Study of Protein Conformation and Orientation in Silkworm and Spider

Biomacromolecules 2004, 5, 2247-2257

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Study of Protein Conformation and Orientation in Silkworm and Spider Silk Fibers Using Raman Microspectroscopy Marie-Eve Rousseau,† Thierry Lefe` vre,† Lilyane Beaulieu,† Tetsuo Asakura,‡ and Michel Pe´ zolet*,† De´ partement de Chimie, CERSIM, CREFSIP, Universite´ Laval, Pavillon Alexandre-Vachon, Que´ bec (Que´ bec) Canada G1K 7P4, and Department of Biotechnology, Tokyo University of Agriculture and Technology, Japan Received May 11, 2004; Revised Manuscript Received July 27, 2004

Raman microspectroscopy has been used for the first time to determine quantitatively the orientation of the β-sheets in silk monofilaments from Bombyx mori and Samia cynthia ricini silkworms, and from the spider Nephila edulis. It is shown that, for systems with uniaxial symmetry such as silk, it is possible to determine the order parameters 〈P2〉 and 〈P4〉 of the orientation distribution function from intensity ratios of polarized Raman spectra. The equations allowing the calculation of 〈P2〉 and 〈P4〉 using polarized Raman microspectroscopy for a vibration with a cylindrical Raman tensor were first derived and then applied to the amide I band that is mostly due to the CdO stretching vibration of the peptide groups. The shape of the Raman tensor for the amide I vibration of the β-sheets was determined from an isotropic film of Bombyx mori silk treated with methanol. For both the Bombyx mori and Samia cynthia ricini fibroin fibers, the values of 〈P2〉 and 〈P4〉 obtained are equal to -0.36 ( 0.03 and 0.19 ( 0.02, respectively, even though the two types of silkworm fibroins strongly differ in their primary sequences. For the Nephila edulis dragline silk, values of 〈P2〉 and 〈P4〉 of -0.32 ( 0.02 and 0.13 ( 0.02 were obtained, respectively. These results clearly indicate that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the β-sheets are oriented parallel to the fiber axis, in agreement with previous X-ray and NMR results. The most probable distribution of orientation was also calculated from the values of 〈P2〉 and 〈P4〉 using the information entropy theory. For the three types of silk, the β-sheets are highly oriented parallel to the fiber axis. The orientation distributions of the β-sheets are nearly Gaussian functions with a width of 32° and 40° for the silkworm fibroins and the spider dragline silk, respectively. In addition to these results, the comparison of the Raman spectra recorded for the different silk samples and the polarization dependence of several bands has allowed to clarify some important band assignments. Introduction The silk filaments produced by orb-weaving spiders and silkworms are among nature’s most highly engineered structural materials, achieving, in some cases, combination of strength and toughness that could not be reproduced by artificial means. Silk exceptional mechanical properties seem to be due to its semicrystalline nature, made of amorphous flexible chains reinforced by small stiff crystallites. According to molecular models of silk organization, the alignment within the fiber of the crystallites that are composed of β-sheets is one of the important factors responsible for the silk tensile strength.1-4 It is thus of fundamental importance to be able to quantify the level of orientation of the β-sheets in natural fibers produced by silkworms and spiders, to establish structure-property relationships in these systems. Such information is not only relevant to improve the artificial spinning techniques but also for the design of new synthetic materials inspired from the organization of natural fibers.5 * To whom correspondence should be addressed. Telephone: (418) 6562481. Fax: (418) 656-7916. E-mail: [email protected]. † Universite ´ Laval. ‡ Tokyo University of Agriculture and Technology.

