mezzenga R. Polymorphism complexity and handedness inversion in serum albumin amyloid fibrils. ACS Nano 2013;7:10465–10474. 40. Blanch EW, Morozova-Roche LA, Cochran DAE, Doig AJ, Hecht L, Barron LD. Is polyproline II helix the killer conformation? A Raman optical activity study of the amyloidogenic prefibrillar intermediate of human lysozyme. J Mol Biol 2000;301:553–563. 41. Blanch EW, Gill AC, Rhie AGO, Hope J, Hecht L, Nielsen K, Barron LD. Raman optical activity demonstrates poly(l-proline) II helix in the Nterminal region of the ovine prion protein: Implications for function and misfunction. J Mol Biol 2004;343:467–476. 42. Yamamoto S, Watarai H. Raman optical activity study on insulin amyloidand prefibril intermediate. Chirality 2012;24:97–103. 43. Kuroda R, Harada T, Shindo Y. A solid-state dedicated circular dichroism spectrophotometer: Development and application. Rev Sci Instrum 2001;72:3802–3810. 44. Kaminksy W, Jin LW, Powell S, Maezawa I, Claborn K, Branham C, Kahr B. Polarimetric imaging of amyloid. Micron 2006;37:324–338. 45. Bustamante C, Tinoco I, Maestre MF. Circular differential scattering can be an important part of the circular dichroism of macromolecules. Proc Natl Acad Sci U S A 1983;80:3568–3572. 46. Harada T, Kuroda R. CD measurements of β-amyloid (1-40) and (1-42) in the condensed phase. Biopolymers 2011;95:127–134. 47. Babenko V, Harada T, Yagi H, Goto Y, Kuroda R, Dzwolak W. Chiral superstructures of insulin amyloid fibrils. Chirality 2011;23:638-646. 48. Andersen CB, Hicks MR, Vetri V, Vandahl B, Rahbek-Nielsen H, Thøgersen H, Thøgersen IB, Enghild JJ, Serpell LC, Rischel C, Otzen DE. Glucagon fibril polymorphism reflects differences in protofilament backbone structure. J Mol Biol 2010;397:932–946. 49. Manning MC, Illangasekare M, Woody RW. Circular dichroism studies of distorted α-helices, twisted β-sheets, and β-turns. Biophys Chem 1988;31:77–86. Chirality DOI 10.1002/chir
50. Hamley IW, Nutt DR, Brown GD, Miravet JF, Escuder B, RodríguezLlansola F. Influence of the solvent on the self-assembly of a modified amyloid beta peptide fragment. II. NMR and computer simulation investigation. J Phys Chem B 2010;114:940–951. 51. Moyer TJ, Cui H, Stupp SI. Tuning nanostructure dimensions with supramolecular twisting. J Phys Chem B 2013;117:4604–4610. 52. Benditt EP, Eriksen N, Berglund C. Congo red dichroism with dispersed amyloid fibrils, an extrinsic Cotton effect. Proc Natl Acad Sci U S A 1970;66:1044–1051. 53. Khurana R, Uversky VN, Nielsen L, Fink AL. Is Congo red an amyloidspecific dye? J Biol Chem 2001;276:22715–22721. 54. Stsiapura VI, Maskevich AA, Kuzmitsky VA, Uversky VN, Kuznetsova IM, Turoverov KK. Thioflavin T as a molecular rotor: Fluorescent properties of thioflavin T in solvents with different viscosity. J Phys Chem B 2008;112:15893–15902. 55. Dzwolak W, Pecul M. Chiral bias of amyloid fibrils revealed by the twisted conformation of thioflavin T: an induced circular dichroism / DFT study. FEBS Lett 2005;579:6601–6603. 56. Dzwolak W, Loksztejn A, Galinska-Rakoczy A, Adachi R, Goto Y, Rupnicki L. Conformational indeterminism in protein misfolding: chiral amplification on amyloidogenic pathway of insulin. J Am Chem Soc 2007;129:7517–7522. 57. Loksztejn A, Dzwolak W. Chiral bifurcation in aggregating insulin: An induced circular dichroism study. J Mol Biol 2008;379:9–16. 58. Loksztejn A, Dzwolak W. Vortex-induced formation of insulin amyloid superstructures probed by time-lapse atomic force microscopy and circular dichroism spectroscopy. J Mol Biol 2010;395:643–655. 59. Kondepudi DK, Kaufman RJ, Singh N. Chiral symmetry breaking in sodium chlorate crystallization. Science 1990; 250:975–976. 60. Viedma C. Chiral symmetry breaking during crystallization: Complete chiral purity induced by nonlinear autocatalysis and recycling. Phys Rev Lett 2005;94:art.no. 065504. 