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1 Natural Protective Amyloids Current Protein and Peptide Science, 2008, 9, Vassiliki A. Iconomidou and Stavros J. Hamodrakas* Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens , Greece Abstract: Amyloidoses are a group of diseases including neurodegenerative diseases like Alzheimer s disease and also type II diabetes, spongiform encephalopathies and many others, believed to be caused by protein aggregation and subsequent amyloid fibril formation. However, occasionally, living organisms exploit amyloid fibril formation, a property inherent into amino acid sequences, and perform specific physiological functions from amyloids, in differing biological contexts. Some of these functional amyloids are natural protective amyloids. Here, we review recent evidence on silkmoth chorion protein synthetic peptide-analogues that documents the function of silkmoth chorion, the major component of the eggshell, a structure with extraordinary physiological and mechanical properties, as a natural protective amyloid. Also, we briefly discuss the reported function of other natural, protective amyloids like fish chorion, the protein Pmel17 which forms amyloid fibrils that act as templates and accelerate the covalent polymerization of reactive small molecules into melanin, the hydrophobins and the antifreeze protein from winter flounder. Molecular self-assembly is becoming an increasingly popular route to new supramolecular structures and molecular materials and the inspiration for such structures is commonly derived from self-assembling systems in biology. Therefore, a careful examination of these studies may set the basis for the exploration of new routes for the formation of novel biocompatible polymeric structures with exceptional physico-chemical properties, for potentially new biomedical and industrial applications. Keywords: Amyloids, natural protective amyloids, silkmoth chorion protein peptide analogues, amyloid fibrillogenesis, liquid crystals, -pleated sheet, fish chorion, Pmel17, hydrophobins, antifreeze protein. INTRODUCTION Proteins or peptides convert under certain conditions from their soluble forms into ordered fibrillar aggregates, called amyloid fibrils. Protein aggregation and occasionally ensued amyloid fibril formation are believed to be the cause of an intriguing group of neurodegenerative diseases including Alzheimer s disease, prion diseases, Parkinson s and Huntington s and, also, type II diabetes and many others, that are referred to as amyloidoses [1,2]. However, in addition, occasionally, living organisms take advantage of the inherent ability of proteins and peptides, to form such structures under certain conditions and generate novel and diverse biological functions [2-4 and references therein], which were noted following our proposal for the existence of natural protective amyloids [5]. There is no apparent similarity between amyloidogenic proteins or peptides in aminoacid sequence, molecular weight, morphology or surrounding conditions. Furthermore, many proteins not implicated in conformational disease have also been shown to form amyloids in vitro, leading to the hypothesis that the potential for amyloidogenesis may be a near universal feature of proteins [6]. However, recent evidence indicates that there is a sequence propensity for amyloid formation [7,8 and references therein], sometimes inherent in sequence after millions of years of molecular evolution, as we have shown [5,9]. *Address correspondence to this author at the Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Panepistimiopolis, Athens , Greece; Tel: ; Fax: ; veconom@biol.uoa.gr, shamodr@biol.uoa.gr The major component of the eggshell (90-95%) of many insect and fish eggs is chorion. Proteins account for more than 95% of its dry mass. This proteinaceous shell forms the outer layer of the eggshell and has extraordinary mechanical and physiological properties, protecting the oocyte and the developing embryo from a series of environmental hazards such as temperature variations, mechanical pressure, proteases, bacteria, viruses etc [10]. It also allows for sperm entry and fertilization and for the exchange of the respiratory gases [10]. Fig. (1a) shows an electron micrograph of a thin transverse section of a silkmoth chorion. A lamellar ultrastructure of packed fibrils is seen: silkmoth chorion is a biological analogue of a cholesteric liquid crystal [11,12]. The X-ray diffraction pattern of a silkmoth chorion shown in Fig. (1b) indicates that -sheet is the dominant secondary structure of its constituent proteins. ATR FT-IR (Fig. 1c) and laser- Raman spectroscopy [12 and references therein] suggest that the -sheets are antiparallel. About 200 proteins have been detected in the silkmoth chorion [17]. These proteins have been classified into two major classes, A and B [10]. The gene families encoding these proteins are related and constitute a superfamily with two branches, the -branch and the -branch [18]. Sequence analyses and secondary structure prediction revealed that chorion proteins consist of three domains [19]. The central domain is conserved in each of the two classes. The flanking N- and C-terminal domains are more variable and contain characteristic tandem repeats (ref. 19; see also Fig. 2). A and B central domains show distant similarities suggesting that the chorion genes constitute a superfamily derived from a single ancestral gene [18] /08 $ Bentham Science Publishers Ltd.

2 292 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Fig. (1). (a) Transmission electron micrograph of an oblique section through the helicoidal proteinaceous chorion of the silkmoth Antheraea polyphemus. Arrays of protein fibrils (ca. 100 Å, F, arrowheads) are seen in each lamella (L). Arrows point towards the outer (O) and inner (I), closest to the oocyte, surfaces of chorion. Bar 0.4 m. Reproduced from [5] with permission. (b) X-ray diffraction pattern from an almost flat fragment of a silkmoth Antheraea polyphemus chorion. The flat fragment of chorion was irradiated with the X-ray beam parallel to the outer and inner surfaces of chorion (Fig. 1a corresponds to a cut face of chorion that would be encountered by the beam). The presence of reflections corresponding to periodicities of 4.6 and 9.1 Å suggests abundance of -sheet in chorion proteins. The elliptical scattering at ca. 30 Å -1 indicates a helicoidal architecture for silkmoth chorion and most probably arises from ca. 30 Å protofilaments, constituents of the ca. 100 Å fibrils [12]. Reproduced from [5] with permission. (c) ATR FT-IR spectra taken from the outer (red) and inner (blue) surfaces of an Antheraea polyphemus silkmoth chorion. 2 nd derivatives are included. The amide I, II and III bands at 1626, 1514 and 1230 cm -1, respectively, clearly indicate a -sheet type of structure for silkmoth chorion proteins. The shoulder in the amide I region, at 1694 cm -1, suggests that the -sheets are antiparallel [25, 27-30]. The study of the properties of chorion proteins has long been hampered by the fact that it has proven very difficult to purify individual chorion proteins in large enough amounts of sufficient purity for structural studies. We therefore synthesized several chorion protein peptide-analogues and studied their structural and assembly properties under various conditions. These studies, reviewed in detail in this work, suggest that silkmoth chorion is a natural protective amyloid. The function of other natural, presumably protective amyloids is also briefly discussed. AMYLOID CHARACTERISTICS OF SILKMOTH CHORION PEPTIDES A. Family Peptide-Analogues ca Peptide Initially, a 51-residue peptide was synthesized [20], that can be considered as a generic central domain of the A class of silkmoth chorion proteins (Figs. 2a, 2b). This peptide, referred to below as ca peptide, is representative for about 20-30% of all the proteinaceous material in the eggshell. We chose this peptide because the central domains of the A class chorion proteins are highly conserved in both sequence and length and because this conservation indicates that this domain plays an important functional role in the formation of chorion structure [5]. The ca peptide forms, structurally uniform, amyloid-like fibrils by self-assembly in an astonishing variety of various solvents, ph values, ionic strengths and temperatures (V.A.I and S.J.H., In preparation). The fibrils were judged to be amyloid-like from their tinctorial and structural characteristics: they bind Congo-red showing the characteristic for amyloids red-green birefrigence when seen under crossed polars [21 and refs. therein] (Figs. 3a, 3b) as well as Thioflavin-T (data not shown). Electron micrographs (Figs. 4a, 4b) show that they are straight, unbranched double helices of indeterminate length and uniform in diameter (~90 Å). Each double-helical fibril consists of two protofilaments wound around each other. The protofilaments both have a uniform diameter of approximately Å. The pitch of the double helix (Fig. 4a, arrows) is approximately 920 Å.

