Conformation and Orientation of Proteins in Various Types of Silk

Biomacromolecules 2009, 10, 2945–2953

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Conformation and Orientation of Proteins in Various Types of Silk Fibers Produced by Nephila clavipes Spiders Marie-Eve Rousseau, Thierry Lefe`vre, and Michel Pe´zolet* Centre de recherche sur les mate´riaux avance´s, De´partement de chimie, Universite´ Laval, Pavillon Alexandre-Vachon, 1045 avenue de la me´decine, Que´bec (Que´bec) G1 V 0A6, Canada Received July 13, 2009; Revised Manuscript Received August 25, 2009

Silk fibers harvested from the web, cocoon, and prey wrapping of the spider Nephila claVipes have been studied by polarized Raman spectromicroscopy. The technique is efficient to differentiate the various types of silk by probing monofilaments produced by the major ampullate (MA), minor ampullate (MI), cylindriform, flagelliform, and aciniform glands. The spectra show that the MA, MI, and cylindriform silks belong to the same structural class and are composed of highly oriented β-sheets (35-37%) with other slightly oriented secondary structures. Spectral markers of particular motifs involved in the β-sheets have been identified. The flagelliform silk represents a second, very peculiar structural class. It displays a heterogeneous disordered conformation without any preferential orientation. Such characteristics certainly play a role in the large extensibility of this silk. The aciniform silk represents a third class of silk dominated by moderately oriented β-sheets (∼30%) and R-helices (∼24%). Such a structure seems important in explaining the high toughness of this silk.

Introduction The silks produced by orb-weaving spiders display various mechanical properties to fulfill diverse functions for reproduction, food, and locomotion. They constitute a very interesting ensemble to investigate the structure-function relationships in protein-based materials. Spider silk fibers are produced by a set of abdominal sericigene glands, the principal ones being the major ampullate (MA), minor ampullate (MI), flagelliform (Flag), cylindriform (Cyl; also named tubuliform), and aciniform (Ac). The different types of silk are commonly named after their respective secreting gland. The MA glands produce fibers of various diameters that are used as lifeline, radii, and frame of the web, as well as mooring thread. The biological function of the MI silk is unclear. It is often associated with MA fibers, especially in the radii, and it has been proposed to act as a reinforcement to the dragline or to form a temporary spiral during the web’s construction.1,2 Recently, this silk has been identified in the wrapping net spun around preys.3 The Flag silk makes the core spirals of the web in which flying animals are caught, and is often called capture spiral or viscid silk. Finally, the Cyl silk forms the cocoon that protects the eggs, while the Ac silk is involved in the wrapping of preys. The MA silk is commonly recognized as a remarkable material due to an exceptional combination of strength and extensibility, which in turn provides a high toughness. The mechanical properties of the MI silk are close to those of the MA silk, although its maximum strength and strain at rupture are smaller and larger, respectively.4 Like MA and MI silks, the cocoon silk shows a behavior typical of a viscoelastic polymer. It has the highest Young modulus of all spider silk fibers but exhibits a low stiffness after the yield region, resulting in a low global toughness.4,5 It has been pointed out recently that Ac silk is almost twice as tough as the MA silk, even though the latter is stronger.4,6 There is thus a strong interest in understanding the origin of such striking mechanical resistance. * To whom correspondence should be addressed. Phone: 418-656-2481. Fax: 418-656-7916. E-mail: [email protected].

The capture spiral is recognized to be a highly extensible protein elastomer.7,8 In comparison to the other spider silks, it has a strain at rupture an order of magnitude higher, a property well adapted to absorb the kinetic energy of a flying prey that hits the web.7 Among the seven types of silk of orb-weaving spiders, the dragline fiber has been by far the most studied. This interest is due to its mechanical properties and to its availability, as the silk produced by the MA gland can easily be obtained in large amounts by forced silking. This silk is made of two proteins, MaSp1 and MaSp2 (which stands for Major ampullate Spidroin 1 and 2), whose sequences have been determined, in particular for the spider Nephila claVipes (N. claVipes).9,10 These proteins are rich in alanine and glycine residues. They share the same sequence repeat pattern, with a hard sequence composed of polyalanine runs (A)p (p ) 2-10) and a soft sequence rich in glycine residues. Due to this block copolymer structure, the MA proteins form a semicrystralline biomaterial made of amorphous flexible chains reinforced by small stiff crystallites.11,12 The crystalline regions, formed by the alanine stretches arranged in antiparallel β-sheets,13-15 are responsible for the high tensile strength of this silk. The protein motifs in which glycine residues are abundant are proposed to form the amorphous domains that provide extensibility to the thread. In the past 10 years, some sequences of spidroins found in other silks have been determined. The MI silk of N. claVipes is also composed of two spidroins, MiSp1 and MiSp2.16 The repetitive sequence of MiSp1 contains GGXGGY blocks (X ) Q or A) that alternate with (GA)n(A)p motifs, where n ) 3-6 and p ) 2-5. The repeat unit of MiSp2 is made of (GGX)m modules (X ) Y, Q, or A; m ) 1-3) separated by GAGA blocks. Unlike the MaSps, both MI spidroins contain nonrepetitive spacer regions composed of 137 amino acids separated by 10 repeat units. The sequence of the Flag silk has been determined for N. claVipes.17 It is dominated by the GPGGX pentapeptide, where X ) A, S, Y, or V. It also contains a tripeptide motif (GGX) and a 28 amino acid spacer. The main protein component of the cocoon silk of different species has