61. Babenko V, Dzwolak W. Amino acid sequence determinants in selfassembly of insulin chiral amyloid superstructures: Role of C-terminus of B-chain in association of fibrils. FEBS Lett 2013;587:625–630. 62. Babenko V, Piejko M, Wójcik S, Mak P, Dzwolak W. Vortex-induced amyloid superstructures of insulin and its component A and B chains. Langmuir 2013;29:5271–5278. 63. Nishijima M, Tanaka H, Yang C, Fukuhara G, Mori T, Babenko V, Dzwolak W, Inoue Y. Supramolecular photochirogenesis with functional amyloid superstructures. Chem Commun 2013;49:8916–8918. 64. Ma S, Cao X, Mak M, Sadik A, Walkner C, Freedman TB, Lednev IK, Dukor RK, Nafie LA. Vibrational circular dichroism shows unusual sensitivity to protein fibril formation and development in solution. J Am Chem Soc 2007;129:12364–12365. 65. Kurouski D, Lombardi RA, Dukor RK, Lednev IK, Nafie LA. Direct observation and pH control of reversed supramolecular chirality in insulin fibrils by vibrational circular dichroism. Chem Commun 2010;46:7154–7156. 66. Kurouski D, Dukor RK, Lu X, Nafie LA, Lednev IK. Spontaneous interconversion of insulin fibril chirality. Chem Commun 2012;48:2837–2839. 67. Kurouski D, Dukor RK, Lu X, Nafie LA, Lednev IK. Normal and reversed supramolecular chirality of insulin fibrils probed by vibrational circular dichroism at the protofilament level of fibril structure. Biophys J 2012;103:522–531. 68. Kurouski D, Lu X, Popova L, Wan W, Shanmugasundaram M, Stubbs G, Dukor RK, Lednev IK, Nafie LA. Is supramolecular filament chirality the underlying cause of major morphology differences in amyloid fibrils? J Am Chem Soc 2014;136:2302–2312. 69. Shanmugam G, Polavarapu PL. Isotope-assisted vibrational circular dichroism investigations of amyloid β peptide fragment, Aβ(16-22). J Struct Biol 2011;176:212–219. 70. Nieto-Ortega B, Nebot VJ, Miravet JF, Escuder B, Navarrete JTL, Casado J, Ramírez FJ. Vibrational circular dichroism shows reversible helical handedness switching in peptidomimetic L-valine fibrils. J Phys Chem Lett 2012;3:2120–2124. 71. Kurouski D, Kar K, Wetzel R, Dukor RK, Lednev IK, Nafie LA. Levels of supramolecular chirality of polyglutamine aggregates revealed by vibrational circular dichroism. FEBS Lett 2013;587:1638–1643. 72. Fulara A, Lakhani A, Wójcik S, Nieznaska H, Keiderling TA, Dzwolak W. Spiral superstructures of amyloid-like fibrils of polyglutamic acid: An infrared absorption and vibrational circular dichroism study. J Phys Chem B 2011;115:11010–11016.
CHIRALITY OF AMYLOID FIBRILS 73. Chi H, Welch WRW, Kubelka J, Keiderling TA. Insight into the packing pattern of β2 fibrils: A model study of glutamic acid rich oligomers with 13 C isotopic edited vibrational spectroscopy. Biomacromolecules 2013;14:3880–3891. 74. Measey TJ, Smith KB, Decatur SM, Zhao L, Yang G, Schweitzer-Stenner R. Self-aggregation of a polyalanine octamer promoted by its C-terminal tyrosine and probed by a strongly enhanced vibrational circular dichroism signal. J Am Chem Soc 2009;131:18218–18219. 75. Measey TJ, Schweitzer-Stenner R. Vibrational circular dichroism as a probe of fibrillogenesis: The origin of the anomalous intensity enhancement of amyloid-like fibrils. J Am Chem Soc 2011;133:1066–1076.
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76. Welch WRW, Kubelka J, Keiderling TA. Infrared, vibrational circular dichroism, and Raman spectral simulations for β-sheet structures with various isotopic labels, interstrand, and stacking arrangements using density functional theory. J Phys Chem B 2013;117:10343–10358. 77. Welch WRW, Keiderling TA, Kubelka J. Structural analyses of 13 experimental C edited amide I’ IR and VCD for peptide β-sheet aggregates and fibrils using DFT-based spectral simulations. J Phys Chem B 2013;117:10359–10369. 78. Yang Z, Zhang P, Xie P, Wu L, Lu Z, Zhao M. Polarization properties in helical metamaterials. Front Optoelectron 2012;5:248–255. 79. Solin N, Inganäs O. Protein nanofibrils balance colours in organic whitelight-emitting diodes. Isr J Chem 2012;52:529–539.
