Diversity of Molecular Transformations Involved in the Formation of Spider Silks

doi:10.1016/j.jmb.2010.10.052

J. Mol. Biol. (2011) 405, 238–253 Contents lists available at www.sciencedirect.com

Journal of Molecular Biology j o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

Diversity of Molecular Transformations Involved in the Formation of Spider Silks Thierry Lefèvre 1 , Simon Boudreault 2 , Conrad Cloutier 2 and Michel Pézolet 1 ⁎ 1

Département de Chimie, CERMA, PROTÉO, Université Laval, Pavillon Alexandre-Vachon, Québec, Québec, Canada G1V 0A6 2 Département de Biologie, Université Laval, Pavillon Alexandre-Vachon, Québec, Québec, Canada G1V 0A6 Received 12 September 2010; received in revised form 26 October 2010; accepted 27 October 2010 Available online 2 November 2010 Edited by M. Moody Keywords: Raman spectromicroscopy; spider silk proteins; spinning dope; secondary structure; sericigene glands

Spiders that spin orb webs secrete seven types of silk. Although the spinning process of the dragline thread is beginning to be understood, the molecular events that occur in spiders' opisthosomal glands, which produce the other fibers, are unknown due to a lack of data regarding their initial and final structures. Taking advantage of the efficiency of Raman spectromicroscopy in investigating micrometer-sized biological samples, we have determined the secondary structure of proteins in the complete set of glands of the orb-weaving spider Nephila clavipes. The major and minor ampullate silks in the sac of their glands have identical secondary structures typical of natively unfolded proteins. Spidroins are converted into fibers containing highly oriented β-sheets. The capture spiral represents a distinct structural singleton. The proteins are highly disordered prior to spinning and undergo no molecular change or alignment upon spinning. The cylindrical, aciniform, and piriform proteins are folded in their initial state with a predominance of α-helices, but whereas the cylindrical gland forms a fiber similar to the major ampullate thread, the aciniform and piriform glands produce fibers dominated by moderately oriented β-sheets and α-helices. The conformation of the proteins before spinning is related to intrinsic characteristics of their primary structure. Proteins that are unfolded in the gland have repeat sequences composed of submotifs and display no sequence regions with aggregation propensity. By contrast, the folded proteins have neither submotifs nor aggregation-prone sequence regions. Taken together, the Raman data show a remarkable diversity of molecular transformations occurring upon spinning. © 2010 Elsevier Ltd. All rights reserved.

Introduction *Corresponding author. E-mail address: [email protected]. Abbreviations used: Ma, major ampullate; Mi, minor ampullate; Cyl, cylindriform; Flag, flagelliform; Ac, aciniform; Pir, piriform; MaSp, major ampullate spidroin; PPII, polyproline II; AcA, aciniform A; AcB, aciniform B; MiSp, minor ampullate spidroin.

Spider silk is a secretion produced by the abdominal glands that transforms a viscous and concentrated protein aqueous solution into an insoluble solid fiber at ambient temperature. Unlike other arthropods, spiders can produce various types of silk intended for diverse biological functions such as

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.

Molecular Transformations in Spider Silk Formation

239

Fig. 1. Top: Ecological functions and repetitive protein sequences of the different silks spun by the orb-weaving spider N. clavipes. Silks are named after their secreting glands: Ma, major ampullate; Mi, minor ampullate; Flag, flagelliform; Cyl, cylindrical (also named tubuliform); Ac, aciniform; Pir, piriform. Sequences with the following IDs and corresponding literature sources were derived from UniProt (http://www.uniprot.org/): P198371 (MaSp1), P468042 (MaSp2), O174343 (MiSp1), Q9NHW44 (Flag), and Q3BCG25 (Cyl). Since the sequence of the wrapping silk for N. clavipes is unknown, the sequence of the wrapping silk for a related spider (A. trifasciata) is presented as a representative example (Q64K55).6 Red amino acids are assumed (or found experimentally) to be involved in β-sheets in the fiber. Green amino acids are the socalled spacer regions. Bottom: The sericigene glands of the orb-weaving spider Araneus diadematus are presented to illustrate the different morphologies. The tail, sac, and duct are shown for the Ma gland.

reproduction, feeding, and locomotion (Fig. 1). Spinning silk is thus one of the major activities of spiders and is involved in each step of their life. Each type of silk is adapted to its function, with specific mechanical characteristics that are completely dictated by the structural organization of the fiber (crystallinity, degree of molecular orientation, sec-

ondary structure, and microstructure), which in turn results from two main determinants: the protein primary structure and the mechanism of spinning. In recent years, various sequences of spider silk proteins have been determined. These proteins, also called spidroins, are high-molecular-weight biopolymers composed of a repetition of numerous