10.1021/bm9007919 CCC: $40.75  2009 American Chemical Society Published on Web 09/28/2009

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been revealed recently.18-23 Although their sequence is made of repetitive units (about 200 amino acids long), they are not subdivided into smaller modules like the MA, MI, and Flag spidroins. Nevertheless, a few motifs that sparsely appear in the sequence have been noted, such as Sn, (SA)n, and GX (where X ) Q, N, I, L, A, V, F, and D).21,22 The sequence of the Ac silk has also been determined for Latrodectus hesperus6 and Argiope trifasciata.24 Like the tubuliform spidroin 1 (TuSp1), AcSp1 has a long, highly homogeneous repetitive sequence without any of the small modules found in other silks. Little is known about the secondary structures adopted by these proteins within the silk materials. X-ray diffraction and NMR spectroscopy have shown that the MI silk contains β-sheets, as MA silk.25,26 Half of the alanine residues would be involved in the β-sheet, with the remaining residing in nonβ-sheet structures.27 The Cyl silk was also shown to be composed of β-sheets, although with a different spacing so that larger side-chain residues can be accommodated.25 Based on NMR spectroscopy, approximately 70-80% of the Ala residues of the Cyl spidroins would be involved in β-sheets, while the remaining could adopt coiled structures.21 Flag silk has been suggested to be composed of successive type II β-turns that would form the so-called β-spirals.17 However, a recent NMR analysis has shown that the (GPGGA)6G Flag model peptide adopts a statistical distribution of torsion angles and not a unique conformation.28 To our knowledge, no data is available regarding the structure of the Ac silk. Raman spectromicroscopy is among the rare techniques that allow the study of single native silk filaments of only a few millimeters in length. Natural silk specimens can be directly harvested from the spider’s webs, cocoons or wrapped preys, and readily analyzed. In this work, we have characterized the molecular conformation and orientation of various natural silk fibers of the spider N. claVipes using polarized Raman spectromicroscopy.

Experimental Section Samples. Silk filaments were produced by mature N. claVipes females obtained from Florida and raised in the laboratory in 20 × 50 × 60 cm cages at 58 ( 5% relative humidity (RH) and 24 ( 2 °C. The spiders were fed four times a week with small crickets and with 3 drops of 10% w/v glucose solution per week. Most MA samples were collected directly from the web. The mooring threads correspond to the large diameter fibers that ensure the attachment of the web to the cages. The frame threads constitute the outer periphery of the web. The radii connect the frame to the hub of the web. MA samples were also collected by allowing the spiders to walk freely horizontally onto a glass microscope slide. Finally, all these fibers were compared with threads obtained by forced silking at a reeling speed of 0.5 cm · s-1. The silk extruded from the MI gland was obtained together with the dragline (horizontal displacement or forced silking) and identified on the auxiliary spirals of the web. The latter was collected from the web and directly stuck to a glass microscope slide without deformation. The glue that embeds the spiral threads and that is responsible for its stickiness appears as visible globules along the thread. These globules were directly probed with the laser beam. The spirals were subsequently washed with deionized water (4-5 consecutive washes) followed by air-drying.29 Cyl fibers were obtained from freshly made cocoons. Wrapping silk was taken from the tangle made by the spiders around crickets. Two sets of fibers of each type were obtained from five spiders. The silk samples were studied within 1 month of collection and were kept at 60% RH by keeping the samples in a sealed chamber containing a saturated NaBr solution. Methods. Raman spectra were recorded at 22 ( 0.5 °C and 60 ( 5% RH using a LABRAM 800HR Raman spectrometer (Horiba Jobin