Chirality DOI 10.1002/chir
Review Article Chirality and Chiroptical Properties of Amyloid Fibrils WOJCIECH DZWOLAK* Biological and Chemical Research Centre, Department of Chemistry, University of Warsaw, Warsaw, Poland
ABSTRACT Chirality of amyloid fibrils-linear beta-sheet-rich aggregates of misfolded protein chains-often manifests in morphological traits such as helical twist visible in atomic force microscopy and in chiroptical properties accessible to vibrational circular dichroism (VCD). According to recent studies the relationship between molecular chirality of polypeptide building blocks and superstructural chirality of amyloid fibrils may be more intricate and less deterministic than previously assumed. Several puzzling experimental findings have put into question earlier intuitive ideas on: 1) the bottom-up chirality transfer upon amyloidogenic self-assembly, and 2) the structural origins of chiroptical properties of protein aggregates. For example, removal of a single amino acid residue from an amyloidogenic all-L peptide was shown to reverse handedness of fibrils. On the other hand, certain types of amyloid aggregates revealed surprisingly strong VCD spectra with the sign and shape dependent on the conditions of fibrillation. Hence, microscopic and chiroptical studies have highlighted chirality as one more aspect of polymorphism of amyloid fibrils. This brief review is intended to outline the current state of research on amyloid-like fibrils from the perspective of their structural and superstructural chirality and chiroptical properties. Chirality 26:580–587, 2014. © 2014 Wiley Periodicals, Inc. KEY WORDS: fibril symmetry; handedness; superstructural chirality; beta-sheet twist; filament; vibrational circular dichroism INTRODUCTION
A common generic route of self-assembly of polypeptide chains leads to formation of so-called amyloid fibrils.1 Occurrence of amyloid deposits in vivo is recognized as a histopathological hallmark in dozens of degenerative maladies, such as diabetes type II, Alzheimer’s, Parkinson’s, and Creutzfeldt-Jakob diseases.2,3 On the other hand, there are many examples of biologically functional amyloid fibrils. These include nonpathogenic yeast prions formed by Ure2p protein which are involved in regulation of nitrogen catabolism4 and Pmel17 amyloid-based scaffold of melanosomal ultrastructures in animals.5 A variety of unrelated proteins and peptides can be induced to form amyloid-like fibrils in vitro. Globular proteins,6,7 short synthetic peptides,8 polyα-amino acids,1 as well as poly-ε-amino acids9 are found among precursors competent to form fibrils which—regardless of the chemical composition—share many structural and physicochemical traits such as the characteristic cross-beta X-ray diffraction pattern.10 Because their noncrystallizable character makes fibrils often inaccessible to high-resolution methods (with several exceptions11–13), early concepts of amyloid structure were based entirely on the cross-beta fiber diffraction signature, leading to the suggestion that stacked β-sheets are the main conformational component of fibrils— corroborated by circular dichroism and infrared spectroscopy.10 The two reflections routinely captured for well-oriented amyloid samples appear at 4.8 Å (strong) and 10–11 Å (diffuse and dependent on the type of amino acid residue in the case of homopolypeptides1), and correspond to the hydrogen bonding distance between strands within a β-sheet and intersheet spacing, respectively.14 Implicating β-sheet as the main secondary structural components of amyloid fibrils (with interstrand © 2014 Wiley Periodicals, Inc.
hydrogen bonds running parallel, and main polypeptide chain perpendicular to the fibril axis) was a starting point to develop more detailed models of fibrils. In particular, it provided a simple conceptual link between the molecular chirality of amino acid residues and the higher-order chirality of amyloid fibrils that have been observed in electron micrographs (TEM and SEM) and atomic force microscopy (AFM) images: the helical twist. The vertical chiral transfer typically observed between protein primary and secondary structures is highly deterministic: a polypeptide chain built of L-amino acid residues is expected to fold either into a right-handed α-helix or a β-sheet with a left-handed twist.15,16 It should be stressed that while the sense of twist of β-sheets found in globular proteins is always the same, historically, there are two conflicting schools of categorizing it as either left- (according to the angle at which neighboring strands cross each other), or right-handed (twist of the peptide plane viewed alongside β-strand), as explained by Richardson.16 Although this would not necessarily hold true for fibrils built of β-helices,17 stacking of multiple β-sheets into superstructures should conserve the left-handedness on tertiary and quaternary levels of the amyloid architecture.14 This concept has been elegantly depicted by Aggeli et al. who looked into amyloidogenesis of synthetic peptides.18 The hierarchical self-assembly of chiral monomer units led to formation of tapes, ribbons, and fibrils—all of which were left-handed Contract grant sponsor: National Science Centre of Poland; Contract grant number: DEC-2011/03/B/ST4/03063. *Correspondence to: Wojciech Dzwolak; Department of Chemistry University of Warsaw, Pasteur 1, 02-093 Warsaw, Poland. E-mail: [email protected] Received for publication 06 January 2014; Accepted 28 March 2014 DOI: 10.1002/chir.22335 Published online 10 May 2014 in Wiley Online Library (wileyonlinelibrary.com).