240 consecutive sequence units (Fig. 1) and highly conserved nonrepetitive C-terminal and N-terminal parts. Repeat units are often composed of small submotifs, such as An, (AG)n, GGX, or GPGXaXb, that are considered the hallmark of silk proteins. A general paradigm assumes that these motifs adopt particular secondary structures in the fiber. For example, An and (AG)n motifs are involved in β-sheets7 and play a crucial role in silk strength. GGX (X = L, Q, R, or Y) and GPGXaX b (X aX b = GA, GS, GY, or QQ) modules adopt less ordered conformations, most likely 31-helices and turns, and are important for fiber extensibility.8 More recent data have shown that the repeat units of spidroins are not always subdivided into such amino acid motifs;9 thus, they are likely to have particular intrinsic properties. The second determinant of fiber structure is the spinning process, which is controlled by the gland system and the animal's behavior. Much of the knowledge in this area has been obtained from studies on the major ampullate (Ma) gland due to its relatively large size and the outstanding tensile performance of the fiber that it produces.10 This sericigene gland is basically composed of three parts: a tail in which the proteins are synthesized, a sac (or ampulla) in which they are stored in aqueous solution (the so-called spinning dope), and a duct that is viewed as a die in which the dope flows and where conversion into fiber occurs (Fig. 1). The process is not simply an extrusion but is rather driven by tension.11,12 The forces involved are shear stress due to the flow resistance of the dope against the luminal wall of the duct and an extensional flow occurring in the distal part of the canal, which is induced when the spider (or its interactions with natural forces such as airflow or gravity) pulls on the fiber. The mechanical constraints developed during the spinning process are strongly affected by the animal's behavior, such as its displacement speed. These constraints, in the case of the Ma silk, lead to a conformational and orientational transition from disordered/31-helix structures into highly oriented crystalline β-sheets.12,13 The resulting extensive protein aggregation is concomitant with dehydration of the material. Several characteristics of the sericigene gland, among which is its anatomy, determine the mechanism of spinning. Spider gland morphology and complexity vary depending on the type of silk (Fig. 1) and spider species. The glands can be ampullaceal, spherical, tubular, or pear-shaped, and they can be singular or multiple; the ducts display variability in length and probably in lateral profiles. For example, the duct diameter of the Ma gland follows a decreasing two-stage exponential function in its distal part, which has a favorable impact on molecular elongation and crystallization.14 The environment of the solution along the duct (pH and ionic gradients) is also important, as it promotes protein–protein interactions.15,16 It is thus expected that the anatom-

Molecular Transformations in Spider Silk Formation

ical diversity and physicochemical parameters of the glands are associated with differing spinning processes. In this respect, Dicko et al. proposed an interesting relation between the degree of disorder of spider silk proteins and the complexity of the spinning apparatus.15 The viscoelastic dope itself, in particular its rheological properties, is critical for the spinning mechanism. As a matter of fact, the sericigene solution has a liquid crystalline (i.e., of non-Newtonian nature) that gives it shear-thinning properties that facilitate the spinning process.16 Due to high protein concentration, intermolecular interactions within this complex fluid are expected to be extensive17 and to be influenced by their secondary and tertiary structures. The protein conformation is thus expected to have an impact on the rheological properties. Such an influence of the secondary structure has been proposed to explain the differences between the shear viscosity of the cocoon silk of Bombyx mori silkworm and the shear viscosity of the cocoon silk of Samia cynthia ricini silkworm.18 From what precedes, it seems that the primary structure of silk proteins has been optimized with respect to two very different—if not opposed— constraints: on one hand, the spidroin has to be water-soluble at high concentration and has to constitute a solution with appropriate conformation and rheological properties; on the other hand, it has to eventually form specific secondary structures and microstructures in a solid material. For the Ma silk, it seems that the switching from the storage form to the assembly form is partly triggered by the C-terminal end of the spidroins,19 in particular its pH sensitivity.20 Apart from studies on the Ma thread, a few studies have been devoted to the structure of spider silk fibers. A recent Raman investigation has reported the spectra of the complete set of silk fibers of the spider Nephila clavipes.21 Minor ampullate (Mi) and cylindrical (Cyl) silks appeared to have a structure very similar to that of the Ma silk, whereas the flagelliform (Flag) and aciniform (Ac) silks differed markedly. The former are characterized by the absence of molecular orientation and a very heterogeneous conformation, whereas the latter are dominated by a mixture of moderately oriented β-sheets and α-helices. The protein conformation in the glands has been the subject of even less work and, furthermore, requires the solubilization of silk dope in water.22 It shows that the Ma, Mi, Flag, and Cyl proteins exhibit different folds in solution and that they respond differently to temperature. Nevertheless, there is clearly a lack of data regarding the structure of silk dopes in their native state. This is mainly due to the difficulty in studying and manipulating silk samples, which are metastable and whose sizes lie in the micrometer range (a few microliters and even tens of nanoliters for liquid silks).

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Molecular Transformations in Spider Silk Formation

Fortunately, Raman spectromicroscopy allows direct probing of such specimens. Since Raman spectra are composed of vibrational bands due to amino acid side chains and backbone peptide bonds (so-called amide bands), amino acid composition and polypeptide chain arrangement can be investigated, respectively. In this work, the lumens of a complete set of sericigene glands of the spider N. clavipes have been probed to determine the spidroin conformation. Since they can affect the spinning mechanism, the intrinsic properties of protein sequences (such as their propensities to be unfolded, to adopt particular secondary structures, and to form β-sheet crystallites) have also been characterized.