Rousseau et al. Yvon, Villeneuve d’Ascq, France) coupled to an Olympus BX30 fixed stage microscope. Silk filaments were studied using the 632.8 nm line of a He-Ne laser (Melles Griot, Carlsbad, CA) as the excitation light source, except the capture spiral and the wrapping silk that were studied using the 514.5 nm line of an Ar+ laser (Coherent, INNOVA 70C Series Ion Laser, Santa Clara, CA). The beam was focused with a 100× objective, generating an intensity of approximately 5-10 mW at the sample. The confocal hole and the entrance slit of the monochromator were optimized for each type of silk to obtain the best signal-to-noise ratio. The diameter of the confocal hole was adjusted between 200 and 400 µm, while the entrance slit was fixed at 200 or 100 µm. Data were collected by an one-inch open electrode Peltier-cooled CCD detector. To obtain information about molecular orientation, four polarized spectra, labeled XX, XZ, ZX, and ZZ, were recorded.30 The first and second letters indicate the polarization of the incident and scattered radiation, respectively, where Z corresponds to the fiber axis and X is the perpendicular direction. In the following, it is assumed that the Raman tensor associated to the amide I mode, that is mainly due to the carbonyl stretching vibration, is cylindrical and aligned along the CdO group. A half-wave plate (Melles Griot) was used to rotate the polarization of the incident laser beam. A polarizer was placed before the entrance slit of the monochromator to allow the detection of the polarized scattered light. A broadband quarter-wave plate was placed after the polarizer to eliminate the polarization dependence of the grating. The samples were irradiated for a period varying between 500 s to 15 min, depending on the type of sample, to stabilize the signal and quench the fluorescence prior to the spectral acquisition. Average values of the polarized spectra were obtained over at least two samples for each type of silk for different integration periods varying from 60 to 180 s, depending on the samples. No degradation occurred under the chosen experimental conditions. The spectra were first corrected to account for the polarization dependence of the instrument based on the intensity of the polarized spectra of liquid chloroform as an isotropic sample. Spectra were then corrected for the fluorescence background over the 400-1800 cm-1 spectral region by subtracting a polynomial baseline. They were smoothed following the Savitzky-Golay method with seven or nine points, except the Flag and the wrapping silk. For these small diameter fibers, the spectra presented are the average of 12 and 14 measurements, respectively. Wavenumber shifts due to experimental conditions of the spectrometer were corrected using the Tyr band at 1615 cm-1. To obtain semiquantitative information relative to the level of orientation, the parameter R ) 1 - IXX/IZZ31 was calculated using the peak-height intensity in the XX and ZZ polarization. Following a method described in detail elsewhere,30 the order parameters P2 and P4 were also calculated assuming a cylindrical Raman tensor and a uniaxial symmetry of the fiber. To adequately characterize the protein conformation, the four raw (not smoothed) average polarized spectra were used to calculate the so-called orientation-insensitive spectrum.32 To estimate the content and order parameters of the different secondary structures, a spectral decomposition of the amide I band was carried out for the different silks, except the Flag silk for which not enough information relative to its structure is available and that appears to have a very specific conformation. A set of components that has previously been shown to successfully decompose the amide I band of MA spider and silkworm cocoon silks was used.33 The curve-fitting procedure makes use of five band components located near 1639 (disordered structures), 1655 (R- or 31-helix), 1669 (β-sheets), and 1682/ 1695 (turns) cm-1. A sixth weak component near 1710 cm-1 was added in some cases because it was necessary to satisfactorily fit the experimental spectra. This component accounts for 3% or less and is most probably associated to carbonyl side chains from glutamine and asparagine.ThebandsusedforthedecompositionhadaGaussian-Lorentzian shape. The initial values of the band intensities were fixed manually based on the best curve-fitting of each experimental spectrum. The variation of the initial band position was constrained within (1 cm-1

Proteins in Silk Fibers of Nephila clavipes Spiders


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observed at 1400 cm-1 in the spectrum of cocoon silk of the silkworm Bombyx mori (B. mori) and was assigned to the combination mode of CH2 wagging, HR bending, and CR-C stretching vibrations from normal mode calculation performed on the synthetic polypeptide poly alanylglycine.37,38 In the case of poly-L-alanine, the corresponding mode is predicted to be located at 1399 cm-1,38 in good agreement with the value found for the MA silk (1397 cm-1, see Figure 1). Similarly, the band at 1368 cm-1 has also been observed for B. mori and was assigned to the combination of CH3 symmetric bending, HR bending, NH in-plane bending, and CH2 wagging modes.37 It is predicted to arise at the same position in β-poly-L-alanine,38 in good agreement with the present Raman data.

Figure 1. XX- and ZZ-polarized spectra of the MA (frame of the web), MI, and Cyl fibers. The spectra of each type of silk are normalized relative to the peak height of the amide I bands in the XX-polarization.

during the curve-fitting calculation. The initial values of the bandwidths of the amide I components was set at 15 cm-1 and were allowed to vary during the calculation without exceeding 22 cm-1. However, none of the bands reached the limit except the component at 1639 cm-1. The area of each component divided by the sum of the total area of the amide I was used to determine the secondary structures content, assuming that the Raman scattering cross section is the same for all structures. The intensity of each component of the fit was used to calculate the order parameters of each structure using the depolarization ratios of the different components given in a previous work.33 For the R-helix component of the Ac silk, a depolarization ratio of 0.17 was used as it was determined from an isotropic film of dried fibroin of the wild silkworm Samia cynthia ricini (S. c. ricini), as explained elsewhere.34 As a matter of fact, this type of fibroin is recognized to be composed of a majority of polypeptide chains that adopt an R-helix conformation.34 All spectral manipulations were performed using GRAMS/AI 8.0 (ThermoGalactic, Salem, NH).

Results and Discussion Different Silks Produced by the MA Gland. The MA glands produce fibers for different functions. These include the lifeline, used to escape predators in case of a free fall, the radii and frame of the web, and the mooring threads. Considering that each fiber has a specific use and that the spider can control its spinning speed and, thus, vary the thread diameter,35 the fibers spun by the same glands are likely to display some variations in molecular structure and orientation. We have thus studied various fibers secreted by the MA glands and have compared them with the silk obtained by forced silking at a reeling speed of 0.5 cm · s-1. A typical example of the XX- and ZZ- polarized Raman spectra of a MA fiber (frame of the web) is given in Figure 1. The spectra of all the MA fibers investigated are shown in Figure S1. As can be seen, they display the same polarization dependence and have almost identical band positions and band shapes, indicating that the molecular structure is nearly the same. The band assignment of the spectra of such fibers has been detailed elsewhere.30,36,37 The spectra display conformationsensitive bands such as the amide I (1669 cm-1), amide III (1230 cm-1), and “skeletal” (1093 and 1068 cm-1) bands that are all associated with the β-sheet structure. The polarized mode at 1393 cm-1 is also due to the β-sheet conformation. It has been