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along the longer axis (Fig. 1). Another aspect of the compatibility between a precursor’s molecular chirality and higher-order chirality of amyloid fibril was captured by Wadai et al.: for an effective autocatalytic seeding of fibrils from β2-microglobulin K3 fragment, chiral types of amino acid residues within the amyloid template and in the seeded monomer must match.19 This contrasts with our earlier findings pointing to preferential self-assembly of racemic fibrils in mixed solutions of poly(L-lysine) and poly(D-lysine) compared to pure enantiomers.20,21 In the case of β2-microglobulin K3 amyloid, an L/D mismatch prevents proper docking interactions between monomer and seed and therefore precludes elongation of the latter one, while for polylysine an alternative stacking of L-only and D-only chains is preferred due to reduced thermodynamic frustration of peptide-solvating water.20,21 However, this mechanism cannot be generalized for homopolypeptides, as an ensuing study on mixed aggregates of poly(L-glutamic) and poly(D-glutamic) acids proved.22 Namely, α-helical samples of pure enantiomers of polyglutamic acid convert swiftly to very stable β2-fibrils (additionally stabilized by bifurcated hydrogen bonds), whereas co-aggregation of poly (L-glutamic) and poly(D-glutamic) acids leads to formation of metastable β1-sheet-rich aggregates which subsequently undergo slow spontaneous transition to the β2-aggregate.22 These studies have contributed to establishing the deterministic concepts of superstructural “chiral transfer” and “chiral matching” as the leading paradigms in research on amyloid handedness. HANDEDNESS OF AMYLOID FIBRILS
Periodic helical twist along fibril’s axis (i.e., handedness) is the most tangible chiral aspect of amyloid morphology and as such is expected to be intimately linked to molecular chirality of its building blocks. In their AFM investigation of fibrils from Aβ1-40 peptide—the main component of senile plaques hallmarking Alzheimer’s disease—Harper et al. found that
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all-L Aβ1-40 fibrils are left-handed while the opposite is true for fibrils from synthetic all-D Aβ1-40 peptide (Fig. 2).23 Other works on amyloid fibrils from naturally occurring and therefore all-L proteins such as SMA (fragment of immunoglobulin light-chain),24 or human amylin25 showed further evidence of morphological left-handedness. In an extensive electron microscopic study on amyloidogenesis of human calcitonin, Bauer et al. probed handedness of amyloid polymorphs appearing at different stages of the process.26 The lefthandedness was observed in early aggregates, protofibrilribbons and fibrils, and remained conserved when those merged, through distinct pathways, into higher-order structures: fibril-ribbons and cable-like entities.26 Rather unsurprisingly, all these works led to the notion that helical twist in amyloid fibrils composed of all-L proteins (or peptides) is always left-handed.27 Meanwhile, experimental evidence suggesting a more intricate nature of the relationship between molecular chirality and handedness in amyloid fibrils began to emerge. While examining the effects of growth conditions on morphology of α-synuclein amyloid, Hoyer et al. realized that certain combinations of pH and monomer concentration promotes formation of heterogeneous aggregates in which fibrils with two different helical periodicities, 45 nm and 95 nm, are prominent.28 According to those authors, the two types of fibrils had opposite handedness (fibrils with 45 nm periodicity being right-handed). It should be stressed, however, that this conclusion was primary based on conventional transmission electron microscopy (TEM)—a method ill-suited to assess the chirality of fibrils. Namely, in TEM images, unlike in scanning electron microscopy (SEM) or AFM, fibril’s back- and front sides are superimposed and the two images cannot be unambiguously told apart afterwards—the key structural information necessary to define handedness is irreversibly lost. That amyloidogenic aggregation of an all-L peptide may, indeed, produce right-handed fibrils was proven only several
Fig. 1. Conservation of left-handedness upon chiral transfer in amyloidogenic self-assembly. Model of hierarchical self-assembly of chiral rod-like units. Local arrangements (c–f) and the corresponding global equilibrium conformations (c’–f’) for the hierarchical self-assembling structures formed in solutions of chiral molecules. Adapted with permission from Ref. 18. Copyright 2001 National Academy of Sciences of the United States of America. Chirality DOI 10.1002/chir
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Fig. 2. AFM images of left-handed amyloid fibrils grown from all-L Aβ1-40 peptide (a) and right-handed fibrils from synthetic all-D Aβ1-40 peptide (b). Scale bar = 50 nm. Adapted with permission from Ref. 23. Copyright 1997 Elsevier.