Results All native silks of the spider N. clavipes [Ma, Mi, Flag, Cyl, Ac, and piriform (Pir)] have been

investigated in the sac of their sericigene glands. The details of the Raman data regarding the Ma silk have been published elsewhere.13,23 The silk feedstock of the glands has been probed in both hydrated and dry states. As shown in Supplementary Data, Raman spectra show that the secondary structure of the proteins is not modified by drying. Therefore and to be able to analyze the amide I band without interferences from water, we presented the spectra of the silk dope in the dry state. Mi silk Figure 2a compares the Raman spectra of the silk contents of the Ma and Mi glands. The inset to the figure shows the amide I band in detail. The spectra of both silks are nearly indistinguishable in the amide I (1658 cm− 1), amide III (~1260 cm− 1), and C–C stretching (1103 cm− 1) regions, showing that the two proteins have nearly identical overall

Fig. 2. Raman spectra of (a) the content of the sac of the Ma and Mi glands of N. clavipes, and (b) the Mi silk before (gland content) and after (fiber) spinning. The insets show the (a) amide I and (b) C–C stretching regions in detail. The spectra are normalized with respect to the area of the amide I band.

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Molecular Transformations in Spider Silk Formation

Table 1. Parameters characterizing the amide I band of the silks of the different silk glands of N. clavipes before and after spinning Position (cm− 1) Secreting gland Ma Mi Flag Cyl AcA AcB Pir a

Bandwidth (cm− 1)

Before spinning

After spinninga

Before spinning

After spinninga

1658 1658 1668 1658 1657 1657 1657

1670 1670 1668 1670 1669 + 1657 (shoulder) 1669 + 1657 (shoulder)

59 ± 1 57 ± 1 61 ± 1 38 ± 1 39 ± 1 39 ± 1 42 ± 1

33 ± 1 34 ± 1 57 ± 1 35 ± 1 44 ± 1 47 ± 1

Values are given for the orientation-insensitive spectra.

secondary structures despite their different amino acid sequences. Major ampullate spidroins (MaSps) are known to be mostly unfolded in the gland,24,25 but vibrational circular dichroism and NMR spectroscopies have clearly shown that they contain a significant amount of left-handed 31-helices [also known as polyproline II (PPII) helices], which have been suggested to be important for the formation of β-sheets during the spinning process.23 From the similarity of the Raman spectra of the Ma and Mi silks, it can be hypothesized that the Mi proteins also contain a significant amount of PPII helices. The large bandwidth of the amide I band (Table 1) reflects the high degree of disorder of these proteins, while the band maximum at 1658 cm− 1 is an indication of the presence of α-helices. The presence of α-helices is clearly confirmed by the weak contribution at 525 cm− 1, a band that is very strong for polyalanine in the α-form.13,26,27 The smaller intensity of this band for the Mi silk is consistent with the fact that this protein has shorter (A)n motifs (Fig. 1). The amide III band has at least two components at 1260 and 1244 cm− 1, reflecting various conformational elements. The latter is confidently assigned to unordered structures,13,28–31 whereas the former is likely due to 31-helices and α-helices. The position of the band at 1103 cm− 1 is also consistent with unordered chains. In conclusion, despite the presence of some α-helices, Mi and Ma proteins belong to the natively unfolded protein family, a class of proteins characterized by the absence of structural order under physiological conditions, although they often contain local and residual structures.32 The presence of 31-helices in MaSp supports this conclusion, since this structural element is recognized as a widespread local structure of unfolded proteins.33 The main differences between the spectra of Fig. 2a are due to amino acid side chains (see the band assignment in Table 2). The higher intensities at 829, 851, 1174, 1208, and 1615 cm− 1 for the Mi silk reflect

Table 2. Position and assignment of the main Raman bands of spider silks Band position (±1 cm− 1)a 525 621 642 748 829 and 851 875 904 922 939 1003 1028 1045 1055 1083 1102 or 1105 (1094 and 1068)c 1126 1175 1155 1207 1260 or 1273 (1242 and 1228)c 1305 1335 1390 1416 1452 1526 1550 1587 1603 1615 1658 or 1668 (1670)c

Assignment Polyalanine (α-helix) Phe Tyr C2b, Trp Tyr Pro Polyalanine Pro α-Helix Phe Phe Pro Ser Ser Skeletal Cα–Cβ stretching Leu Tyr C1b Phe, Tyr Amide III Ala Ala C2b Gly CH3 asymmetric bend, CH2 stretching C1b C2b, Trp Phe Phe, Tyr-protonated form Tyr Amide I

a

Values may vary slightly depending on the type of silk. C1 and C2 are carotenoid and isoquinoline compounds, respectively.13 c Values in parentheses correspond to β-sheet conformation. b

the higher Tyr content of this fiber. The higher amount of Ala residues for the Mi silk probably contributes to the intensity difference at 851 cm− 1, whereas the higher intensity at 904 cm− 1 for the Ma silk is associated with the longer segments of its An motifs. Finally, the small band at 1126 cm− 1 is assigned to Leu (which is present in higher amounts in the Ma silk), whereas the weak bands at 1056 and 1082 cm− 1 are due to Ser. In Fig. 2b, the spectrum of the Mi spinning dope is compared with that of the fiber. To remove any bias due to the anisotropy of the latter, we present the orientation-insensitive spectrum. 34 The amide I, amide III, and C–C skeletal regions display dramatic modifications due to spinning. The peak maxima for the fiber are found at 1670, 1233, and 1091 cm− 1, and are characteristic of β-sheet conformation.21 The band at 940 cm− 1 almost disappears for the fiber, showing that it is related to the native state, probably to α-helices.13 These changes are the same as those undergone by the Ma silk.13 The