3 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No Fig. (2). (a) Alignment of several chorion proteins of the A class [12 and references therein]. The names of the proteins are to the left of each sequence. The designations Bm and Ap refer to B. mori and A. polyphemus respectively. By convention 'pc' numbers refer to sequences derived from cdna clones, whereas the initials ERA and AL refer to 'early' A and 'late' A proteins correspondingly. Numbers to the right of each sequence are the total number of residues of each protein. The borders of the central conservative domain are marked at the top (by arrows). The numbering at the top is arbitrary. Black-boxed residues are identical and gray-boxed residues represent conservative substitutions. The ca peptide sequence is shown at the bottom. Asterisks at the bottom mark invariant Gly (G) residues. Reproduced from [5] with permission. (b) A schematic representation of the tripartite structure of silkmoth chorion proteins of the A family. A highly conservative central domain of invariant length, and two more variable flanking arms constitute each protein. Characteristic, tandemly repeating peptides are present both in the central domain and in the arms [12 and references therein]. The aminoacid sequence and relative position of the synthetic ca peptide (one letter code), designed to be an analogue of the entire central domain of the A family, is shown. Invariant glycines (G) repeating every six-residues are boxed and marked with an asterisk below the sequence. Reproduced from [26] with permission. Fig. (3). (a) Photomicrographs of ca peptide fibrils stained with Congo red: (a) Bright field illumination, (b) crossed polars. The red-green birefringence characteristic for amyloid fibrils is clearly seen. Bar 200 m. Reproduced from [26] with permission. Suspensions of these fibrils form oriented fibres, which give characteristic cross- X-ray diffraction patterns (Fig. 4c) [22]. In these oriented fibres, the long axes of the amyloid-like fibrils seen in the electron micrographs of Figs. (3a) and (3b) are oriented more or less parallel to the fibre axis. The oriented X-ray pattern (Fig. 4c) taken from these fibres indicates the presence of oriented -sheets in the amyloid-like fibrils of peptide ca. The presence of reflections corresponding to periodicities of 4.66 and Å indicates the existence of -sheets [23]. The strong meridional reflec-

4 294 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas tion at 4.66 Å suggests that the -sheets are oriented so that their -strands are perpendicular to the fibre axis and thus also to the long axis of the amyloid-like fibrils. The strong equatorial reflection at Å, which corresponds to the inter-sheet distance, suggests that the packing of the -sheets is parallel to the fibre axis and preferentially oriented. This X-ray pattern closely resembles typical cross- patterns [22] taken from amyloid fibres [21 and refs. therein]. A list of the meridional and equatorial reflections observed in the X-ray pattern (Fig. 4c) is given in Table 1. Reflections along the meridian are orders of the 115 Å spacing (116±1 Å) of the -helix of the amyloid protofilament model proposed by Blake & Serpell [24], which supports this model. Reflections along the equator are orders of the Å inter-sheet distance indicating a long range order of packed -sheets in the fibre. The ATR FT-IR ( cm -1 ) spectrum of ca peptide amyloid fibrils, cast on a Au mirror (Fig. 4d), clearly suggests that the -sheets present in the structure of the ca peptide amyloid fibrils (evident from the existence of the amide I and III bands at 1628 and 1234 cm -1, respectively) are antiparallel (shoulder at 1692 cm -1 in the amide I band) [25,26]. ca_m1 and ca_m2 Peptides Further, we have designed and synthesized, two peptides, mutants of the ca peptide. The first, called hereinafter Fig. (4). (a) Electron micrograph of amyloid-like fibrils derived by self-assembly, from a 9 mg.ml -1 solution of the ca peptide in a sodium acetate 50mM buffer, ph 5. Fibrils are negatively stained with 1% uranyl acetate. They are of indeterminate length (several microns), unbranched, approximately 90 Å in diameter and have a double helical structure. The pitch of the double helix is ~ 920 Å (marked with arrows). A pair of protofilaments each Å in diameter are wound around each other, forming the double-helical fibrils. Bar 800 Å. (b) Electron micrograph of amyloid-like fibrils derived from a solution of the ca peptide (conditions as in Fig. 4a). Fibrils are rotary shadowed with Pt/Pd at an angle of 7 degrees under high vacuum. Bar 1000 Å. (c) X-ray diffraction pattern from an oriented fibre of ca peptide amyloid-like fibrils. The meridian, M (direction parallel to the fibre axis) is horizontal and the equator, E, is vertical in this display. The X-ray diffraction pattern is a typical cross- pattern showing a 4.66 Å reflection on the meridian and a Å reflection on the equator. This indicates a regular structural repeat of 4.66 Å along the fibre axis (meridian) and a structural spacing of Å perpendicular to the fibre axis. The structural repeat of 4.66 Å along the fibre axis corresponds to the spacing of adjacent -strands (which should be perpendicular to the fibre axis, a "cross- " structure) and the Å spacing perpendicular to the fibre axis corresponds to the face-to-face separation (packing distance) of the -sheets. (d) ATR FT-IR ( cm -1 ) spectrum of ca peptide amyloid fibrils, cast on a Au mirror. 2 nd derivative spectra are included. Errorbar equals in the IR spectrum. Reproduced from [5] with permission.