years later by Addadi and colleagues who employed SEM to show exclusively right-handed fibrils assembled from SAA1-12 peptide—an N-terminal fragment of serum amyloid A (SAA) protein.29 Sticking with the argument that a hierarchical organization of β-sheets would (for all-L proteins) always lead to left-handed twists, the authors suggested that β-helix—a fold more ambiguous in terms of superstructural chiral transfer—could be the actual secondary motif of SAA1-12 fibrils’ architecture. In their follow-up work, Addadi et al. compared the morphology of fibrils grown from several different peptides including SAA1-12—all of them obtained through different truncation scenarios of SAA protein.17 Interestingly, while the aforementioned SAA1-12 peptide (with the amino acid sequence: RSFFSFLGEAFD) produced uniformly righthanded fibrils, truncation of the single aspartic residue (D) from the C-terminus resulted in fibrils (formed by peptide now labeled as SAA1-11) adapting exclusively left-handed symmetry. Moreover, SAA2-9 octapeptide truncated at both termini formed heterogeneous mixtures of right-handed and left-handed (dominating) fibrils. These findings show that molecular all-L chirality of monomer does not warrant fibrils’ left-handedness, and the relationship between the two depends on very subtle effects controlled on the level of single amino acid substitutions (without L/D isomerization). Furthermore, the behavior of SAA2-9 peptide indicates that, for certain peptides, the superstructural chiral transfer can be under less strict deterministic control. This is likely to reflect coexistence of multiple competing aggregation pathways leading to fibrils with distinct handedness. The structural complexity of amyloid fibrils and scarcity of high-resolution data make the task of uncovering mechanisms of chirality transfer from the molecular to morphological levels a very difficult one. Recent works employing coarse-grained models are helpful in understanding different aspects of dynamics of chiral superstructural self-assembly. For example, Wales and colleagues have shown how lefthanded particles (dimers of Paramonov-Yaliraki ellipsoids) can self-assemble into right- and left-handed helices.30 Our own recent study pointed to low-barrier pathways to establishing dominant superstructural chirality in an aggregate of several chiral protofibrils.31 In spite of the insights provided by such models, the case of SAA1-12 / SAA1-11 peptides17 wherein truncation of a single amino acid residue changes the chiral outcome of the supermolecular self-assembly underscores the limitations of the coarse-grained approach. As this problem is becoming recognized, efforts are being made to elucidate links between chirality on different structural levels Chirality DOI 10.1002/chir
employing more realistic models. Using molecular dynamics, Ghattyvenkatakrishna et al. analyzed how internal organization of β-hairpins within an Aβ amyloid fibril affects its handedness.32 It was demonstrated that only negative staggers in the periodic alignment of β-hairpins would lead to the experimentally detected left-handedness of fibrils. A comprehensive model linking chirality on molecular and superstructural levels must address the polymorphism of amyloid fibrils, i.e., the existence of manifold of kinetically stable types of fibrils assembled from covalently (though not conformationally) identical monomers.33 From the energetic standpoint, fibril’s handedness is the outcome of competition between the intrinsic chiral bias of polypeptide chains and a mechanical strain suppressing twisting.18,34 Balance between these forces will depend not only on the amino acid sequence of the constituent peptide, but also on environmental factors such as pH and ionic strength that influence, for example, repulsive interactions within the fibril. This reasoning is best illustrated by the work of Adamcik and Mezzenga revealing that helical pitch of left-handed β-lactoglobulin fibrils grown in the absence of salts can be tuned by adding NaCl afterwards.35 In other words, the periodic twisting pitch reflects a balance between Coulombic interactions and torsional elastic energy of the fibril with NaCl affecting the former, and hence shifting the balance. Further AFM studies on aggregation of β-lactoglobulin36,37 and lysozyme37,38 by Mezzenga and collaborators have depicted later stages of amyloidogenesis as dominated by lateral association of filaments into left-handed multistranded ribbons (with the helical pitch increasing with the number of filaments involved) followed by bending of the ribbons into a helical superstructure which would ultimately close into a smooth nanotube concealing its helical sense. More recently, while exploring fibrillation of bovine serum albumin using AFM-based methodology combined with statistical analysis, the same group came across a fascinating reversal of the fibril’s handedness occurring at higher levels of structural organization.39 Formation of early flexible left-handed twisted ribbons was identified as a critical stage of the fibrillation process followed by splitting of the aggregation pathway into two: one in which a twisted ribbon transforms into helical ribbons and ultimately nanotubes (similar to the case of β-lactoglobulin and lysozyme), another in which two lefthanded ribbons merge into a right-handed ribbon subsequently transforming into a rigid right-handed helical ribbon (Fig. 3). The right-handed helical ribbon in Fig. 3B does not close into a nanotube, making its surface handedness