Silks from different species are composed of proteins of particular primary structures and are expected to exhibit variability of microstructures and properties. One of the most studied silk is that from the domesticated silkworm Bombyx mori (B. mori). It contains about 60% of crystallizable repeating sequences (GAGAGS)n that adopt the β-sheet structure.6,7 Also widely studied is the dragline from the orbweaving spider Nephila edulis (N. edulis). Like in B. mori silk, spider dragline is known to contain well-aligned β-sheets that would be responsible for the high tenacity of the thread. However, the crystallizable regions that are composed of repeated poly(alanine) regions of 5 to 7 residues are quite different from those of B. mori cocoon silk.8,9 Samia cynthia ricini (S.c. ricini) is a wild silkworm that produces an interesting fibroin because its primary sequence consists of repeated poly(alanine) segments of 12 to 13 residues10 followed by glycine-rich domains containing bulky residues, which is reminiscent of primary structure of spider dragline silk. Although it has been shown that R-helices are present in the liquid silk extracted from the silk glands of S.c. ricini,11-14 antiparallel β-sheets are found in the native fibroin as previously observed for spider dragline and B. mori cocoon silks.

10.1021/bm049717v CCC: $27.50 © 2004 American Chemical Society Published on Web 09/03/2004

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Biomacromolecules, Vol. 5, No. 6, 2004

The orientation of proteins in silk has been investigated using different experimental techniques. For samples with uniaxial symmetry, orientation is defined by an orientation distribution function N(θ) that can be expanded in a series of even Legendre polynomials. The first coefficients 〈Pl〉 of the series, the so-called order parameters, can be determined experimentally, but the number of accessible coefficients depends on the technique used. X-ray diffraction is a powerful method to analyze the crystal structure of silk, the size of the crystals, and their distribution of orientation.15-17 All of the coefficients of the Legendre series can be obtained by using this technique.18,19 However, the information is only restricted to the crystalline regions within the sample. Nuclear magnetic resonance (NMR) spectroscopy is also a powerful method for structure determination of semicrystalline biopolymers such as silk, but it requires a large quantity of sample.1,4,20 The order parameters 〈P2〉, 〈P4〉, 〈P6〉, and 〈P8〉 can potentially be determined using this technique. However, it has not yet been done on silk since it is a rather laborious and complex method. Finally, infrared spectroscopy is a simple, fast, and efficient method to characterize the molecular structure and orientation, but it cannot be applied on a single fiber and only the second term 〈P2〉 of the Legendre polynomial series can be determined. Thus, despite their advantages, these techniques are not all well-suited to characterize the molecular structure and orientation of a single monofilament having a diameter of a few micrometers. On the other hand, Raman microspectroscopy is a powerful nondestructive technique to investigate the molecular structure of a unique silk fiber.13,21-28 Since a microscope is used to focus the laser beam on the sample and to collect the scattered light, it allows the in situ recording of high quality spectra of single silk filaments having a diameter of less than 10 µm. In addition, polarization measurements provide information about the orientation of the structural units (secondary structure elements, amino acid side chains) in the samples. The first two order parameters 〈P2〉 and 〈P4〉 can be calculated, but the determination of the orientation is complex from both the experimental and theoretical point of views, so that only a few quantitative measurements have been performed so far on azobenzene derivatives.29,30 In this paper, we have done a detailed analysis of the polarized spectra obtained by Raman microspectroscopy of single filaments of B. mori and S.c. ricini fibroins and N. edulis dragline silk. Regenerated films of B. mori were also studied. The results have first allowed to refine the assignment of several bands in the Raman spectra of silk. Since the equations for calculating the order parameters 〈P2〉 and 〈P4〉 using Raman microspectroscopy for a vibration with a Raman tensor having a cylindrical symmetry were not available, they have been derived and applied to quantify the orientation in silk. Thus, we have been able to determine quantitatively the orientation of the β-sheets in different types of silk from the analysis of the amide I band. The values of 〈P2〉 and 〈P4〉 obtained show that the level of orientation is identical for B. mori and S.c. ricini fibroins and is slightly lower for N. edulis dragline silk. The calculation of the most probable distribution using the information entropy30-32 has

Rousseau et al.