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Molecular Transformations in Spider Silk Formation

band at 525 cm− 1 is less intense for the fiber than before spinning, but is still slightly visible. This is also true for the band at 1103 cm− 1, which can be seen as a shoulder for the fiber (inset to Fig. 2b). The persistence of these bands after spinning reveals that residual native-like structures are present in the Mi thread, in contrast to the Ma silk.13 Such structures may partly explain the higher breaking strain of the Mi filament as compared to the Ma filament.35 Abovementioned data show that the Ma and Mi proteins have identical conformations in the spinning dopes and that the two fibers share the same structural pattern. More particularly, the β-sheet contents of these two threads are equal or very close, and their molecular orientations are similar (mainly parallel with the fiber axis), although the degree of orientation is lower for the Mi silk.21 Consequently, the molecular events that occur during the spinning process of these silks are qualitatively identical and quantitatively similar. This is consistent with the fact that these filaments are produced by glands that have similar morphologies (the ampullaceal form of the sac and the long narrowing S-shaped ducts), although the Ma gland is bigger. The structural identity of the Ma and Mi silks, both before and after spinning, is intimately related to the similarity of their sequences (Fig. 1). No other spider silk proteins share more common points than the Ma and Mi spidroins. Thus, the common features of the sequences, on one hand, and gland anatomies, on the other hand, lead to equivalent molecular transformations upon spinning and similar fiber structures. This in turn explains why these silks have fairly resembling tensile properties.35

Nevertheless, subtle structural differences—in particular the presence of residual conformational elements reminiscent of the dope and a lower degree of orientation of β-sheets in the Mi fibers— remain. Flag silk Flag silk is used by orb-weaving spiders as the core thread of the capture spiral of the web and is well-known to behave like elastomeric materials due to its high strain at rupture.10 These tensile properties, in conjunction with those of the frame and radii threads, provide the web with particular attributes to efficiently absorb the kinetic energy of flying insect prey. Its repetitive sequence is dominated by the GPGXaXb submotif. Figure 3 shows the spectrum of the silk probed in the sac of the gland compared with the orientation-insensitive spectrum of the fiber. The capture spiral threads were harvested directly from the web, and the sticky glue that coats the fiber was removed by washing with water. Apart from some intensity differences, the two spectra are surprisingly similar. The amide I band has the same shape, and the peak maximum is located at the same position before and after spinning. The same observation is made for the amide III band, which is composed of at least two components located at 1250 and 1263 cm− 1. Thus, the Flag silk undergoes no conformational change during the spinning process, as opposed to all other spider silks. The Flag silk fiber has no crystalline fraction.36 The secondary structure of this protein has recently

Fig. 3. Raman spectra of the Flag silk of N. clavipes before (content of the gland sac) and after (fiber) spinning. The spectra are normalized with respect to the area of the amide I band. The spectrum of the fiber has been obtained with the He–Ne laser so that the spectral range investigated is smaller than that for the gland content.

244 been described as a distribution of conformations.21,37 This structure is reflected by the large bandwidth of the amide I band (Table 1). No β-sheet is present, as a large proportion of Pro residues, distributed regularly along the whole repetitive sequence, prevent the formation of this secondary structure. The fiber also has no preferential molecular orientation.21 This silk is thus unique, as the spinning involves neither conformational transformation nor molecular alignment. It seems that the formation of the fiber mainly consists in the dehydration and densification of the material with the development of interchain bonds. Interestingly, in this case, dehydration is not associated with—and does not result from—β-aggregation. Thus, the formation of β-sheets is not necessarily required to induce the dehydration of silk. In addition, like other spider silk fibers, the Flag filament is not water-soluble (as opposed to its sticky coating), although it can also be partly penetrated by water as it acts as a plasticizer.38,39 Therefore, the spinning process is more than just a dehydration of the silk dope. The development of interchain interactions during the spinning process appears crucial in constituting a network that makes the capture thread insoluble and resistant to atmospheric humidity. What particular assembly features are formed remains to be clarified. Incidentally, the large extensibility of the Flag silk has been proposed to rely on water plasticization by the viscid glue coating.38,39 However, although plasticization is crucial in weakening intermolecular interactions and thus in providing a large extensibility to this silk, the present Raman data reveal that

Molecular Transformations in Spider Silk Formation

its effectiveness has to be associated with a disordered protein conformation and the absence of molecular orientation. Consequently, one of the requirements of thread formation seems to be the development of interchain interactions with minimal conformational ordering and polypeptide alignment. Cyl silk Cyl silk is produced by three pairs of tubular glands and is used by spiders to build the outer sac that protects the eggs. Figure 4 shows the spectra of the fiber and silk contained in the sac of the Cyl gland. As judged from the small width of the amide I band (Table 1), the conformation before spinning is much more homogeneous than that of the Ma, Mi, and Flag silks. The maximum at 1658 cm− 1 is due to α-helices present in high amounts. The amide III band has a very low intensity and is located at 1273 cm− 1—two features that are well-known to be the signature of α-helical proteins.26,28,40 These characteristics differ dramatically from the intense and broad amide III band of the Ma, Mi, and Flag silks, which underlines their fundamental conformational dissimilarity. The position of the C–C stretching mode at 1105 cm− 1, slightly higher than that for Ma and Mi (1103 cm− 1), and the presence of the peak at 524 cm− 1 are also consistent with αstructures.26,27,41 Cyl protein can thus be classified as a folded (i.e., globular-like) protein in the gland. The estimate of the α-helix content from the spectral decomposition of the amide I band is 51 ± 3% (Fig. S4). Other secondary structures are probably random

Fig. 4. Raman spectra of the Cyl silk of N. clavipes before (content of the gland sac) and after (fiber) spinning. The inset shows the amide I region in detail. The spectra are normalized with respect to the area of the amide I band.