5 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No Table 1. Spacings of the Meridional and Equatorial Reflections, Observed in the X-Ray Diffraction Pattern taken from an Oriented Fibre of ca Peptide Amyloid-like Fibrils (Fig. 4c). Indexing was done Assuming a Repeat Period of 116±1 Å for the Meridional Reflections. The Equatorial Reflections are Orders of the Å Inter-Sheet Packing Distance Meridian Equator d(obs) Å Index d(obs) Å Index ca_m1, is approximately half the size of the ca peptide (Fig. 5), whereas the second, called ca_m2, is a variant of the first, having three hydrophobic residues (two valines, V, and an alanine, A) replaced by glutamates (E) at specific positions (Fig. 5). The logic behind this synthesis was the following: We were interested to find out (a) whether amyloid-like fibril formation is sustained by fractions of the ca peptide, which contains tandemly repeating hexapeptides, in other words to determine whether amyloid fibril formation ability is inherent into the hexapeptide repeating structure of the central domain of chorion proteins, and, (b) whether specific mutations in the model -strands of ca, at locations where former residues exist, would influence the structure and assembly of the formed super-structures [9]. ca_m1 peptide (Fig. 5) folds and self-assembles, forming mature amyloid-like fibrils (Fig. 6) after 1-2 weeks incubation, also in a variety of solvents and conditions as the ca peptide. In contrast, solutions of ca_m2 (Fig. 5), under the same conditions and after incubation for several months, do not form amyloid-like fibrils and only very rarely show some tubular-like structures (data not shown). The fibrils formed by the ca_m1 peptide are very similar in structure and properties to the fibrils formed by the ca peptide [5, 9]. They are of indeterminate length, they have a thickness of ca. 100 Å and they appear to be double helical in structure (Fig. 6). They also bind Congo red showing the characteristic redgreen/yellow birefringence when seen under crossed polars (Fig. 7), and they give characteristic cross- -like X-ray diffraction patterns (Fig. 8) from oriented fibers. Therefore, they display all the features that characterize amyloid fibrils as well. The X-ray diffraction patterns of oriented fibers from ca_m1 exhibit several reflections (Fig. 8). Most of these reflections appear as rings due to rather poor alignment of the constituent fibrils. The strong reflections corresponding to periodicities of 4.67 Å and Å may be attributed to Fig. (5). Another schematic representation of the tripartite structure of silkmoth chorion proteins of the A family. A highly conservative central domain of invariant length, and two more variable flanking arms constitute each protein. Characteristic, tandemly repeating peptides are present both in the central domain and in the arms [12 and references therein]. The aminoacid sequence and relative position of the synthetic ca peptide (one letter code), designed to be an analogue of the entire central domain of the A family, is shown and also the sequences of the designed synthetic mutant peptides ca_m1 and ca_m2. ca_m1, 24-residues in length, has approximately half the size of the ca peptide (51 residues). Peptide ca_m2 differs from ca_m1 at three positions, where glutamates (E) have replaced two valine (V) and one alanine (A) residues. Conserved residues are shaded. Invariant glycines (G) repeating every six-residues are marked with an asterisk below the sequence. Reproduced from [9] with permission.

6 296 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Fig. (6). Electron micrograph of amyloid-like fibrils derived by self-assembly, from a 10 mg.ml -1 solution of the ca_m1 peptide in a sodium acetate 50mM buffer, ph 5. Fibrils were negatively stained with 1% uranyl acetate. They are of indeterminate length (several microns), unbranched, approximately 90 Å in diameter and have a double helical structure. A pair of protofilaments each Å in diameter are frequently wound around each other, forming the double-helical fibrils. Bar 0.2 m. Reproduced from [9] with permission. Fig. (7). Photomicrographs of ca_m1 peptide fibrils stained with Congo red: (a) Bright field illumination, (b) crossed polars. The red-green/yellow birefringence characteristic for amyloid fibrils is clearly seen. Bar 100 m. Reproduced from [9] with permission. Fig. (8). X-ray diffraction pattern from an oriented fibre of ca_m1 peptide amyloid-like fibrils. The meridian, M (direction parallel to the fibre axis) is horizontal and the equator, E, is vertical in this display. The X-ray diffraction pattern resembles a cross- pattern showing a 4.67 Å reflection as a ring and and 11.5 Å reflections on the equator. This indicates a regular structural repeat of 4.67 Å and structural spacings of and 11.5 Å perpendicular to the fibre axis. These two equatorial reflections usually merge into one, in other diffraction patterns from oriented fibres of ca_m1 peptide amyloid-like fibrils. The structural repeat of 4.67 Å corresponds to the spacing of adjacent -strands and the Å spacing parallel to the fibre axis corresponds to the face-to-face separation (packing distance) of the -sheets. Possible origin and measured spacings of the other reflections have been discussed previously [5]. Reproduced from [9] with permission. the inter-strand and inter-sheet distances of -sheet arrangements, respectively. These reflections are characteristic of the "cross- " conformation [22], and are observed for several amyloid-like fibrils [5, 21 and references therein], in which the -strands (if oriented) are perpendicular to the fibre axis and the sheets are packed parallel to the fibre axis. This conformation would produce oriented patterns from oriented samples with a meridional reflection at ca. 4.7 Å and the corresponding equatorial reflection at ca. 10 Å, i.e. very similar to the patterns obtained from fibres of the chorion ca peptide [5]. As seen in Fig. (8), only the Å reflection of the oriented fibers produced from the amyloid fibrils of the ca_m1 peptide shows preferred orientation. Similarly, preferred orientation is observed for the very sharp 5.22 Å and 3.46 Å reflections, which are the second (10.45/2) and third (10.45/3) orders of the Å reflection. This probably indicates that the packed -sheets exhibit long range order in a direction perpendicular to the fibre axis, that is the sheets are packed parallel to the fibre axis. The fact that the 4.67 Å reflection is not preferentially meridional (perpendicular to the fibre axis) but instead is a ring, indicates a more random orientation of the packed -sheets (and concomitantly of the -strands) with respect to the fibre axis. It is perhaps interesting to note that diffraction patterns obtained from not very well oriented fibres of the ca peptide