Figure 1. Coordinate system used for the recording of the Raman spectra of a silk fiber.

also revealed that the distribution of orientation of the β-sheets is broader in the dragline silk than in silk fibroins. Theoretical Section Orientation Determination. For a system of uniaxial or fiber type symmetry (i.e., uniaxial orientation of the molecules around the fiber axis), the molecular orientation can be described by an orientation distribution function N(θ) that can be expanded in a series of even Legendre polynomials in cos θ33 even

N(θ) )

( ) 1

∑l l + 2

〈Pl〉 Pl(cos θ)

(1)

where Pl(cos θ) are the Legendre polynomials and 〈Pl〉 are the coefficients of the series, often called order parameters, that are given by 〈Pl〉 )

∫0π sin θ Pl(cos θ)N(θ) dθ

(2)

These order parameters are thus the average values over the distribution of orientation of the Legendre polynomials. By using polarized Raman spectroscopy, only the second, 〈P2〉, and the fourth, 〈P4〉, coefficients of the orientation function can be determined. By replacing P2(cos θ) and P4(cos θ) in eq 1, we have N(θ) )

5 1 1 + P2(3 cos2 θ - 1) + 2 2 9 P (35 cos4 θ - 30 cos2 θ + 3) + ... (3) 8 4

[

]

The general procedure to determine 〈P2〉 and 〈P4〉, hereafter called P2 and P4, from polarized Raman measurements has been developed in details by Bower18 and Jen et al.34 This method has successfully been used to determine the order parameters P2 and P4 in several systems including highly oriented samples of high-density polyethylene.35,36 In the case of Raman microscopy, Turrell has shown that the depolarization of the incident electric field in the focal plane of the objective and the solid angle of collection of the scattered light have to be taken into account in orientation measurements when using high numerical aperture objectives.37,38

Biomacromolecules, Vol. 5, No. 6, 2004 2249

Conformation and Orientation in Silk Fibers

By considering these two factors, the Raman intensity, I, is obtained by integration of the intensity over the volume of scatterers, V, and over the solid angle of light collection, Ω37,38 I)

∫V ∫Ω |E+i 〈R〉Es|2 dV dΩ

(4)

where Ei + is the transposed vector of the incident electric field, Es is the vector of the scattered electric field, and R is the Raman tensor of rank two expressed in the frame fixed in the laboratory (Figure 1). The brackets indicate the average of the element Rij of R over the orientations of the molecules18,34 〈(Rij) 〉 ) 2

∫0 ∫0 ∫- 1 2π

2π

+1

(6)

Izx A〈(Rzx)2〉 + B〈(Rzy)2〉 R1 ) ) Izz A〈(R )2〉 + B〈(R )2〉

(7)

Ixz A〈(Rxz)2〉 + B〈(Rxy)2〉 ) Ixx A〈(R )2〉 + B〈(R )2〉

(8)

zy

xy

The constants A and B come from the integration over Ω in eq 437,38 A) π

∫0

4 1 (cos β + 1) sin β dβ ) π - cos θm - cos3 θm 3 3 (9)

(

2

∫0θ

B ) 2π2

)

2

m

sin 3β dβ ) 2π2

) R3

R

with a ) R1/R3 (11)

1

it can be shown that the 〈Rij〉 averages in eqs 7 and 8 are related to the principal component of the Raman tensors, R1 and R3, as follows: 2 1 3 c - dP2 + bP4 15 21 35

〈(Rxz)2〉 ) 〈(Rzx)2〉 ) 〈(Rzy)2〉 ) b 〈(Rzz)2〉 )

(32 - cos θ

m

1 + cos3 θm 3

)(10)

(12)

(151 + 211 P - 354 P ) 2

4

(13)

1 8 4 c + dP2 + bP4 15 21 35

(14)

(151 - 212 P + 351 P )

(15)

〈(Rxy)2〉 ) b

2

4

where b ) R32 (1 - a)2

(16)

c ) R32 (3 + 4a + 8a2)

(17)

d ) R32 (3 + a - 4a2)

(18)

At this stage, there are 3 unknowns in eqs 7 and 8, namely P2, P4, and a. The parameter a of the Raman tensor for a given vibration can be directly determined from the depolarization ratio, Riso, obtained from eqs 7 and 8 for an isotropic sample (P2 ) P4 ) 0) Riso ) R1 ) R2

xx

θm

R3

〈(Rxx)2〉 )