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Molecular Transformations in Spider Silk Formation

coils, 31-helices, and turns. This result is consistent with the presence of 57% of α-helices, as determined from the infrared spectra of the Cyl dope dissolved in deuterium oxide.22 The presence of αhelices has also recently been found by circular dichroism for the Cyl dope of N. clavipes dissolved in water15 and by NMR for the recombinant Cyl proteins of Nephila antipodiana.42 X-ray diffraction has shown that the Cyl silk fiber is composed of oriented β-sheets43 such as Ma and Mi silks. Raman analyses indicate that the amount of β-sheets and the level of orientation are almost identical for Ma and Cyl fibers.21 The structure of the Cyl fiber is illustrated in Fig. 4, with the orientation-insensitive spectrum showing the β-sheet bands at 1668 cm− 1 (amide I), 1237 cm− 1 (amide III), and 1088 cm− 1 (C–C stretching). As in Ma and Mi silks and in contrast with the Flag silk, strong molecular modifications are observed upon spinning. However, the spinning of the Cyl silk involves mainly an α-helix-to-β-sheet transition. This process is different from that of the Ma and Mi silks, as the initial secondary structures are different. This means that various initial conformations can lead to fibers with very similar secondary structures. It is noteworthy that the same kind of α-helix-to-βsheet transformation also occurs for the cocoon silk of the wild silkworm S. cynthia ricini.44 Various bands reflect the particular amino acid composition of this silk (Table 2). The very strong contributions at 621, 1003, 1027, 1587, and 1605 cm− 1 arise from Phe. The Cyl silk is recognized for its high amount of Ser, consistent with the significant contributions at 974, 1055, and 1082 cm− 1. The

band at 904 cm− 1 probably originates from the (A)2 and (S)3 motifs of tubuliform spidroin 1, since a similar band appears in polyalanine 26 and polyserine.45 The low amount of Gly residues is reflected by a weak band at 1416 cm− 1. Finally, the low amount of Tyr residues results in weak bands at 642, 829, 852, and 1615 cm− 1. Not only is the Cyl protein amino acid composition specific but also the basic arrangement and structure of its repeat sequence are different (Fig. 1), with only very few submotifs reminiscent of those of Ma, Mi, and Flag. The repeat sequence of N. clavipes Cyl silk (Fig. 1) is a highly conserved 171-aa-long sequence, with only short and randomly distributed (A)2 and (S)3 blocks.5 Thus, its amino acid architecture is fundamentally different from those of the abovementioned silks. Thus, it is not surprising that the protein conformations of the initial states are different. However, it is remarkable that the structures in the fibers of the Cyl and Ma silks are very close. Ac and Pir silks Two types of Ac glands exist in N. clavipes.46 Based on their spherical or elongated shapes and their association with the posterior and median spinnerets, they are labelled as aciniform A (AcA) and aciniform B (AcB), respectively.46 As opposed to the previous glands investigated, AcA and AcB apparatuses are multiple and found as clusters, with AcB glands occurring in fewer cluster numbers (about a dozen) than AcA (tens). Although the function of the silks that they produce has not been totally resolved, Ac fibers are used to wrap prey. The Ac

Fig. 5. Raman spectra of the content of the sac of the AcA and AcB glands of N. clavipes and of the wrapping silk fiber. The inset shows the amide I region in detail compared with the gland content of the Cyl glands. The spectra are normalized with respect to the area of the amide I band.

246 filament of Argiope argentata has been shown to have remarkable mechanical properties, as its breaking energy is 50% higher than that for the dragline thread.6,35 Figure 5 shows the Raman spectra of the luminal contents of these two glands with the orientationinsensitive spectrum of the wrapping silk. The spectra of the feedstock of the AcA and AcB silks are virtually identical. Thus, they are both made of proteins with identical conformations. The amide I band at 1657 cm− 1 (inset to Fig. 5), the weak amide III band at 1276 cm− 1, and the small contribution at 522 cm− 1 share the same features (position and shape) as those of the Cyl proteins, showing that they are basically folded with a very close secondary structure before spinning. The spectral decomposition of the amide I band indicates that 49 ± 3% of the amino acids are in the α-helical conformation (Fig. S4). Thus, spidroins with dissimilar sequences (Cyl and Ac) can have very similar initial secondary structures. However, the wrapping and cocoon silks do not share the same organization in the fibers. Whereas the cocoon silk is dominated by highly oriented βsheets, the wrapping silk belongs to a third structural class of fibers, made of β-sheets (30 ± 3%) and α-helices (24 ± 3%), that are moderately oriented.21 The contribution of the β-sheets and α-helices can be seen in the amide I region in the inset to Fig. 5 at 1667 and 1657 cm− 1, respectively. Thus, the spinning process of the Ac silk also involves an α-helix-to-βsheet conversion, but the transformation is not “complete” as 20–30% of residual α-helices remain. These α-helices seem to be simply constrained to