Several bands characteristic of amino acid side chains are scattered over the investigated spectral range (780-1750 cm-1). The band near 1450 cm-1 arises from CH2/CH3 bending modes, while those at 1615 and 1210 and the doublet at 820-830 cm-1 are due to the Tyr residues. The band at 1003 cm-1 is characteristic of the Phe residues, while the polyalanine motifs give rise to a band at 903 cm-1. The fact that the intensity of the latter band is polarization-dependent indicates that the polyalanine blocks have a preferential orientation,30 which is consistent with the fact that they are involved in the β-sheets. The pattern of all these bands is identical for all the collected MA threads (Figure S1), which ascertains that the amino acid composition is the same, thus ensuring that they are all secreted by the same gland. Such observations demonstrate the capability of Raman spectromicroscopy to efficiently assess the nature of a single silk fiber specimen. The variations of the intensity of the different bands with polarization and the discussion on their relationship to orientation have been described elsewhere30 and show that the β-sheets are mainly aligned along the fiber axis. In particular, the amide I band is much stronger in the XX-polarization than in the ZZpolarization, showing that the carbonyl groups are preferentially oriented perpendicular to the fiber axis. The orientational parameter R ) 1 - IXX/IZZ provides a semiquantitative characterization of the level of molecular orientation. It is equal to zero for an isotropic sample, negative for a Raman tensor preferentially aligned perpendicularly to the fiber axis, and positive when the tensor is mainly aligned along the fiber axis. The R values obtained from the intensities of the amide I band of the MA fibers are given in Table 1. R is negative, showing that the proteins chains are preferentially parallel to the fiber axis for all the fibers, but a large variability is observed, even for various specimens of the same type of thread (i.e., that have the same function). The overall range of values is between -0.9 and -2.0. This variability is far greater than the experimental uncertainty (below 10% of error) and reflects the biological nature of the samples. This conclusion is supported by the fact that values obtained for forcibly spun MA silk are much more precise ((0.03) than for harvested samples. This biological variability may actually be accounted for interindividual and intraindividual variations arising from particular genetic and physiological characteristics or from the spinning production process. The spinning speed, in particular, has a strong impact and may vary as a result of temperature changes or because the spiders have to adapt their displacements to the topography and obstacles present in their habitat.35,39-41 The interindividual and intraindividual variability of the tensile properties of the dragline thread is well documented.35,39-41 For example, a large disparity has been observed for the MA fibers

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Table 1. Order Parameter R Calculated from the Amide I Band of the Raw Spectra and Average Spectra for Each Type of MA Fiber

a

order parameter R ) 1 - (IXX/IZZ)

type of fiber

approximate diameterb (µm)

number of threads

sample 1

sample 2

avg spectrum

mooring thread frame radius horizontal displacementa forced silking (0.5 cm/s)

5 5 4 5 4-5

2 1 1 or 2 2 1 or 2

-1.85 -1.55 -1.82 -0.95 -1.57

-1.19 -0.91 -1.55 -2.01 -1.52

-1.45 -1.20 -1.74 -1.34 -1.50

The spider was allowed horizontal free displacement on a glass slide.

Figure 2. Orientation-insensitive spectra of the MA (frame of the web), MI, and Cyl fibers in the amide I (A) and amide III-skeletal (B) regions. The spectra of each type of silk are normalized relative to the amide I bands.

spun during a horizontal crawling40 and large differences have been observed daily between dragline fibers of the same individual.41 To investigate the protein conformation without interferences from spectral effects due to orientation, the orientation-insensitive spectra of the MA fibers have been calculated in the amide I region. A typical orientation-insensitive spectrum of a MA fiber, the frame of the web, is shown in Figure 2, while those of all MA fibers investigated are compared in Figure S2. These spectra are virtually identical. The variability has the same magnitude as the experimental error, suggesting that these fibers have identical secondary structures. To obtain more information on the protein conformation, a spectral decomposition of the amide I band of a typical MA fiber has been carried out (Figure S3) following a spectral pattern used in a previous analysis where spider dragline and silkworm cocoon silks were compared.33 In this model, the amide I band is decomposed with a set of five components, one at 1669 cm-1 due to the β-sheets, the others, representative of the amorphous phase, are located near 1639 (disordered structures), 1655 (31-helices), and 1682/ 1695 cm-1 (turns). This spectral decomposition was also applied to the polarized spectra. The complete secondary structure content and the order parameters are presented in Table 2. For

b

The average diameter is reported for each single filament.