7 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No give almost identical X-ray diffraction patterns to the patterns obtained from fibres of the ca_m1 peptide, which implies almost identical secondary structures for both ca and ca_m1 peptides (data not shown). Attempts to obtain oriented fibres from solutions of the ca_m2 peptide were not successful since this peptide did not form amyloid fibrils or suitable viscous solutions for fibre formation. Furthermore, solutions of this peptide do not bind Congo red and do not exhibit the characteristic for amyloids red-green/yellow birefringence. The ATR FT-IR spectra of the ca_m1 and ca_m2 peptides are compared in Figs. (9a-c) and Table 2. The spectrum from ca_m1 peptide amyloid fibrils (Fig. 9a and Table 2) shows one prominent band at 1628 cm -1 in the amide I region and an amide III component at 1228 cm -1, which are definitely assigned to -sheet [25, 27-30]. The low frequency of these amide I and III components results from the strong hydrogen bonds in the -sheets, whereas the very narrow width of the amide I band at 1628 cm -1 (ca. 20 cm -1 ) suggests that the distribution of the phi and psi angles in the sheets is narrow and implies a very uniform structure. Thus, ATR FT- IR supports the presence of uniform -sheets in the structure of ca_m1 peptide fibrils, in agreement with the existence of a -sheet structure suggested by X-ray diffraction. It is interesting to note that, the ATR FT-IR spectrum of the ca_m1 peptide obtained immediately after dissolving the peptide in D 2 O (Fig. 9b and Table 2), before any formation of amyloid fibrils, exhibits components characteristic of sheet structure as well: an amide I band at 1633 cm -1 and an amide II band at 1437 cm -1 [31]. Formation of mature amyloid fibrils occurs after a period of approximately 4-5 days, as we have shown previously rather conclusively for solutions of the ca peptide [26] and for solutions of the ca_m1 peptide (our unpublished data) as well. Therefore, in this case, it appears that -sheet structure dictates formation of amyloid fibrils in a rather decisive way, since -sheet structure is the structure that the peptide ca_m1 adopts, directly after solution. On the contrary, thin films cast from ca_m2 peptide solutions on front-coated gold mirrors produce ATR FT-IR spectra characteristic of an unordered conformation of the peptide in these solutions (Fig. 9c). The infrared band at 1653 cm -1 in the amide I region and the absence of any features in the amide III region are indicative of unordered structure [25, 27-30]. A shoulder at ca cm -1 may indicate a small fraction of -sheet structure. In conclusion, the synthesis and study of the structural properties of peptides ca_m1 and ca_m2, provided convincing evidence that a peptide with a length half of that of the central domain of the A family of silkmoth chorion proteins (namely ca_m1) folds and self-assembles into amyloid fibrils, which are very similar in properties to those of the ca peptide (which corresponds to the entire length of the A family central domain). This probably implies that the underlying molecular substructure that dictates proper folding and self assembly of chorion fibrils into the superstructure of silkmoth chorion is encoded into the tandem hexapeptide repeats present in the aminoacid sequences of the central Fig. (9). ATR FT-IR ( cm -1 ) spectrum of : (A) ca_m1 peptide amyloid fibrils, cast as a thin film on a Au mirror, (B) a freshly made solution of ca_m1 peptide in D 2 O (10 mg.ml -1 ) cast as a thin film on a diamond ATR element, and (C) a ca_m2 peptide solution, cast as a thin film on a Au mirror. Errorbar equals in the IR spectra. Reproduced from [9] with permission. domain of silkmoth chorion proteins. Apparently, it is in strong support of our proposal that silkmoth chorion is a natural protective amyloid [5, 26]. Furthermore, we have demonstrated that carefully designed mutations on the sequence of these amyloidogenic peptides (ca_m2) can inhibit self-assembly and amyloid formation. Of course, it remains to be seen which is the shortest possible peptide from the sequences of chorion proteins that folds and self assembles forming fibrils similar to those appearing in vivo in the structure of silkmoth chorion.

8 298 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Table 2. Main ATR FT-IR ( cm -1 ) Peak Maxima of: (a) ca_m1 Peptide Amyloid Fibrils Cast as a thin Film on a Au Mirror, (b) Soluble ca_m1 Peptide in D2O Cast as a thin Film on a Diamond ATR Element and (c) ca_m2 Peptide Solution in Water Cast as a thin Film on a Au Mirror (Fig. 9). Tentative Assignments are Included. For details See Text ca_ml fibrils ca_ml in D 2O ca_m2 solution in H 2O Peak (cm -1 ) Assignment Peak (cm -1 ) Assignment Peak (cm -1 ) Assignment 1228 Amide III ( -sheet) 1279 Amide III ( -turns?) B. Family Peptide-Analogues B Peptide We have also synthesized an 18-residue peptide (B peptide) representative of a part of the central conservative domain of the B family silkmoth chorion proteins (Fig. 10). The B peptide when dissolved in distilled water (ph=5.5) at a concentration of 10 mg ml -1 was found to produce gels [32]. Twisted and non-twisted ribbons are constituents of the gels (Fig. 11). Each ribbon consists of thin protofilaments (fibrils) that have the tendency to coalesce laterally among each other (Fig. 11). The protofilaments have a uniform diameter of approximately Å. They associate sideways to form twisted and non-twisted ribbons of variable width. Similar twisted ribbons have been observed in amyloidfibrils formed by fusion peptides [33], an SH3 domain [34], various Alzheimer's disease peptides [35] and a peptide from the adenovirus fibre shaft [36], to mention a few examples. "Beading" is evident along the protofilaments (fibrils), indicating that they probably have a helical structure [37]. The 1437 CH 2-deformation 1543 Amide II 1437 Amide II ( -sheet) 1541 Amide II 1628 Amide I ( -sheet) 1633 Amide I ( -sheet) 1630 (sh) Amide I ( -sheet?) 1672 Amide I ( -turns?) (TFA?) 1653 Amide I (unordered structure) "beads" have dimensions of the order of ca. 30 Å. Congo red stained B peptide gels showed the red-green birefringence characteristic of amyloid fibrils when viewed under crossed polars (Fig. 12). Suspensions of the fibril-containing gels seen in Fig. (11), form fibres. X-ray diffraction patterns (Fig. 13) taken from these fibres showed four major reflections at 4.64, 5.20, 9.14 and Å. These reflections appear as rings due to the poor alignment of the constituent fibrils. Several attempts were made to obtain oriented specimens, but they were not successful. This is probably due to random packing of the ribbons in the gels, which adopt all possible orientations (see Fig. 11) as confirmed by electron microscopy (data not shown). The strong and relatively sharp reflection corresponding at a periodicity of 4.64 Å and the weaker one at 9.14 Å may be attributed to -sheet structure present in the fibrils (interstrand and inter-sheet distances respectively). These reflections are characteristic of the "cross- " conformation [22] Fig. (10). A schematic representation of the tripartite structure of silkmoth chorion proteins of the B family. A highly conservative central domain of invariant length, and two more variable flanking arms constitute each protein. Characteristic, tandemly repeating peptides are present both in the central domain and in the arms [12 and references therein]. The aminoacid sequence and relative position of the synthetic B peptide (one letter code), designed to be an analogue of a part of the central domain of the B family, is shown. Invariant glycines repeating every six-residues are marked with an asterisk below the sequence. Reproduced from [32] with permission.