Molecular Transformations in Spider Silk Formation

align along the fiber axis as spinning proceeds. They probably contribute to the particular mechanical properties of this type of silk. The bands due to amino acid side chains show the specific amino acid composition of the Ac silks, as one considers the intensity of the bands due to Phe (1605, 1587, 1003, and 621 cm− 1), Tyr (1615, 854, 829, and 643 cm− 1), Pro (921 cm− 1), and Leu (1080 cm− 1). Based on the structure of their sequences, AcSp 1 and Cyl proteins share the same basic features, that is, a long (typically 200 aa) sequence without regular submotifs, except for (A) 2 and (S) n (n = 2–5) motifs.5,47 Similarly to Ac filaments, the Pir silk threads are spun by multiple clustered gourd-like glands. They form attachment disks that allow fibers to stick together or to fix the dragline on substrates. There are no available data on the tensile properties or the structure of this filament. Figure 6 shows the spectra of the Pir silk before and after spinning. They share obvious similarities with the corresponding spectra of the Ac silks, especially in the amide I and amide III regions. The spinning dope of the Pir silk thus displays a predominance of α-helices, with the content being estimated to be 45 ± 3% (Fig. S4). The fiber is also characterized by a mixture of α-helices and β-sheets, so that the spinning process of the Pir silk is totally comparable to that occurring for the Ac silk. From these Raman data, the repeat unit of Pir proteins is anticipated to share similarities with the Cyl and Ac spidroins. As judged from the fingerprint region of the Raman spectra, its amino acid composition seems to be close to those of the Ac

Fig. 6. Raman spectra of the Pir silk of N. clavipes before (content of the gland sac) and after (fiber) spinning. The inset shows the amide I region in detail. The spectra are normalized with respect to the area of the amide I band.

Molecular Transformations in Spider Silk Formation

247

Fig. 7. Prediction of the unordered regions of the repetitive sequence of spider silk proteins using the PONDR® VL-XT algorithm.

spidroins. The sequence of Pir proteins has only been determined for Latrodectus hesperus.48 The repeat unit is dominated by two special submotifs, AAARAQAQAE and AAARAQAQAEARAKAE. However, the discrepancy with the amino acid composition of the gland feedstock suggests that another spidroin is involved in this silk.48 It is thus difficult to establish any correlation between the sequence of this protein and its secondary structure.

Discussion If one considers the protein conformation in the gland, two classes of spidroins are observed: those that are totally disordered (Flag) or mainly unfolded (Ma and Mi), and those that are globular-like (folded) with a high content of α-helices (Cyl, AcA, AcB, and Pir). However, Ma and Mi spidroins might be considered as intermediate between Flag and Cyl/Ac/Pir due to the presence of some α-helices. To support this classification drawn from Raman data, we have used various algorithms that predict structural parameters from the protein primary structure. Since some silk proteins belong to the class of natively unfolded proteins, we used algorithms that have been specifically developed to predict polypeptide segments that are unfolded. A typical prediction is presented in Fig. 7, where the folded/

unfolded character of the repetitive sequence of the different silk proteins is estimated using the PONDR® algorithm, which gives the most consistent results. Complete data obtained using various algorithms are given in Supplementary Data (Figs. S5–S10). MaSp1, MaSp2, minor ampullate spidroin (MiSp) 1, and Flag proteins are predicted to be unfolded. In the case of the spacer region of MiSp1, it seems to contain some folded structures, although it is difficult to conclude due to the variability of the predictions. The Cyl spidroins are predicted to be partially folded, in agreement with the presence of α-helices as found by Raman spectroscopy. Overall, the results of the order prediction algorithms are consistent with the Raman data and confirm the discrimination of the two classes of spidroins. The secondary structure of the proteins has been estimated using secondary structure prediction algorithms. A typical result calculated with the Porter algorithm is presented in Fig. 8 for the different proteins. The complete data set obtained with various methods can be found in Supplementary Data (Figs. S11–S16). The Flag spidroin is predicted to adopt loops or turns. No α-helix and very few β-sheets are anticipated. Such results are consistent with a disordered protein. MaSp1 and MaSp2 are also predicted to adopt mainly loops or turns, with no α-helix or β-sheet. This is again in agreement with an unfolded protein. However, four methods, including the Porter method, predict An

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Molecular Transformations in Spider Silk Formation

Fig. 8. Prediction of the secondary structure of the repetitive sequence of spider silk proteins using the Porter algorithm.

segments to adopt α-helices. A similar situation exists for MiSp1 with the short An and (AG)n modules. These results are probably the expression of the fact that An motifs have a certain propensity to adopt α-helix and PPII conformations, with the probability to adopt the former structure increasing as the polypeptide length increases.49,50 Thus, the conformation of the (A)n motifs can be adequately described as a dynamic equilibrium between the α-helix and the PPII conformation, with the residence time in the α-helix increasing as the (A)n length increases. The length of the polyalanine motifs is not only optimized with respect to the secondary structure that they adopt but also optimized with respect to the size of the β-sheet nanocrystals of the fiber, since the mechanical properties of the Ma thread decrease as the crystal size increases.51 The spacer region of MiSp1 probably contains some α-helix segments. Finally, Cyl and Ac proteins are predicted to adopt significant amounts of α-helices. Depending on the type of algorithms, the α-helix content ranges between 22% and 92% for Cyl, and between 8% and 45% for Ac (see Supplementary Data). These results are consistent with the Raman data and the abovementioned folding/unfolding algorithms. Thus, the two types of structure predictions support the conclusion that two classes of silk proteins exist: unfolded disordered silk proteins and folded ordered silk proteins (mainly α-helical).