Among the Cj coefficients, C1 and C2 are 1 and 2 orders of magnitude smaller than C0, respectively. Therefore, only C0 needs to be considered.39 Since this term vanishes when calculating the intensity ratios, one finally obtains29,37-39

zz

) ( ) R

R1

dψ dφ d(cos θ) (Rij) N(θ) (5)

n sin θm ) NA

2

(

R1

R)

2

where (θ, φ, ψ) are the Euler angles relating the laboratory frame (x, y, z) and the molecular frame. The elements Rij in the laboratory frame can be calculated as functions of the elements of the Raman tensor Rm linked to the molecular frame using an Euler angle transformation matrix. In the backscattering configuration, the propagation direction of the incident and scattered radiation is in the y direction (Figure 1), and four polarized spectra can be measured. The Raman intensities Ikl (k, l ) x or z), when the incident electric field is polarized in the k direction and the scattered light is polarized in the l direction, can be calculated using eq 4. According to Turrell,37,38 the integration over V of the square of the incident electric field leads to the appearance of three coefficients Cj (j ) 0, 1, 2) that depend on the effective halfangle θm of the solid angle inside the sample and on the intensity in the focal plane of the objective. The angle θm is related to the numerical aperture of the microscope objective, NA, and the refractive index of the sample, n, by

R2 )

For B , A, eqs 7 and 8 lead to the conventional expressions of R1 and R2, i.e., without using a microscope.34 For wide-aperture objectives, corrections due to the collection solid angle can be non negligible. For a Raman tensor with a cylindrical symmetry

(19)

Then, P2 and P4 can be determined from the values of R1 and R2 measured experimentally. Information Entropy Theory. Since N(θ) is an infinite expansion in Legendre polynomials, the knowledge of P2 and P4 is not sufficient to completely define the distribution function. Information is missing. However, one can estimate N(θ) by using the information entropy theory.30-32,40 With this method, the most probable orientation distribution (in a statistical sense) is assumed to be the “smoothest” one (that does not vary too sharply). This is most likely the case of natural fibers such as silk. Then, the most probable orientation distribution, Nmp(θ), is derived by maximizing the information entropy of the orientation distribution, S[Nmp(θ)]:30-32 S[Nmp(θ)] ) -

∫-+11 N(θ) ln N(θ) d(cos θ)

(20)

One then obtains Nmp(θ) )

exp(λ2P2 + λ4P4)

∫-1+1 exp(λ2P2 + λ4P4) d(cos θ)

(21)

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Rousseau et al.

where λ2 and λ4 are the Lagrange multipliers. The denominator is a normalization factor so that

∫0π Nmp(θ) sin θ dθ ) 1

(22)

Once P2 and P4 are determined experimentally, λ2 and λ4 are calculated numerically using the constraints given by eq 2. Explicitly P2 )

∫-1+1 P2(cos θ)Nmp(θ) d(cos θ)

(23)

P4 )

∫-1+1 P4(cos θ)Nmp(θ) d(cos θ)

(24)