9 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No Fig. (11). Electron micrograph of amyloid-like fibrils derived by self-assembly from a 10 mg/ml solution of the B peptide in distilled water, ph 5.5. Fibrils are negatively stained with 1% uranyl acetate. The protofilaments (fibrils) are Å in diameter and they selfassemble laterally into twisted and non-twisted ribbons of indeterminate thickness and length (several microns) forming gels (not shown). Individual protofilaments have a "beaded" appearance, suggesting a helical structure. The "beads" have a diameter of ca Å. Reproduced from [32] with permission. observed for several amyloid-like fibrils [5,21 and references therein], in which the -strands are oriented perpendicular to the fibril axis and the sheets run mainly parallel to the fibril axis. This conformation would produce oriented patterns from oriented samples with the ca. 4.7 Å reflection meridional and the ca. 10 Å reflection equatorial, as in the oriented patterns obtained from fibres of the chorion ca peptide [5]. In the case of the B peptide fibrils, all efforts to obtain oriented samples were not successful as described above, and thus the diffraction patterns contained diffraction rings instead of oriented reflections. A similar case to ours, where non-oriented X-ray diffraction patterns were observed, is clearly that of a peptide from the adenovirus fibre shaft [36], which also produces amyloid-like fibrils. The reflection at 26 Å may arise from the lateral packing of the fibrils when forming the ribbons or, perhaps, it is due to the repeating "beaded" structure along the protofilaments (Fig. 11). The origin of the 5.2 Å reflection is not clear. It might simply be the fifth order of the 26 Å reflection. However, no additional data are available to explain its presence. Spectral acquisition by ATR FT-IR and FT-Raman spectroscopy have been shown to yield rich information about the secondary structure of the B peptide, without the drawbacks associated with the more conventional vibrational techniques. Table 3 shows the most prominent bands and their tentative assignments in the FT-Raman spectrum of the B-peptide, utilizing as sample the fibre used for the X-ray diffraction experiment (Fig. 14). In the amide I region of the Raman spectrum, the band at 1669 cm -1 (Fig. 14) suggests the presence of -sheet conformation in the structure of the B peptide, when it forms amyloid-like fibrils in the fibre used for X-ray diffraction experiments [38,39 and references therein]. Analysis of the amide I band of the FT-Raman spectrum (Fig. 14) using the method of Hamodrakas et al. [40] suggests that there is 64% antiparallel -sheet and 30% -turns in the B peptide structure. The presence of a strong band at 1229 cm -1 in the conformationally sensitive amide III region (Fig. 14), a characteristic signature of -pleated sheet [39 and references therein], strongly supports the amide I evidence as well as the X-ray diffraction data. Bands at ca. 620, 1003, 1033 and 1603 cm -1 are most likely due to the two Phe residues present in the B-peptide (Fig. 10). Fig. (12). Photomicrographs of B peptide fibrils stained with Congo red: (a) Bright field illumination, (b) crossed polars. The red-green birefringence characteristic for amyloid fibrils is clearly seen. Bar 150 m. Reproduced from [9] with permission.

10 300 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Fig. (13). X-ray diffraction pattern from a fibre of B peptide amyloid-like fibrils. Four reflections, which correspond to structural repeats of 4.64, 5.20, 9.14 and 26 Å, are marked by arrows. The two most prominent at 4.64 and 9.14 Å, which probably correspond to the spacing of adjacent -strands and to the packing distance of -sheets respectively, indicate the presence of -sheets ("cross- " sheets) in the structure of the fibrils. Due to the poor alignment of fibrils these reflections appear as rings. The reflection at 26 Å is either due to the packing of the fibrils or originates from the "beaded" sub-structure along the fibrils (see Fig. 11). The origin of the 5.2 (=1/5 of 26) Å reflection is not known. Reproduced from [32] with permission. Table 3. Main FT-Raman Bands ( cm -1 ) and Tentative Assignments in the FT-Raman Spectrum of B Peptide Band 620 Phe Assignment 832 v(c-c) 859 v 886 Skeletal stretch 925 v(c-c) of Pro ring 1003 Phe 1033 Phe 1087 v(c-n) 1229 Amide III ( -sheet) 1318 v(c-n) 1341 (CH) 1403 v 5(COO-) 1453 (CH 2, CH 3) Fig. (14). FT-Raman spectrum of B peptide amyloid-like fibrils in the range cm -1. Error bar equals the standard deviation ( ) of the measurement. The spectrum has been acquired from the fibre used to obtain the X-ray diffraction pattern of Fig. 13. Reproduced from [32] with permission. Concomitant evidence for the preponderance of -sheet in amyloid-like fibrils derived from the B peptide is given by ATR FT-IR spectroscopy (Fig. 15). The ATR FT-IR spectrum shows one prominent band at 1623 cm -1 in the amide I region. This band is clearly due to -sheet conformation [28]. Its relatively low wavenumber may be attributed to strong hydrogen bonds in the -sheets [28], whereas the fact that it is sharp (its half-width is ca. 20 cm -1 ) suggests that the distribution of the phi and psi angles in the sheets is rather narrow, which means a very uniform structure. This is also true for the amide I band of the FT-Raman spectrum (Fig. 14). Its apparent half-width is ca. 17 cm -1, lower than that of the very uniform silk fibroin (ca. 27 cm -1 ) [39 and references therein]. The infrared shoulder at 1697 cm -1 is a strong indication that the -sheets are antiparallel [27, 28, 41] in good agreement with the analysis of the amide I band of the FT- Raman spectrum. Thus, both FT-Raman and ATR FT-IR support the presence of uniform antiparallel -sheets in the structure of B peptide fibrils, apparently in agreement with the existence of a "cross- " structure implied by X-ray diffraction and Congo red binding data Phe 1669 Amide I ( -sheet) Fig. (15). ATR FT-IR spectrum of B peptide amyloid-like fibrils in the range cm -1. Reproduced from [32] with permission.