Except for the Flag silk, the formation of βsheets is one of the main events that occur during silk spinning. Its occurrence relies on a universal property of proteins—aggregating in the form of β-sheets. This phenomenon has been a well-known technofunctional property of food proteins since the 1980s52 and has been the subject of major investigations since the late 1980s because amyloidogenic proteins form fibers that are composed of cross-β-sheets involved in neurodegenerative diseases and other amyloidoses.53 An important finding has been made with the recognition that many—if not all—proteins can form cross-β-sheet amyloid-like fibers.54 The importance and universality of β-aggregation have led to the development of algorithms, such as TANGO, that determine the sequence segments of proteins that are prone to form β-amyloids. This algorithm has been used to estimate the β-aggregation propensity of the repeat sequence of the spider silk proteins. Figure S17 shows the results for Cyl and Ac spidroins. For Ac, two sequences have been evaluated: the sequence of Argiope trifasciata and the sequence of L. hesperus. The prediction for the repetitive sequences of the Ma, Mi, and Flag spidroins is not shown, since they have no propensity for aggregation at all. Of course, this does not mean that the formation of β-sheets is prohibited for these proteins, especially with regard to the spinning process of spiders. This result only indicates that the repeat sequence of these proteins does not have a

Molecular Transformations in Spider Silk Formation

“natural” propensity for β-aggregation. Cyl and Ac spidroins have two segments that display β-aggregation propensity reminiscent of amyloid proteins. A comparison of the sequences of these segments reveals remarkably common features such as the marked presence of Asn, Leu, and Val amino acids. There is thus a fundamental difference between Ma, Mi, and Flag, on one hand, and Cyl, AcA, and AcB, on the other hand. The former have a repeat sequence that has no propensity for aggregation, whereas the latter have a repeat sequence that has propensity for aggregation. Interestingly, the former are mainly unfolded in the gland, while the latter are folded. Such coupled folding/aggregation properties could have been predicted, since the formation of a globular structure comes at the cost of a higher β-aggregation propensity.55 This is due to the fact that the formation on an ordered native structure and β-aggregates obeys very similar physicochemical rules and interactions.55 A similar distinction between both groups of proteins has been made based on the Pro and Gly contents.56 These two protein families are also characterized by another particularity: whereas the repeat sequence of Ma, Mi, and Flag is composed of small motifs that are the hallmark of silk proteins, the repeat sequence of Cyl, AcA, and AcB is characterized by the absence of submotifs.

249 The discrimination between spidroin structural families that share intrinsic and conformational characteristics in the spinning dope should help to understand the spinning process of spider silks. The secondary structure adopted by the proteins may have an impact on the rheological properties of the silk dope, as the process of β-sheet formation may be affected by the secondary structure adopted prior to spinning.18 Moreover, the generation of β-sheets from a totally disordered protein or an α-helical protein may be different, as the latter structure requires an unfolding step with the breaking of intramolecular hydrogen bonds, while the former does not. The former process may thus require a higher activation step, while the latter may be less energy-demanding. In particular, the occurrence of a PPII-to-β-sheet transition of the Ma and Mi silks may be advantageous, since the dihedral angles of the left-handed PPII structure are close to those of the β-sheets. In addition, for proteins that display a strong aggregation propensity, the formation of the β-sheets may be more rapid and/or disorganized (chaotic). In turn, such spinning processes may require shorter ducts, whereas proteins that have no particular propensities to aggregate may need more time (i.e., longer ducts) and may result in a smoother and more ordered aggregation phenomenon. Thus, the

Fig. 9. Schematic models of the initial and final protein secondary structures of the different silks spun by the spider N. clavipes showing the structural conversion induced by the spinning process. Spinning dopes and fibers that belong to the same structural family are placed together and identified by colors. Images of the glands of the related spider Ara. diadematus are also shown.

250 structural properties of spidroins should be intimately related to the anatomical characteristics of the glands.

Conclusion The initial and final secondary structures of the spidroins of orb-weaving spiders and the molecular events occurring during the spinning process display an interesting diversity that is summarized in Fig. 9. The structure of silk proteins can be classified into three different families: (i) mainly unfolded proteins characterized by PPII helices, unordered segments, and some α-helices (Ma and Mi); (ii) folded proteins dominated by α-helices (Cyl and Ac); and (iii) the unordered Flag protein. Three families of silk fibers were also identified, leading to four different mechanisms of spinning (Fig. 9). Although the spinning process generally involves a dramatic transformation, it can imply only slight molecular transformations, as shown by the Flag silk. Silk spinning often results in the formation of βsheets, but the final secondary structure obtained may vary: Ma, Mi, and Cyl fibers are dominated by β-sheets, whereas Ac and Pir fibers contain both βsheets and α-helices. A similar initial conformation can also lead to different final structures (Cyl versus Ac/Pir). By contrast, a different native initial conformation can lead to a similar secondary structure in the fiber (Ma/Mi versus Cyl). These different strategies of spinning observed at the protein structure level are only one of the aspects of the process and are obviously associated with various opisthosomal glands. Further work is required to better understand the anatomical features that transform these various viscoelastic dopes (and their specific structures) into their corresponding structural organizations in spider silks.