CHIRALITY OF AMYLOID FIBRILS
Fig. 3. Schematic representations of the two main pathways in fibrillation of bovine serum albumin. (A) The single-fibril pathway occurs when the l flexible left-handed twisted ribbons F transform into the rigid nanotube-like 0 structure R . (B) The double-fibril pathway occurs when two left-handed l twisted ribbons 2F wind together forming the rigid right-handed helical r ribbon R . Adapted with permission from Ref. 39 (Usov et al., ACS Nano 2013;7:10465-10474). Copyright 2013 American Chemical Society.
accessible to AFM microscopy. Arguably, should the elastic energy of such fibril allow formation of a continuous nanotube, its surface twist would be no longer detectable, as is the case of the left-handed-helical-ribbon-turned-nanotube shown in panel A of Fig. 3. The inversion of amyloid handedness taking place on higher levels of superstructural selfassembly only stresses the complexity of these systems and the urgent need to develop more adequate mechanistic models to particular cases of superstructural chiral transfer. CHIROPTICAL PROPERTIES OF AMYLOID FIBRILS
While Raman optical activity (ROA) was employed to study amyloidogenic intermediate states of some proteins,40,41 its use in research on amyloid fibrils has been much more limited (e.g.,42). Meanwhile, applications of circular dichroism (CD) spectroscopy, especially in the infrared range (i.e., vibrational circular dichroism, VCD), has led to several fascinating findings on fibrils and their chirality. The conventional far-UV CD is routinely used to probe secondary structure of globular proteins in solution. However, when used to examine amyloid samples, the technique often turns out to be vulnerable to artifacts arising from optical anisotropy and light scattering on insoluble fibrils. Linear birefringence and linear dichroism of oriented samples overlap much weaker benign CD signals, which results in distortion of CD spectra43 (also see44 for a brief review). Similarly, a CD spectrum will be distorted when light scattering on chiral particles (resulting in the phenomenon of circular differential scattering45) takes place. Therefore, particular care must be taken when CD spectra of amyloid fibrils are collected: it often calls for advanced chiroptical solid state CD spectrometers.43,46,47 Navigating through the plethora of studies using conventional far-UV CD to simply assess the secondary structure of amyloid fibrils is beyond the scope of this review. It is noteworthy, however, that analysis of CD data (along with infrared spectra) allowed Andersen et al. to show that pronounced differences in the relative β-sheet/β-turn content characterize two morphologically distinct types of glucagon fibrils.48 Twisting of β-sheets has been predicted to affect the far-UV CD signature of proteins (e.g., by causing spectral shift and increasing CD intensity),49,50 but so far these findings have been only marginally exploited in the field of protein fibrils.51
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Congo Red (CR) is a diazo dye with a high affinity to amyloid fibrils. The CR molecule is flat and therefore achiral, yet upon binding to fibrils—either obtained from diseased tissues52 or in-vitro-grown insulin and SMA amyloids53—an extrinsic Cotton effect in the stain (induced circular dichroism, ICD) was observed. Molecular mechanisms of such chiral transfer may involve twisting of CR conformation upon docking at amyloid surface moieties, arranging flat CR molecules into a helical staircase, or a combination of both. Thioflavin T (ThT) is another amyloid-specific organic stain with the characteristics of molecular-rotor-type fluorophore.54 Quantum yield of fluorescence of ThT increases dramatically when its intramolecular rotation is hindered—for example, in highly viscous media or through intercalation in amyloid’s stacked β-sheets. Because all rotamers of ThT are chiral (except those twisted at the right angle or perfectly planar), we sought to use ICD coupled to ThT to probe the surface chirality of amyloid fibrils.55 Namely, surface ThT-binding moieties of amyloid fibrils would imprint their local chirality in flexible molecules of the stain. Eventually, the net bias of amyloid surfaces, as probed by docking interactions with ThT, should manifest in nonzero-induced CD at the wavelength corresponding to the electronic transition in ThT. Hence, ICD of amyloid-bound ThT is a spectroscopic tool complementary to the microscopic means of studying amyloid fibril chirality. Certainly, as for