11 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No MODEL STRUCTURE OF SILKMOTH CHORION PEPTIDES Taking into account all experimental and theoretical evidence accumulated previously for silkmoth chorion proteins [12 and refs. therein] and their synthetic peptide analogues [5, 9, 32], and the hexapeptide periodicities in the sequences of the A and B families of silkmoth chorion proteins [42, 43], the models shown in Figs. (16a,b and 17a,b), for the peptide ca, are the most probable models for the structure of silkmoth chorion peptides. Fig. (16). (a) Antiparallel twisted -sheet model proposed for the ca peptide. Sequence should be read continuously, beginning at the bottom. Invariant glycines (G) occupying the 2 nd position in the turns are black boxed. Tentative II' -turns alternate with fourresidue -strands. (b) A ribbon representation of the antiparallel twisted -sheet model proposed for the ca peptide. View approximately along the central -strands denoted as arrows. Reproduced from [5] with permission. The model shown in Figs. (16a,b) is an antiparallel twisted -pleated sheet of four-residue -strands alternating with type II' -turns. Invariant Gly (G) residues occupy the second position of the -turns, a location especially favourable for Gly in II' turns of twisted -sheets of globular proteins [44]. Another interesting possibility for the structure of the ca peptide might be that of the left-handed parallel -helix (Figs. 17a,b), similar to that found in the structure of UDP- N-acetylglucosamine acyltransferase [45] and other lefthanded parallel -helical proteins [4]. This protein shows hexapeptide sequence motifs. It is interesting to note that right-handed parallel -helices similar to those found in the pectate lyases have been postulated as the main molecular components of amyloid protofibrils, although no detailed molecular models were presented [46]. Characteristic hexapeptide periodicities of both Gly and hydrophobic residues also appear in the sequence of the ca peptide [12, 43]. Its sequence shows structural similarities with the sequence of UDP-N-acetylglucosamine acyltransferase (Fig. 17a), and the peptide would have a nice hydrophobic core when folded this way (Fig. 17b). Another attractive feature of the lefthanded parallel -helix model of Fig. (17b) are the hydrophobic faces of the triangular prism-like -helix. Nevertheless, the "edges" of this prism are occupied by charged, polar residues and glycines and this makes 3-D packing difficult, unless there are very specific interactions. On the contrary, the hydrophobic faces of the antiparallel -sheet structure shown in Fig. (16b) facilitate uniform 3-D packing of the sheets, leaving the polar and charged residues on both lateral "edges" of the sheet for favourable lateral interactions. Although we were the first, to our knowledge, to propose a detailed left handed parallel -helix structural model at atomic resolution, as a possible structure underlying amyloid fibrils [5], this publication remained unnoticed. However, several groups also proposed recently that -helices might dictate amyloid fibrillar structure [4, 47-52]. Fig. (17). (a) Structurally based alignment of the sequence of the ca peptide with the sequence of the N-terminal domain of UDP-N- Acetylglucosamine Acyltransferase (LpxA-E.coli) [45]. The one letter code is used. Sequences should be read continuously beginning at the top. The structure of the N-terminal domain of LpxA-E.coli is a left-handed parallel -helix [45]. C1 to C10 rows identify individual turns of the helix. PB1, PB2 and PB3 denote the parallel -strands of each turn. T1, T2 and T3 denote turn residues. Conserved hydrophobic residues which have their side-chains pointing towards the interior of the left-handed parallel -helix to form the hydrophobic core, are black boxed. (b) A ribbon representation of the ca peptide in a left-handed parallel -helix conformation. Arrows denote -strands. View almost perpendicular to a face of the left-handed parallel -helix. Reproduced from [5] with permission.

12 302 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Preliminary calculations of X-ray diffraction patterns from the models presented in Figs. (16b and 17b) and comparison with the experimental diffraction pattern of Fig. (4c) (data not shown), as well as analysis of the amide I band of FT-Raman spectra [32 and data not shown] and the presence of the high frequency component in the cm -1 in the ATR FT-IR spectra taken from samples containing amyloid-like fibrils formed from all, similar to natural, chorion peptides synthesized so far, clearly support the antiparallel twisted -pleated sheet model shown in Fig. (16b) (however, see also below). Unfortunately, as the study of Khurana and Fink [53] has conclusively shown, proteins that adopt a parallel -helix structure do not exhibit a unique infrared signature. Fig. (18). (a) A schematic antiparallel twisted -sheet model ("cross- " structure) for the ca_m2 peptide, with the side-chains of the three glutamate (E) residues that have replaced two valines (V) and one alanine (A) residues, in the ca_m2 peptide (cf. Fig. 5) as ball and sticks. Arrows represent -strands. Four-residue -strands alternate with tentative type II' -turns. View along the -strands. It is clear that this structure is not favoured because of strong repulsive electrostatic interactions of the glutamate side chains in close proximity. (b) A ribbon representation of the ca_m2 peptide in a left-handed parallel -helix conformation with the side-chains of the glutamates (as in (a) above) added as balls and sticks. Fourresidue -strands alternate with tentative type II -turns (the two middle residues of the -turns comprise the Gly (G) residues, tandemly repeating every six residues in Fig. 5, and the subsequent, usually polar or charged residue). Arrows represent -strands. View parallel to the axis of the helix. It is evident that this structure is also not favourable because the side chains of two glutamate residues are packed in the hydrophobic interior of the left-handed parallel -helix. Reproduced from [9] with permission. In contrast, none of the models shown in Figs. (16b, 17b) seem to be favourable structures for peptide ca_m2, which is not similar to natural chorion peptides (it contains three non-conservative mutations (Fig. 5)). As shown in the ladder model of Fig. (18a), glutamates would be very close to each other leading to unfavorable electrostatic interactions. Similarly, in the left-handed helix model of Fig. (18b), two glutamates would occupy the hydrophobic interior of the helix producing a very unstable structure. These results explain why ca_m2 adopts a random coil structure (Fig. 9c) and are clearly compatible with the fact that the ca_m2 peptide does not form amyloid fibrils even after very long incubation periods. Fig. (19). Photomicrographs of a part of a silkmoth chorion from Bombyx mori stained with Congo red: (a) Bright field illumination, (b) crossed polars. The red-green/yellow birefringence characteristic for amyloids is clearly seen. Bar 400 m. Thirty years ago, it was suggested that, because of the inherent twist of the -sheets in the monomer, the polymeric amyloid protofilaments might form long spacing helical structures in which the protofilaments are intertwined to produce 100 Å doubly helical amyloid fibrils [54]. The verbal description of this model, reminiscent of the -helix structure of transthyretin amyloid protofilament produced twenty years later by high-resolution X-ray studies [24], fits well with our data. We postulate that successive ca units form continuous twisted antiparallel -sheets (the so-called sheet helices following the nomenclature proposed by Blake and Serpell [25]), along the protofilaments, with their strands perpendicular to the long axis of the protofilaments ("cross- " structures). The thickness of each individual ca unit is of the order of Å, similar to the thickness of the individual protofilaments. Suspiciously, the pitch of the double helical amyloid fibril formed by the two intertwined protofilaments is 920 Å (Fig. 4a), a multiple of the 115 Å spacing of the -helix in the transthyretin amyloid protofilament [24]. Furthermore, the antiparallel twisted -pleated sheet model of the ca peptide is an eight-stranded antiparallel sheet, in contrast to the six-stranded -sheet of transthyretin. According to Sunde and Blake [55], an eight-stranded sheet could well form a ' -sheet helix' instead of a sixstranded -sheet. SIKMOTH CHORION: A NATURAL PROTECTIVE AMYLOID We have shown that ca-peptide fibrils have an amyloid nature and also all the other synthesized peptide analogues of silkmoth chorion proteins, but we have so far silently assumed that the peptide fibrils are truly representative of the structure of chorion proteins in the eggshell. The ca peptide alone corresponds to about 25-30% of the total chorion mass. Its self-assembly mechanisms produce amyloid-like fibrils under a great and diverse variety of conditions (see above), which strongly suggests that it should fold in an amyloid fashion also in the physiological state. Concomitant evidence for this assumption can be found in Fig. (1a). Lamellae (layers) of fibrils with the same dimensions ( Å; see also [10] and [11]) as the ca peptide double-helical fibrils shown in Fig. (4a), constitute the helicoidal architecture of silkmoth