the benefit of the reader, we recall that the theoretical limit values of P2 of a given vibrational mode are -0.5 and 1.0. If P2 ) -0.5, all the molecules are perfectly aligned, the Raman tensor being perpendicular to the fiber axis, whereas P2 ) 1.0 indicates that all the molecules are perfectly aligned with the Raman tensor parallel to the fiber axis. Finally, if P2 ) 0, the sample is isotropic. The results obtained are in good agreement with earlier results.33 They show that β-sheets represent about 37% of the secondary structure and that they are highly oriented (P2 of -0.39), and the remaining of the secondary structures are slightly oriented (-0.10 e P2 e 0.11). Minor Ampullate and Cylindriform (Tubuliform) Silks. In addition to the spectra of a typical MA fiber, Figure 1 shows the XX- and ZZ-polarized spectra of the MI and Cyl silks. The similitudes in the position, shape, and polarization dependence of the bands, especially the amide I, amide III, and “skeletal” bands indicate that these fibroins share the same type of structural pattern despite their different primary structures. This pattern is characterized by a significant proportion of highly oriented β-sheets dispersed in an amorphous matrix composed of polypeptide chains that adopt less rigid secondary structures (turns, unordered segments, and 31-helices). Thus, the MA, MI, and Cyl threads can be classified within the same family of silk fibers which also includes the cocoon silk of silkworms such as those produced by B. mori and S. c. ricini. Although these fibers globally share the same molecular structure (orientation and conformation), differences also exist. A first spectral difference is the wavenumber position of the above-discussed β-sheet marker around 1400 cm-1 that is observed at 1397 cm-1 for the MI silk compared to 1393 cm-1 for the MA silk and 1400 cm-1 for B. mori silk. The intermediate position of this band suggests that blocks of both polyalanine and poly alanineglycine are involved in the β-sheets, which is in good agreement with the presence of (GA)n(A)p motifs in MI spidroins.16 This motif has been suggested to be involved in β-sheets16,25 and our Raman data clearly confirm this assumption. In the case of the Cyl silk, the presence of a shoulder at 1368 cm-1 indicates that some polyalanine motifs are involved in the β-sheets. The repeat sequence of TuSp1 is not composed of small motifs like the MA, MI, and Flag spidroins, but it contains very short segments of (A)p (p ) 3).22 Thus, it seems that these short segments are part of the β-structures, as already suggested by Hu et al.21 Another spectral difference concerns the narrow skeletal bands that is found at 1093 and 1068 cm-1 in the MA silk and that are due to the (A)p motifs. In the case of the MI silk, a unique broad and asymmetric band with its maximum at 1093 cm-1 is observed. This spectral feature results from the fact that the (GA)n motif of the MI spidroins gives a single band at 1085 cm-1, as previously observed in B. mori cocoon silk.30 As a result, the contribution of the (GA)n overlaps with the contribution of the (A)p motif, which gives rise to the observed broad asymmetric band. This observation is thus also related to the fact that the β-sheets in this fiber are composed of both (GA)n



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Table 2. Full Width at Half-Height (FWHH) of the Amide I Band of the Orientation-Insensitive Spectra of Each Type of Silk Fiber, and Order Parameters and Secondary Structure Content as Obtained by Spectral Decomposition

type of fiber

a

component

FWHH (cm-1) ((1 cm-1)

MA

33.1

MI

34.2

Cyl

34.8

Ac

44.3

Flag

57.3

1

2

3

4

5

position assignment

1695 turn

1682 turn

1669 β-sheet

1655 helix

1639 unordered

P2 ((0.02) P4 ((0.02) content ((0.03) P2 ((0.02) P4 ((0.02) content ((0.03) P2 ((0.02) P4 ((0.02) content ((0.03) P2 ((0.02) P4 ((0.02) content ((0.03) P2 P4 content

-0.07 0.03 0.11 -0.10 -0.11 0.12 -0.01 0.05 0.09 0.05 -0.18 0.09 nda nda nda

-0.10 -0.02 0.22 -0.04 0.07 0.18 -0.17 0.04 0.14 0.09 -0.05 0.20 nda nda nda

-0.39 0.25 0.37 -0.22 0.09 0.35 -0.35 0.20 0.37 -0.13 -0.05 0.30 nda nda nda

0.01 -0.17 0.17 0.01 -0.09 0.20 -0.07 -0.02 0.22 0.13 -0. 40 0.24 nda nda nda

0.11 -0.20 0.12 0.05 -0.01 0.15 -0.06 -0.01 0.18 0.10 -0.13 0.18 nda nda nda

Not determined.

and (A)p modules. In the Cyl fiber, the shoulder at 1068 cm-1 also confirms the presence of polyalanine motifs in the β-sheets. Overall, the analysis of these small bands demonstrates the capability of Raman microspectroscopy to identify particular sequence motifs of proteins that are involved in β-sheets. In addition to the “skeletal” bands due to the β-sheets at 1068 and 1093 cm-1, a shoulder at 1105 cm-1 is present for the MI and Cyl silks, a band not observed in the spectrum of the MA silk. Other studies carried out in our laboratory have confirmed that this component is characteristic of the silk proteins in their native state before spinning since it has been observed in the sac of the MA,42 MI, and Cyl glands (data not shown). Consequently, the MI and Cyl proteins seem to contain a small but significant amount of disordered native-like structures after the spinning process, as opposed to the MA proteins. This is consistent with a higher amount of unordered structures of the MI and Cyl fibers (18 and 15%, respectively) compared to the MA silk (12%). Such disordered conformational elements may play a role in the higher extensibility of the MI and Cyl silks with respect to the MA silk. By contrast, it seems that the absence of the 1105 cm-1 component in MA silk suggests that the spinning process in the complex MA gland is particularly optimized to convert all initial native structures into new secondary structure elements. Other bands are specifically representative of the amino acid composition. For example, the intensity of the bands due to the Tyr side chains (1615, 850, and 830 cm-1) indicates that the Tyr content increases in the order MI > MA > Cyl. Moreover, the 830/850 cm-1 intensity ratio of Cyl silk is completely reversed with respect to the intensity ratio observed for the MA and MI silks. Based on the work done on the Raman spectra of Tyr-based compounds by Siamwiza et al.,43 a 830/850 cm-1 intensity ratio inferior to 1 is an indication that Tyr hydroxyl groups are proton donors for hydrogen bonds in Cyl silk, whereas hydroxyl groups are both proton donors and acceptors in MA and MI silks (ratio greater than 1). Thus, Tyr residues would be more buried (i.e., in a more hydrophobic environment) in the Cyl thread.44 The spectra of the Cyl fiber are dominated by a strong band at 1003 cm-1, which is a very sensitive marker of Phe. The small and narrow bands, appearing at 1604, 1585, and 1027 cm-1 in the spectra of the Cyl silk, are also due to Phe. The strong intensity of these bands with respect to that of the MA and MI silks suggests a high content of Phe residues,