Experimental Section Materials. Cocoon fibers from the silkworms B. mori and S.c. ricini were supplied by the Insects Production Unit of the Canadian Forest Service (Sault-Ste.-Marie, Ontario, Canada) and by Prof. T. Asakura, respectively. B. mori cocoons were emptied, and the raw silk was degummed in boiling water containing sodium bicarbonate (0.05% w/v) for 15 min.41,42 The resulting material was rinsed thoroughly with deionized water and dried under vacuum. To prepare regenerated films, degummed fibers were solubilized with 9 M LiBr at 40 °C for 20 min.7 The salt was then removed by dialyzing the solution against deionized water using cellulose tubing (12 to 14 kDa cutoff). The regenerated films (thickness of about 25 µm) were obtained by casting the dialyzed solution onto polyethylene films and drying for 48 h in dry air. The treated or coagulated film was obtained by immersing the regenerated film in methanol for 30 min followed by drying in air. To remove the sericin coating, S.c. ricini cocoons were degummed three times with a sodium peroxide solution (0.1% w/v) alternated with boiling distilled water.43 Dragline silk samples from the golden orb-weaving spider N. edulis were provided by Prof. Marie E. Herberstein (Department of Biological Sciences, Macquarie University, Australia) and Prof. Frances Separovic (Department of Chemistry, Melbourne University, Australia). Samples came from adult female spiders collected from Queensland in Australia and dragline was reeled directly from fully awake spiders.44 The samples were studied more than one month after collection and were rinsed with acetone prior to their shipment. Upon reception, all silk samples were kept in a dry environment at 4 °C away from sunlight in order to avoid silk biodegradation. Methods. For polarized Raman measurements, monofilaments of cocoon silks and spider dragline silk, having less than 20 µm in diameter, were gently mounted on glass microscope slides with double-sided tape and aligned toward the z axis, as illustrated in Figure 1. The same procedure was used for the films. Spectra were recorded under controlled conditions of temperature (22.0 ( 0.5 °C) and relative humidity (20 ( 5% RH) in the backscattering configuration using a LabRam 800HR Raman spectrometer (Jobin Yvon Horiba, Villeneuve d′Ascq, France) coupled to a Olympus BX 30 fixed stage microscope. The B. mori samples were studied using the 514.5 nm line of an argon

ion laser (Spectra-Physics, model 2020, Mountain View, CA). The 632.8 nm line of a He-Ne laser (Melles Griot, Carlsbad, CA) was used for all other samples. The use of the red laser was particularly important for the spider silk samples to avoid high fluorescence background generated by the golden pigments present in dragline silk. In all cases, the laser beam was focused down with a 100× objective (0.9 NA-Olympus) to a diameter of approximately 1 µm, generating an intensity of 5 mW (green line) and 6.5-7.5 mW (red line) at the sample. The confocal hole and the entrance slit of the monochromator were fixed at 200 and 100 µm, respectively. By using a 600 lines/mm holographic grating, spectral windows of approximately 2000 and 1000 cm-1 with the green and red laser lines, respectively, were collected for each exposure by the one-inch open electrode Peltier-cooled CCD detector (1024 × 256 pixels) (Andor Technologies, Belfast, Northern Ireland). The order parameters were calculated from the absolute peak-height intensity of the amide I band in the polarized spectra. Average values were obtained over 9 to 12 samples for different integration periods varying from 30 s to 10 min depending on the samples. No sign of structural deterioration was observed under these experimental conditions. A half-wave plate (Melles Griot, Carlsbad, CA) was used to orient the polarization of the incident laser beam either parallel (z) or perpendicular (x) to the fiber axis. A polarizer was placed before the entrance slit of the monochromator to allow the detection of the polarized scattered light along the z and x directions. A broad-band quarter-wave plate was placed after the polarizer to eliminate the polarization dependence of the grating. A total of four polarized Raman spectra were thus obtained and identified as IXX, IXZ, IZX, and IZZ (see Theoretical Section). This method for acquiring polarized spectra avoids the displacement or rotation of the sample, thus ensuring that the same point is irradiated on the fiber for the four polarized spectra. The unnormalized intensities Ikl (k, l ) x or z) were obtained by measuring the maximum of the peaks considering a linear baseline in the 1750-1500 cm-1 region. The spectra were also corrected to account for the polarization dependence of the optical elements of the Raman microscope. The correction factor was determined experimentally from the ratio of the spectra in the IXX and IZZ configuration obtained for isotropic samples such as isotropic films of globular proteins or regenerated silk. The isotropic refractive index of silk used for the orientation calculations was 1.5, which is very close to what was previously determined for B. mori silk45 and to our own measurements on N. edulis (data not shown). All spectral manipulations were performed using GRAMS/ AI 7.0 (ThermoGalactic, Salem, NH). The spectra were 5-9 points smoothed and corrected for a slight fluorescence background using a polynomial baseline. Results and Discussion Band Assignments. Comparison between Different Types of Silk. Figure 2 shows the Raman spectra obtained from B. mori and S.c. ricini fibroins and from N. edulis dragline. As it can be seen, the use of state-of-the-art Raman