the aforementioned case of CR, the observed spectral effects could correspond to twisting of single dye molecules, arranging them into helical staircases of coupled chromophores, or both. This approach allowed us to discover that agitation of acidified insulin solutions in the presence of NaCl triggers rapid formation of superstructures of insulin amyloid fibrils with strong chiroptical properties manifesting through ICD a sign of ThT-stained fibrils around 450 nm, as well as in far-UV CD spectra.56–58 Moreover, within a certain temperature range of fibrillation, the ICD sign of insulin amyloid superstructures cannot be predicted, i.e., a microscopic fluctuation results in a macroscopic excess of chiral aggregates with either positive (+ICD fibrils) or negative (–ICD fibrils) chiroptical properties (Fig. 4). This fascinating phenomenon bearing all the hallmarks of chiral bifurcation draws a few analogies to symmetry-breaking observed upon agitation-assisted formation of chiral crystals in achiral solutions.59,60 We have shown recently that formation of the + ICD/–ICD type of amyloid superstructures may be rather rare among amyloidogenic proteins.61 A covalent modification of insulin which decreases its capacity to form dimers through C-termini of B-chains prevents self-assembly of these superstructures.61 We have also shown that out of two component chains of insulin, A and B themselves amyloidogenic, only the latter one reveals a tendency to form chiral superstructures.62 While the current understanding of structural prerequisites for selfassembly of such amyloid superstructures remains limited, the picture emerging from these two articles accentuates the role of tight and orderly lateral alignment of fibrils, which is the case of insulin amyloid provided by the C-terminal part of B-chains acting as molecular velcros.61,62 More efforts must be made to explain the origins of the observed strong chiroptical properties of insulin amyloid superstructures. In our very recent study, +ICD and –ICD fibrils were used as catalytic molecular hosts for photocyclodimerization of 2-anthracenecarboxylate, a photochemical reaction transforming the achiral substrate into up to four different types Chirality DOI 10.1002/chir
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Fig. 4. Chiral bifurcation: stochastic formation of chiral variants of insulin fibrils of the + ICD and –ICD phenotypes reflected in sign of induced circular dichroism after staining with ThT. The multiple spectra correspond to 10 amyloid samples obtained through parallel agitation of insulin samples 48 h at 1400 rpm and at different temperatures, according to Ref. 57.
of covalent dimers, two of which are chiral.63 Approximately opposite enantiomeric excesses of one of the chiral products (anti-head-to-head dimer) were obtained when + ICD and –ICD insulin fibrils were used as hosts. This result constitutes strong evidence that the opposite supermolecular chiralities of these fibrils are represented at the molecular level. Certainly, more efforts must be made before a satisfactory mechanistic explanation of the chiroptical properties of +/–ICD insulin fibrils is obtained. Some of the most interesting results on chiroptical properties of amyloid fibrils have been obtained using VCD spectroscopy. While ICD of amyloid-bound stains reports on the surface chiral bias, VCD is a more adequate probe of the within aspects of chirality of fibrils—such as twisting of stacked β-sheets. Nafie and colleagues first reported unusually strong VCD spectra of lysozyme and insulin fibrils formed—unlike the previously discussed “+/–ICD fibrils”— under quiescent conditions.64 The amide I vibrational region revealed a characteristic signature consisting of five component peaks with the sign pattern “+ + – + +”: the middle negative peak being the most intensive one (placed at 1627 cm-1 for insulin fibrils; Fig. 5). Even more interestingly, a followup study from the same group showed that when insulin fibrillation takes place at lower pH the precipitating fibrils have a distinct VCD spectrum: less intensive, but most importantly with a quasi-reversed sign pattern.65 Hence, the two types of fibrils were termed ”normal“ (NF) and ”reversed“ (RF). The similarity of deep-UV resonance Raman spectra collected for both the types of aggregates indicated that the NF/RF-type polymorphism is established on the quaternary structural level—i.e., through association of individual protofilaments which are identical in terms of the secondary structure.65 Eventually, ”reverse“ fibrils turned Chirality DOI 10.1002/chir
Fig. 5. VCD (top) and corresponding infrared absorption spectra (bottom) of native lysozyme, centrifuged lysozyme supernatant, and centrifuged lysozyme fibril gel at pH 2. VCD spectrum of fibrils reveals the characteristic “+ + – + +” pattern. The supernatant and the gel were obtained after heating at 60°C for 2 days. Adapted with permission from Ref. 64 (Ma et al., J Am Chem Soc 2007;129:12364-12365). Copyright 2007 American Chemical Society.