13 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No chorion. Chorion fibrils consist of Å protofilaments with a helical structure [12 and references therein]. Furthermore, antiparallel -pleated sheet is the dominant molecular conformation of silkmoth chorion proteins in vivo (12 and references therein and also Figs 1b,c). In addition, silkmoth chorion binds Congo red showing the characteristic redgreen/yellow birefringence when seen under crossed polars (Figs. 19a,b). This, strengthens further our proposal that silkmoth chorion is a natural protective amyloid. Amyloids are generally associated with diseases such as Alzheimer's, spongiform encephalopathies, type II diabetes etc: More than 20 types of human disease are associated with the deposition of protein fibrils forming amyloids and resulting in tissue damage and degeneration [1, 2, 56, 57]. Amyloidogenic proteins appear to be related by their ability to undergo a conformational change and adopt a new amyloidogenic conformation under partially denaturing conditions in vivo, which permits self-assembly into amyloid [57, 58]. Our study [5] was the first to show that not all amyloids are by definition harmful. In chorion protein amyloids, the amyloidogenic conformation is, apparently, the native conformation. Chorion proteins and peptide-analogues provide a model system for the study of amyloid formation, and perhaps we can even extract medically relevant information from the chorion destruction mechanisms [59] used by the embryo upon hatching. Our proposal for the existence of natural, functional (in our case protective) amyloids [5] was followed by a number of examples, which undoubtedly confirmed the existence of several natural, functional amyloids, performing various important functions [see for example 2, 3, 4 and references therein]. However, to our knowledge, this is the first well documented case where amyloid-like fibrils are formed from peptides that have a sequence so clearly folded in an antiparallel -pleated sheet type of structure of the "cross- " type. These amyloid-like chorion peptides play an important functional role, after millions of years of molecular evolution: protect the oocyte and the developing embryo from a wide range of environmental hazards [10, 12]. Chorion proteins selfassemble extracellularly to form the chorion of silkmoths, far-away from the follicle cells that synthesize and secrete them [10, 12]. AMYLOID FIBRILLOGENESIS OF SILKMOTH CHORION PEPTIDE-ANALOGUES The phenomenon of the transformation of proteins into amyloid fibrils is of interest, firstly, because it is related to the protein folding problem, and secondly because it is connected to the so-called conformational diseases, the amyloidoses. Consequently, various attempts have been directed towards an understanding of the fibrillogenesis pathway(s), with the aim of developing inhibitors-drugs of therapeutic benefit. These are summarized in several excellent recent reviews [6, 60-64]. However, the molecular and energetic factors affecting protein misfolding and amyloid fibrillogenesis are still largely unknown [63, 65]. Recently, we presented data, which clearly show that the first main step of amyloid-like fibrillogenesis from silkmoth chorion peptides is the formation of nuclei of liquid crystalline nature [26]. Subsequently, these liquid-crystalline nuclei collapse and they are transformed into amyloid-like fibrils in a time-period, which depends on several factors. The transformation is performed, most probably, as a result of a conformational transition to the structure of chorion peptides, from a left-handed parallel -helix to an antiparallel pleated sheet. Apparently, chorion peptides play suitably this role, after millions of years of molecular evolution. These data can be summarized as follows [26]: ca peptide was found to spontaneously assemble into supramolecular spherical structures, after 1-2 hours incubation, under a great variety of conditions. These structures, when viewed in a polarizing microscope under crossed polars, are seen to have a liquid crystalline texture. They are spherulites with Maltese crosses (Fig. 20). Under a transmission electron microscope, after negative staining, nontransparent spherical structures are seen (Fig. 21), apparently spherulites with smaller diameters (compare with Fig. 20). Spherulites with larger diameters than those shown in Fig. (21) are also seen (not shown). The spherulites appear to have diameters ranging from 0.1 m to 200 m, combining evidence from light and electron micrographs (Figs. 20 and 21). They frequently coexist with readily formed (after an hour or less incubation) fibrils, Å in thickness (Fig. 22). These fibrils frequently seem to be derived from spherulites that collapse or deteriorate (Figs. 23 and 24). Since it might be argued that the association of spherulites with fibrils is not real, it should be mentioned that upon dilution and re-examination of the samples, fibrils are still seen to coexist with micelle-like structures. Furthermore, as seen from Fig. (25), multiple fibrils radiate out from collapsing / deteriorating micelle in all different directions, which means that they are indeed derived from the collapsing / deteriorating micelles. The spherulitic structures are characteristic, (and for the first 2-3 days dominant) features in the electron micrographs, which were taken from the samples, collected from the initial ca peptide incubations, on an every day basis, for approximately two weeks. Figs. (26a-e) show that in a period of one week micelle numbers decrease, with a concomitant increase of straight amyloid-like fibrils. However, quantification is not possible since it appears that there is no obvious statistical relationship between fibrils bound to micelles and free micelles. The exact time of transformation of these spherulitic structures to mature amyloidlike fibrils (Fig. 27) was found to depend mainly on temperature, concentration and type of solution. At room temperature, for most concentrations and types of solutions, the time is approximately one week. The fibrils formed from the ca peptide solutions exhibit all the hallmarks of amyloids, mentioned above. It is interesting to note that, solutions of peptide ca_m1 behave in an almost identical way: amyloid fibrillogenesis occurs also via a liquid-crystalline intermediate phase (our unpublished data). The common structural properties of amyloid fibrils most probably imply similar mechanisms of amyloid fibril formation and, perhaps, common features of amyloid disease pathogenesis. Therefore, much effort has been devoted towards understanding the pathway(s) of fibrillogenesis. Our

14 304 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Fig. (20). Photomicrograph of ca peptide spherulites viewed in a polarizing microscope under crossed polars. These supramolecular spherical structures are formed spontaneously after 1-2 hours incubation, under a great variety of conditions (see Methods). Maltese crosses are clearly seen. Bar 4 m. Reproduced from [26] with permission. Fig. (22). Electron micrograph of ca peptide spherulites along with readily formed fibrils, Å in thickness, after 1-2 hours incubation. The samples were negatively stained with 1% uranyl acetate. Bar 100 nm. Reproduced from [26] with permission. Fig. (23). Electron micrograph of ca peptide spherulites along with readily formed amyloid fibrils, which frequently seem to emanate from spherulites that collapse or deteriorate. The samples were negatively stained with 1% uranyl acetate. Bar 100 nm. Reproduced from [26] with permission. Fig. (21). Electron micrograph of a ca peptide spherulite derived by self-assembly, from a 6.5 mg.ml -1 solution of the ca peptide in distilled water, ph 5.5. The sample was negatively stained with 1% uranyl acetate. Bar 100 nm. Reproduced from [26] with permission. work [26], has shown rather conclusively that the first main step of chorion peptide amyloid fibrillogenesis involves the formation of a liquid crystalline phase, immediately after dissolution of chorion peptides in a variety of solvents and environments (Figs. 20, 21). The spherulitic, liquid crystalline phase formed is preserved for a period of 3-4 days. During this period the spherulites convert gradually into amyloid fibrils in a rather consistent and spectacular manner (Figs ). They are seen to explode producing several short fibrillar components, which, apparently, self assemble to form long fibrils. Apparently, in previous fibrillogenetic studies of the synthetic amyloid A peptide [66], micelles of much smaller dimensions (diameters of the order of 140 Å) have been observed to act as initial nuclei from which fibrils emerge. Nevertheless, more recently, the A peptide, at high concentrations of the order of M, was found to assemble into clearly defined spheres called amy balls, with diameters of ca m; however, it is not certain whether these spherical structures are spherulites [67]. It is also interesting to note that, AFM images of freshly dissolved A (1-42) peptide after its adsorption onto a freshly cleaned mica surface show predominantly monomeric and dimeric globular structures, with diameters of 1.5 to 2.5 nm [68]. No fibrillar structures are observed in AFM images of the freshly dissolved A s. Repeated AFM imaging reveals that A s retain the globular and non-fibrillar shape for an extended period of time [68].