Conformation and Orientation in Silk Fibers Table 1. Band Assignments for the Different Types of Silk Studieda wavenumber (cm-1)

B. mori silk films

fibers

B. mori

N. edulis

S.c. ricini

1693sh 1665 1614 1447 1401

1699sh 1668 1616 1450 1397 1365

1263

1262sh 1241 1226 1206

1698sh 1669 1615 1450 1396 1365 1337 1327 1266 1242 1226 1207 1163

1229 1207

1093

1093

1068 1002 963 904

1067 964 905

827 and 851 642

827 and 851 642

regenerated 1666 1614 1455

1333 1264 1248

881 828 and 853 642

1663 1613 1449 1400

1263sh

1207

1230 1207sh

1103

1102sh

1083 1001

coagulated

1084 1003

1001

827 and 852 642

883 828 and 853 642

assignment

refs

amide I, mainly CdO s in antiparallel β-sheets amide I, CdO s in β-sheets aromatic ring s in F and Y CH3 ab in poly(A), CH2 b in poly(AG) COO- ss in D and E, CH2 w in poly(AG), HR b in poly(A) HR b, N-H ib CH3 sb CH3 sb, HR b amide III: N-H ib, CdO ib, CR-C s amide III, B2 symmetry: HR b, N-H ib, C-N s amide III, A symmetry: N-H ib, CH2 tw aromatic C-H ib HR b CR-Cβ s, CH3 r CH3 r, CR-Cβ s CR-Cβ s, CH3 r CR-Cβ s aromatic ring br in F and Y CH3 r, N-CR s CR-C s, C-N s, CH3 r, C-N-CR d, CdO s CR-C s, C-N s, CdO s, CR-Cβ s Fermi resonance of the Y doublet

46,49,71 46,48,59 72 46,49 46,49,60 46 46 46 46,49 46,49,71 46 72 49 24,46 49 46 49 72 49 49,71 46,59 23,56,57,72

aromatic ring in Y

72

Abbreviations: sh ) shoulder, s) stretching, b ) bending, ab ) anti-symmetric bending, sb ) symmetric bending, w ) wagging, ib ) in-plane bending, tw ) twisting, r ) rocking, br ) breathing, d ) deformation, ss ) symmetrical stretching; A ) alanine, AG ) alanylglycine, F ) phenylalanine, Y ) tyrosine, D ) aspartic acid, E ) glutamic acid. a

Figure 2. Raman spectra (XX polarization) of silk monofilaments of (A) S.c. ricini, (B) N. edulis, and (C) B. mori.

instrumentation with visible light excitation coupled to a microscope allows the in situ recording of high quality spectra of single fiber with a diameter of less than 20 µm in a very short acquisition time. The spectra of Figure 2 are similar to those already published.13,21-28 The assignment of the major bands observed in the spectra of Figure 2 is given in Table 1. These assignments are based on published Raman results on silk, proteins, and polypeptides. As shown in Figure 2, S.c. ricini and N. edulis silks have very similar Raman spectra. In particular, both types of silk give rise to a strong band at around 904 cm-1 which is absent in the spectrum of B. mori. This band is clearly characteristic of the long poly(alanine) sequences found in both S.c. ricini and N. edulis silk, whereas B. mori is rather composed of alternating alanine-glycine motifs. Such a band was also