in agreement with the amino acid content of Cyl silk and with the sequence of TuSp1.22 The band at 903 cm-1 is weaker for the MI than for the MA silks which may be related to the shorter (A)p motifs of the MI spidroin (4-10 successive Ala residues for MaSps, 2-5 for MiSps).16 As observed for the MA fibers, this band is sensitive to polarization, providing an indication that the (A)p motifs of the MI silk are also oriented, in agreement with the fact that they form β-sheets. The orientation-insensitive spectra of the MA, MI, and Cyl silks are compared in Figure 2 in the 1570-1750 cm-1 (amide I) and 1050-1365 cm-1 (amide III-skeletal) regions. In the amide I region, the spectra are very similar. The amide III band occurs at a wavenumber characteristic of the β-sheets, but the band maximum is found at 1231 cm-1 for the MI silk compared to 1236 cm-1 for the MA and Cyl silks. This difference may be due to the amino acid composition (the amide III region is recognized to be particularly affected by amino acid side-chain modes)45 or to the fact that the (A)p motifs are involved in the β-sheets in the MA silk, while both the (GA)n and the (A)p motifs constitute the β-sheets in the MI silk. As a matter of fact, the amide III band of the orientation-insensitive spectrum of the cocoon silk of B. mori is located at 1229 cm-1. Thus, the position of the amide III band of the MI silk is located between that of the MA and B. mori silks, which in turn may also reflect the presence of both (GA)n and the (A)p motifs in the β-sheets of MI silk. The spectral decomposition of the amide I band of the orientation-insensitive spectrum of the MI and Cyl fiber is shown in Figures S4 and S5, respectively, and the secondary structure content is given in Table 2. This decomposition confirms the similarity of the structure of these silks. In particular, the β-sheet content is very close for the 3 fibers (35-37%), the differences being below the experimental error and the uncertainty due to the curve-fitting procedure. The orientation parameter P2 of the different amide I components (Table 2) show that the β-sheets are more oriented than the other secondary structures, that is, those representative of the amorphous phase. However, the P2 value of the β-sheets of the MI thread is less negative (P2 ) -0.22) than for the MA and Cyl fibers (P2 ) -0.35). This result indicates a significantly lower level of orientation for the MI silk. It has to be mentionned that, as opposed to the present Raman results, X-ray diffraction experiments have show no clear orientation in cocoon silk.25 A possible explanation for this

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Figure 4. Orientation-insensitive spectra of the MA (frame of the web), Flag, and Ac fibers in the amide I region. The spectra of each type of silk are normalized relative to the area of the amide I bands. Figure 3. XX- and ZZ-polarized spectra of the Flag fiber before (upper spectra) and after (bottom spectra) washing with water.

discrepancy may reside in the fact that these authors used a bundle of fibers whereas single filaments were probed in our Raman experiments. In conclusion, the presence of some disordered conformations in the MI fiber combined with a lower level of orientation may contribute to the difference in the mechanical properties of the MI silk compared with the MA silk,4 especially the larger extensibility of the former silk. In the case of the Cyl silk, the Raman data show that, apart from some residual native conformational elements, this silk has a very similar molecular structure than the MA silk, having both almost identical β-sheet contents and levels of orientation. The differences found at a molecular level by Raman spectroscopy appear too small to explain the difference in the tensile properties of these silks, especially the low strength at rupture.4 Specific micro- or macrostructural organizations may be part of the explanation and should require additional investigation using complementary characterization techniques. Flagelliform Silk (Viscid Silk or Capture Spirals). The capture spirals of orb-weaving spiders are composed of a core thread coated with a glue that appears as droplets dispersed along the fiber. Figure 3 shows the XX- and ZZ-polarized spectra of the capture spiral before and after washing with water. Prior to washing, the spectra are dominated by a very intense line at 1045 cm-1. The fact that it disappears after the silk filament is washed with water unambiguously associates this component to the glue coating. Among the different compounds found in this aqueous coating, GB amide (4-aminobutyramide) and isethionic acid (2-hydroxyethanesulfonic acid) are the most abundant.46,47 The strong band at 1045 cm-1 probably arises from the symmetric SO3 stretching mode48,49 of isethionic acid. As seen in Figure 3, the amide I and amide III bands of the washed capture spiral are much broader that than those of the fibers discussed so far showing that the protein conformation is mostly disordered. In addition, the XX- and ZZ-polarized spectra are equal in intensity in the amide I region and over the entire spectral range, indicating that the proteins have no preferential (or a very low) molecular orientation. This characteristic is unique among spider silk and is likely to explain at least in part the peculiar mechanical properties of the Flag fiber. As for the other silks studied, the Raman spectra allow the unambiguous identification of the capture spiral when consider-