Materials and Methods N. clavipes spiders from Florida were raised in the laboratory in 20 cm × 50 cm × 60 cm cages at a relative humidity of 58 ± 5% and at a temperature of 24 ± 2 °C. They were fed small crickets and drops of 10% wt/vol glucose solution. Immature spiders were anesthesized and dissected using phosphate-buffered saline. Their glands were extracted, deposited on glass slides or polystyrene Petri dishes, and immersed in the same buffer. The epithelium of the glands was gently removed or pierced to expose the native silk to the laser beam. Great care was taken to perturb the silk material minimally during the dissection procedure. The spectra of the silk content of the ampulla of the glands were recorded in both dry and wet states. Details regarding the Raman spectrometer and the polarization measurements have been given elsewhere.57

Molecular Transformations in Spider Silk Formation The spectra were only corrected for a slight fluorescence background over the spectral range of 400– 1800 cm− 1 using a polynomial baseline. Fourier deconvolutions in the amide I and amide III regions were performed following the method of Griffith and Patiente, using a γ factor of 11 and a smooth factor of 90% with a Bessel function.58 All spectral manipulations were performed using GRAMS/AI 7.0 (ThermoGalactic, Salem, NH). Spectral decompositions of the amide I band were carried out in order to estimate the amount of α-helices of the silk proteins that were found to be mainly folded (Cyl, AcA, AcB, and Pir; see the text). The spectral decomposition of the unfolded proteins (MaSp1, MaSp2, MiSp1, and Flag) is more difficult to perform due to the broadness and featureless shape of the amide I band and the lack of knowledge on the spectral components that compose it. The same set of initial spectral parameters has been used to perform all curve-fittings. Based on second derivative and Fourier deconvolution calculations (data not shown), four bands were present. The components are located at 1638, 1657, 1675, and 1691 cm− 1. The assignment is straightforward for the component at 1657 cm− 1 and is due to the α-helix. The assignment of the other components is unclear, but they originate from disordered structures, loops, and/or turns. A fifth band has been added in some cases, since it was required to obtain reasonable fits. This band accounts for less than 3% and is associated with the carbonyl side chains of glutamine and arginine amino acids. The shape of the components was a mixture of Gaussian and Lorentzian functions. The initial bandwidth of the amide I components was set to 16 cm− 1. All spectral parameters (position, bandwidth, intensity, and Lorentzian character) were free to change during the curve-fitting, except for the bandwidth that was limited to 20 cm− 1 and the Lorentzian character that was limited to 30%. Such constraints are necessary; otherwise, these parameters will spontaneously go beyond these values during the calculation without physically based reasons. The α-helix content was evaluated from the ratio of the area of the 1657 cm− 1 component to the sum of the areas of all amide I components. Various algorithms have been used to estimate the propensity of the repetitive sequence regions of spider silk proteins as ordered or disordered. To obtain reliable results, we have used several algorithms based on different disorder criteria, 59 including Disprot†, 60 FoldIndex©‡, 61 GlobPlot 2.3§, 62 NORSp‖, 6 3 and PONDR® VL-XT¶.64,65 Some algorithms rely on amino acid properties (hydrophobicity and charge), whereas others rely on data sets of known disordered proteins. The default parameters of all predictors have been used to perform the calculation. To complete the results provided by disorder predictive algorithms, we have similarly predicted the secondary structure of silk proteins using

† http://www.ist.temple.edu/disprot/Predictors.html ‡ http://bip.weizmann.ac.il/fldbin/findex § http://globplot.embl.de/ ‖ http://cubic.bioc.columbia.edu/services/NORSp/ ¶ http://www.pondr.com/

251

Molecular Transformations in Spider Silk Formation various algorithms, including Portera,66 NNPREDICTb,67 HNNc,68 SSpro8d,69 and JUFOe.70 The algorithms have been applied to all silk proteins of the spider N. clavipes, whose sequence is known (MaSp1, MaSp2, MiSp1, tubuliform spidroin 1, and the Flag protein). The spidroin MiSp2 has been discarded because the sequence given in the literature is too fragmentary. Moreover, since the sequence of the Ac protein for N. clavipes is unknown, we have used the sequence determined for the spider A. trifasciata. As a matter of fact, we expect greater similarity between the homologous proteins of the two orb-weaving spiders N. clavipes (Tetragnathidae) and A. trifasciata (Araneidae) than between the different silks of a given species. The sequence of a Pir protein has been determined very recently for the black widow spider L. hesperus (Theridiidae).48 Discrepancies with the amino acid compositions of the gland content suggested that another protein component should be present48 (not considered here).

4. 5. 6.

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8. 9.

Acknowledgements Funding for the Raman spectrometer was obtained through a grant from the Canadian Foundation for Innovation. This work was also supported by the Natural Sciences and Engineering Research Council of Canada and by the Fonds Québécois de Recherche sur la Nature et les Technologies. The authors express their gratitude to Jean-François Rioux-Dubé for his valuable technical support and to Marie-Eve Rousseau for the recording of the polarized spectra of the Pir fiber.

Supplementary Data

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