out to be metastable—a readjustment of pH of grown RF triggers an irreversible RF/NF transition.66 Intensity differences apart, VCD spectra of RF and NF are nearly mirror reflections. Yet although NF are left-handed in AFM, RF are flat.66,67 The authors have put forward a hypothesis that aggregating insulin first forms protofilaments which are lefthanded (stable, formed at pH 2.4–3.2), or right-handed (unstable, formed at pH 1.1–2.1). The handedness of protofilaments may manifest only in VCD; however, it is inaccessible to AFM.67 It is only the subsequent hierarchical lateral association of left-handed protofilaments that would produce thicker left-handed proto-fibrils and fibrils recognizable in AFM. Importantly, due to chiral conflict, a righthanded protofilament could only merge into flat tape-like assemblies retaining the RF signature in VCD spectra.67 A very recent study from the same group has indicated that the correlation between AFM/SEM-accessible morphological patterns and VCD signatures may in fact be quite universal for amyloidogenic proteins.68 These studies on insulin amyloid64–68 not only proved that VCD spectroscopy may be a very insightful approach to protein fibrils, but they have also made a strong argument that at least certain types of polymorphism of fibrils are defined on quaternary structural levels. Finally, these works provide evidence that higher-order chirality of protein aggregates can escape the deterministic control of the molecular chirality of building blocks—in agreement with the thesis of our earlier studies based on the ICD of ThT-stained fibrils.56,57
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VCD has also been useful in distinguishing between parallel and anti-parallel β-sheets in fibrils of Aβ16-22 peptide,69 monitoring handedness of fibrils from peptidomimetic compounds,70 and detection of supramolecular assembly of polyglutamine peptides.71 The technique will become an even more powerful tool for amyloid research when the link between spectral features and the amyloid structure is better understood. This concerns the sign pattern but also the remarkable enhancement of VCD absolute intensity described not only for fibrils from insulin,64,65 lysozyme,64 polyglutamine,71 but also poly-L-glutamic acid,72,73 and short synthetic octapeptide (termed AKY8) whose fibrils yielded a particularly strong VCD signal.74 Measey and SchweitzerStenner, in their theoretical work based on an excitonic coupling model, proposed that intrasheet vibration coupling and helical twisting of stacked β-sheets are primarily responsible for the VCD enhancement.75 More recently, Density Functional Theory (DFT) has been used to model VCD spectra of amyloid fibrils.76,77 Welch et al. suggested that a mutual rotation of stacked β-sheets could be another structural feature conducive to magnified VCD signals.76
CONCLUSIONS
Recent years have witnessed several exciting and often entirely unexpected discoveries regarding chirality and chiroptical properties of amyloid fibrils. The microscopic studies have demonstrated that fibrils formed from all-L peptides could—depending on the primary structure and aggregation conditions—be left- or right-handed. Hierarchical assembly of amyloid fibrils does not have to preserve the left-handedness of protofilaments. These two morphological aspects of the “loosened” vertical chiral transfer in fibrils are supported by chiroptical evidence of amyloid polymorphs with quasi-opposite VCD/ICD spectra. The intrinsic structural complexity and polymorphism of fibrils, and the fact that the different research groups do not work on the same samples, leave many fundamental questions unanswered: 1. Does the chiroptical dimorphism of insulin fibrils contradict the structural models based on stacked βsheets, since β-sheets, unlike β-helices, are expected to conserve the left-handedness upon hierarchical selfassembly? 2. Could anomalously tilted (and therefore metastable) stacked β-sheets underlie the reversed VCD spectra detected for some amyloid species? 3. How does the reversal of fibril’s chirality to a microscopically detectable right-handedness caused by amino acid truncation or supermolecular intertwining affect the corresponding VCD/ICD spectra? 4. What are the origins of ICD signals of insulin fibrils and why agitation of aggregating, but not already aggregated, insulin samples favors so strongly the formation of the chiral superstructures? These are just few out of many questions facing scientists in this field. Addressing them will not only deepen current understanding of the biological role of amyloid chirality, but will also enable exploration of possible nanotechnological and optical applications of chiral amyloid fibrils.78,79
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