ing the signature bands associated to the amino acid side-chains. The particularly visible bands at 1044, 920, and 875 cm-1 are due to the high content in Pro residues.17 Also, the band at 1414 cm-1, assigned to the CH2 wagging mode,38 reflects the presence of a high amount of Gly, in agreement with the known protein sequence (54% of amino acids are Gly).17 The orientation-insensitive spectrum of the capture spiral is compared with that of the MA fiber in Figure 4 (the spectrum of the Ac fiber is also shown and is discussed in the next section). As seen for the spectra of the MA, MI, and Cyl silk shown in Figure 2, the orientation-insensitive spectrum of the Flag silk displays an amide I band centered at 1667 cm-1, a position compatible with β-sheets. However, it is very unlikely that this structure is present in Flag silk. First, the amide I band is broad (Table 2), which is typical of the presence of various secondary structures. Second, similar positions of the amide I band have often been found in the Raman spectra of disordered proteins such as R-casein and poly-L-lysine in its ionized form.50 In fact, among the different silks investigated, the orientationinsensitive spectrum of Flag silk shows, by far, the broadest amide I band (Table 2), indicating that the protein conformation is very heterogeneous. Third, the amide III band is broad and composed of at least two components at 1248 and 1261 cm-1 that do not correspond to β-sheets (Figure 3). Amide III bands near 1248 cm-1 have often been observed for unordered proteins,45,50 whereas the maximum of the amide III band of the MA silk before spinning is located at 1262 cm-1.51 Thus, these two amide III components likely originate from disordered (random) conformations and 31-helices. Finally, no theoretical model has proposed β-sheets in the Flag silk, especially because of its very low tensile strength and extraordinary extensibility, and also because of its high content in Pro residues, which prevent the formation of β-sheets. This complex multicomponent conformation of Flag silk revealed by Raman spectromicroscopy is in line with the conclusions obtained with a model peptide studied by NMR by Asakura and colleagues.28 This unordered conformation has to be related to the high Gly content of the Flag protein because this particular amino acid is acknowledged to provide flexibility in proteins.52 It is noteworthy that the amide I band of the Flag silk shares strong similarities with that of elastin.45,53 This biomaterial is very elastic, has a high resilience, and a high strain at rupture. The Flag silk has comparable mechanical


Figure 5. XX- and ZZ-polarized spectra of three different fibers identified in wrapping silk: (A) MA, (B) MI, and (C) Ac silk. The spectra of each type of silk are normalized relative to the peak height of the amide I bands in the XX-polarization.

properties, but it is stronger.54 The present results emphasize the structural similitudes of both materials. In conclusion, Flag silk has no (or a very low) molecular orientation that is associated with a heterogeneous and disordered conformation. Both characteristics are critical to explain the origin of the molecular reorganization and the related large extensibility of the Flag silk, because they provide mobility to the polypeptide chains to enable them to change their conformation and their alignment under the application of an external load. It has been shown that the viscid coating glue plasticizes the core thread of the spirals and that elasticity of this fiber arises from a water-induced molecular mobility.55,56 However, the molecular mobility of viscid silk due to the water coating should clearly be associated with molecular characteristics such as isotropy and unordered conformation. Aciniform Silk. Wrapping nets spun around crickets by the N. claVipes spiders were investigated. Three types of silk, each having a specific diameter and a distinctive Raman spectrum, were identified and their polarized spectra are shown in Figure 5. Based on the spectral signatures of the larger and intermediate diameter fibers over the spectral range investigated, there is no doubt that their spectra (labeled A and B) correspond to the MA and MI silks, respectively (compared with Figure 1). This is a typical example that demonstrates the efficiency of Raman spectromicroscopy to simply and rapidly identify the nature of silk filaments. The third silk has a smaller diameter and is present in large amounts in the nets wrapping the preys. Both characteristics suggest that this silk comes from the Ac gland. This conclusion is strongly supported by the fact that the general shape of the spectra of the content of the Ac gland (not shown) and those of the Ac fiber are quite similar. More particularly, the shape and intensity of the bands due to amino acid side chains, especially Tyr and Phe, are totally equivalent and are different from those of the other types of silk investigated. The presence of three fibers in wrapping nets spun by N. claVipes is in good agreement with Hayashi and colleagues who have also distinguished three types of thread in the wrapping silk of Latrodectus hesperus.3 Two of them were shown to be mainly composed of the MI and Ac spidroins, while the assignment to MA of the larger thread was based on the fiber’s diameter.3 The latter assignment is thus consistent with the present Raman data. Based on their