15 Natural Protective Amyloids Current Protein and Peptide Science, 2008, Vol. 9, No Fig. (24). A snapshot of spherulite collapse / deterioration after 2 days incubation. The sample was negatively stained with 1% uranyl acetate. Bar 0.2 m. Reproduced from [26] with permission. Fig. (25). A snapshot whereby multiple fibrils radiate out in all different directions from a collapsing / deteriorating micelle (arrow), which means that they are derived from the collapsing / deteriorating micelle. Bar is 400 nm. Reproduced from [26] with permission. In spiders, soluble proteins are converted to form insoluble silk fibres, stronger than steel and, recently, the amyloidogenic nature of spider silk has been documented [69]. An important lesson to learn from the spider is how it stores protein molecules in a highly concentrated liquid crystalline state and then extends these in the spinning duct to form a supremely tough thread [70]. Apparently, this is another clear case where formation of insoluble, very tough material with amyloidogenic properties, initially passes through a liquid crystalline state. Observing closely both cases, the transformation of liquid crystalline soluble spider silk into fibres stronger than steel and also the transformation of soluble silkmoth chorion proteins into silkmoth chorion, a structure with extraordinary mechanical and thermal properties, via a liquid crystalline phase, may help us to gain insight, through lateral thinking, into the sudden but unwanted assembly of other proteins into amyloids, in various amyloidoses. Following our proposal that amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues proceeds via a liquid-crystalline intermediate phase [26], it has nicely been shown that the formation of amyloid-containing spherulitelike structures has been observed in several instances of amyloid diseases, as well as in amyloid fibril-containing solutions in vitro [71-73 and references therein]. At this stage, it is obviously natural to wonder what is the molecular denominator of the spherulitic and of the mature fibrillar state. The view that is currently valid is that amyloid fibrillogenesis requires partial unfolding of globular proteins or partial folding of disordered proteins [60]. However, in the case of chorion peptides this view may not necessarily be true. Apparently, when chorion peptides adopt their final structure forming mature amyloid fibrils, they have a characteristic antiparallel -pleated sheet structure strongly supported by X-ray diffraction, ATR FT-IR, FT-Raman spectroscopy and modelling data (Fig. 16b) and this is obviously their dominant molecular structure in the amyloid fibrillar state. It is tempting to speculate that in the spherulitic micelles formed directly after the solution of chorion peptides into various solvents, the peptides adopt a left-handed parallel -helix type of structure (Fig. 17b), which, as a model of chorion peptide structure, has several attractive features as well. We propose here that the transition from the spherulitic liquid crystalline phase to the mature fibrillar amyloid state involves a transformation of the left-handed parallel -helix type of structure to the antiparallel -sheet type of structure present in the mature fibrils of the amyloid state. This molecular conformational switch can easily be achieved by a transition of a type II -turn (in the structure of the lefthanded parallel -helix) to a type II -turn (in the structure of the antiparallel -pleated sheet) every six residues along the sequence of the ca peptide. Whether this is actually true remains to be seen by more refined experimental future work (In preparation). If it is true, it will be the first case whereby a peptide capable to form amyloid fibrils may adopt two, well defined, -sheet structures of a different kind. Apparently, after millions of years of molecular evolution, in a rather unique way, these dual-structure, amyloid-like chorion peptides play an important functional role: protect the oocyte and the developing embryo from a wide range of environmental hazards. It is also clear that, the results of these studies may have important morphogenetic implications for silkmoth chorion structure as well: silkmoth chorion, a biological analogue of a cholesteric liquid crystal [12 and references therein] selfassembles from its constituent proteins a long distance away from the follicle cells which secrete these proteins on the surface of the oocyte. From our work [26], we have shown

16 306 Current Protein and Peptide Science, 2008, Vol. 9, No. 3 Iconomidou and Hamodrakas Fig. (26). ca peptide dissolved in a methanol-distilled water 1:1 mixture, at a concentration of 6.5 mg.ml -1 observed for a period of approximately one week. It is clearly seen that micelle numbers decrease gradually, with a concomitant increase of straight amyloid-like fibrils. (a) Day 0. Only micelles exist. Bar 10 m (b) Day 2. Co-existence of micelles with some fibrils. Bar 100 nm (c) Day 5. The numbers of fibrils have considerably increased whereas those of micelles decreased. Bar 100 nm (d) Day 6. Total disappearance of micelles. The field is full of immature amyloid like fibrils. Bar 100 nm (e) Day 7. Only immature amyloid-like fibrils are seen. Bar 100 nm. Reproduced from [26] with permission. Fig. (27). Electron micrograph of mature ca peptide amyloid-like fibrils, after one week incubation. The sample was negatively stained with 1% uranyl acetate. Bar 0.2 m. Reproduced from [26] with permission. rather conclusively that the ca peptide, a representative of the entire central domain of the A class of chorion proteins, that is a representative of 25% of the entire chorion mass approximately, self-assembles under a great variety of conditions into spherulitic liquid crystalline structures, obviously precursor substructures of silkmoth chorion itself, which are transformed into amyloid-like fibrils constituting chorion. Thus, it appears that in vitro formation of such an important biological structure can be studied in detail. Since it might be argued that the peptide concentrations which lead to the liquid crystalline intermediates cannot occur in vivo, it should be mentioned that similar spherulitic structures are formed at very low (nm) concentrations of the ca peptide in the same solvents [26]. These structures seem to retain their shape for a prolonged, not very well defined, period of time before their conversion into amyloid fibrils. It remains also to be seen whether the results of this work can be generalized into other pathological cases involving amyloidoses and their, relevant in each case, proteins. OTHER NATURAL PROTECTIVE AMYLOIDS The generic ability of polypeptide chains to form amyloid fibrils, which can vary dramatically with sequence [2, 6], has been exploited by living organisms for specific purposes, and it has been found that certain organisms, during