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observations, Hayashi and colleagues proposed that the function of the Ac silk is to hold larger threads together in cocoons and wrapping nets.24 The Ac silk exhibits a particular spectral pattern, from both the conformation and amino acid composition point of view. Based on the shape of the amide I band of the XX spectrum and its maximum at 1669 cm-1, Ac silk clearly contains β-sheets. This band being more intense in the XX spectrum than in the ZZ one, the β-sheets are preferentially aligned parallel to the fiber. The amide III maximum of the ZZ spectrum at 1230 cm-1 and the polarized band at 1403 cm-1 are also consistent with this conclusion. However, the ZZ spectrum reveals in the amide I region the presence of another strong component located at 1657 cm-1, which is a wavenumber typical of R-helices.50,57,58 The higher intensity of this component in the ZZ spectrum than in the XX one indicates that the carbonyl groups of these polypeptide chains are preferentially aligned along the fiber axis. Because the carbonyl groups of R-helices are aligned along the helix axis, it appears that these R-helices are mainly oriented parallel to the fiber. Thus, the Ac silk would be mainly composed of β-sheets and R-helices both preferentially parallel to the fiber axis. The presence of the small band at 525 cm-1 strongly supports the presence of R-helices because this component has been observed in polypeptides or proteins rich in polyalanine sequences that adopt an R-helix conformation, especially the cocoon silk of Samia cynthia ricini.34 Its small intensity is consistent with the fact that the known aciniform spidroins contain only a small proportion of short length polyalanine segments.6,24 The orientation-insensitive spectrum in the amide I region and its spectral decomposition are shown in Figure 4 and Figure S6, respectively. These figures clearly show the predominance of the β-sheets and R-helices. The other amide I components indicate that the Ac silk contains additional less-ordered structures (turns, random chains). The orientation-insensitive spectrum is broader than its MA counterpart (Table 2), which reflects the more heterogeneous conformation of Ac silk. It is nevertheless narrower than the amide I band of Flag silk. The spectral decomposition (Table 2 and Figure S6) reveals that 30% of the peptide bonds adopt β-sheets and 24% adopt R-helices that account together for more than half of the total secondary structures. The order parameters calculated for the different components of the amide I band show a negative value of P2 for the β-sheets and a positive one for the R-helix, which indicates that these secondary structures are indeed preferentially aligned along the fiber axis. Because the magnitude of the P2 value of the β-sheet (P2 ) -0.13) and R-helix (P2 ) 0.13) structures is closer to zero than for the corresponding value found for the β-sheets of the MA and Cyl silks (typically, P2 ) -0.37), the global level of orientation of Ac silk appears to be moderate. Additional information can be obtained from the Raman spectra of the Ac fiber (Figure 5). No (A)p or (GA)n motifs are detected, although the band at 1403 cm-1 supports the presence of oriented β-sheets. From the intensity of the band at 1003 cm-1, one can estimate that the Phe content in the Ac silk is higher than in the MA and the MI silks but smaller than in the Cyl silk. Two small bands at 1048 and 922 cm-1 indicate the presence of Pro residues, which seem to be in lower amount than in the viscid silk, but in higher amount than in the MA and MI silks. Two other bands at 1026 and 960 cm-1 are consistent with the presence of Leu. This work constitutes the first experimental evidence that R-helices are found in spider silk fibers, although this secondary

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Innovation. This work was also supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Fonds Que´be´cois de Recherche sur la Nature et les Technologies (FQRNT). The authors express their gratitude to Jean-Fanc¸ois Rioux-Dube´ for his valuable technical support. Supporting Information Available. XX- and ZZ-polarized spectra of all the MA fibers in the 800-1750 cm-1 region, and spectral decomposition of the amide I band of the orientationinsensitive spectra of the MA, MI, Cyl, and Ac silks. This material is available free of charge via the Internet at http:// pubs.acs.org.

References and Notes Figure 6. Schematic representation of the three classes of structure found in the silks spun by spiders. Model A corresponds to the MA, MI, and Cyl silk fibers. Red structural motifs represent the β-sheets, while green lines represent the amorphous phase (unordered segments, 31-helices and turns). Model B corresponds to the aciniform silk. Dark blue helices represent the R-helices. Model C corresponds to the flagelliform silk. The violet lines represent the unordered and heterogeneous conformation of this silk.

structure has been observed in the silk fiber of a few insects59 and in the MA gland.51,60 It remains to be clarified whether these R-helices are formed during the spinning process or whether they already exist in the Ac gland. It may be hypothesized that the β-sheets of Ac are formed during the spinning process, as observed for the MA silk. The presence of R-helices in Ac silk is likely to have a strong impact on the fiber’s mechanical properties, particularly on its large toughness which results from a high extensibility and a reasonable tensile strength.4 As a matter of fact, the deformation and extension of R-helices under a longitudinal draw may contribute or facilitate the elongation of the fiber. The moderate degree of molecular alignment probably also contributes to the extensibility of Ac silk. On the other hand, the β-sheets, although present in a lower amount than in the MA, the MI, and the Cyl silks, certainly play a similar role with regard to the tensile strength.

Conclusions The above results show that Raman spectromicroscopy is a very efficient technique for determining quantitative parameters for the characterization of the secondary structure and the molecular orientation of single silk filaments. It also provides a rapid and straightforward way to identify the different types of silk fibers. Based on their molecular structure, the five spider silk fibers investigated can be divided into three groups, as represented schematically in Figure 6. The first group is composed of the fibers coming from the MA, MI, and Cyl glands and also includes the cocoon silk of silkworms. These fibers are characterized by a large amount of oriented β-sheets, but they also contain disordered structures, 31-helices, and β-turns. These silk fibers are totally devoid of R-helices. The second and third groups are represented by the Ac and the Flag silks. The Ac silk exhibits a particular structure made of R-helices and β-sheets that are moderately aligned parallel to the fiber axis, while the Flag silk displays little or no molecular orientation with a conformation that is heterogeneous and mostly disordered. Acknowledgment. Funding for the Raman spectrometer was obtained through a grant from the Canadian Foundation for

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Conformation and Orientation of Proteins in Various Types of Silk

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