observed in the Raman spectrum of β-poly(L-alanine)46 and was assigned from normal mode calculations to a combination of vibrational modes including the CR-C and C-N stretching modes of the peptide backbone and the rocking vibration of the methyl side chains.47-49 The high intensity observed for the 904 cm-1 band in the spectrum of S.c. ricini is due to the fact that this type of silk is composed of longer alanine sequences varying between 12 and 13 residues,10 compared to 5-7 residues for spider silk.8 From the position (near 1665-1669 cm-1) and the small width of the amide I band, the spectra of all three silk fibers clearly show that the β-sheet content of the silk proteins is quite high, as expected. The amide I band appears at 1668 and 1669 cm-1 for N. edulis and S.c. ricini, respectively, whereas it is located at 1665 cm-1 for B. mori. This frequency difference is most likely associated with the primary sequence of the silk proteins. A similar difference has been observed for the spectra of β-poly(L-alanine) and β-poly(L-alanylglycine).46 Figure 2 shows that the amide I band is asymmetric for all fibers. A shoulder appears at around 1693 cm-1 for B. mori and 1699 and 1698 cm-1 for N. edulis and S.c. ricini, respectively. This high-frequency amide I component was also observed for poly(L-alanylglycine) and for β-poly(Lalanine)46,48 and was assigned to an infrared-active mode of the antiparallel pleated β-sheets that becomes Raman-active due to the loss of symmetry of the peptide unit cell in the case of high molecular weight macromolecules such as silk. Comparison between the Regenerated Film, the Coagulated Film, and the Fiber of B. mori. Since it has been shown that methanol is very efficient to induce the β-sheet

2252


Figure 3. Raman spectra (XX polarization) of three different silk samples of B. mori, (A) a regenerated film, (B) a regenerated film coagulated in methanol, and (C) a fibroin silk thread.

conformation in silk proteins,12,23,50 the treatment of a regenerated film with methanol was used to make clear band assignments. Figure 3 shows the Raman spectra of regenerated silk films before and after coagulation in methanol, as well as the spectrum of the silk fibroin of B. mori for comparison. In the spectrum of the regenerated film, both the amide I and amide III bands are broad and centered at 1666 and 1248 cm-1, respectively, showing that the conformation of the protein in the film is predominantly unordered. The comparison of this spectrum, which was obtained from a freshly dialyzed solution, with that obtained for undrawn silk fibroin extracted directly from the silk gland of B. mori23,28 reveals that our regenerated film has a lower content of β-sheets. Upon treatment of the film with methanol (Figure 3B), the amide I band shifts to 1663 cm-1 and its width at half-height drastically decreases from 57 to 25 cm-1. In addition, the broad amide III band is narrower and centered at 1230 cm-1 in the spectrum of the coagulated film. These features are characteristic of polypeptide chains that adopt a β-sheet conformation. Therefore, the present data indicate an obvious conformational change from random coil in the regenerated film to β-sheet in the treated film. In addition, the spectra of the treated film and the fiber are very similar, which confirms the high β-sheet content in the treated film. Besides the amide bands, other bands are affected by the methanol treatment, particularly in the 800-1110 cm-1 region. For example, the band due to the C-C stretching vibration at around 1103 cm-1 in the spectrum of the regenerated film clearly shifts to 1083 cm-1 in the spectrum of the treated film and in the fiber spectrum. In the treated film, the band at 1103 cm-1 appears as a small shoulder. These results undoubtedly show that the band at 1083 cm-1 is associated with the β-sheet conformation as first suggested by Magoshi et al.51 and Zheng et al.28 Even though the 1103 cm-1 band is also due to a C-C stretching mode, its precise assignment to a given secondary structure is still unclear. It has been associated to both R-helical conformation and random coil structures.23,28,51 More recently, Monti et al.24 have suggested that it should be used as a Raman marker of silk I, a structure containing β-turns of type II.52,53 Interestingly, this band is also observed in the spectrum of S.c. ricini films obtained from the casted liquid silk which is known to contain R-helices.12,54,55 Even if our results do not rule

Rousseau et al.

out the presence of R-helices in the regenerated film, the shape of the amide I and amide III bands strongly suggest that the silk fibroin is mostly in the random coil conformation. As seen in Figure 3, a band at around 881 cm-1 is observed in the spectra of the treated film and fibroin fiber but is absent in the spectrum of the regenerated film. This band has not yet been assigned for silk but according to the results obtained on β-polypeptides,46 and it mainly originates from the combination of the CR-C and C-N stretching modes and confirms the presence of β-sheets adopted by the poly(alanylglycine) sequences. Figure 3 reveals differences in relative intensity of the tyrosine doublet at 828 and 853 cm-1. It is well-known that the relative intensity of the tyrosine doublet can be used as a spectral marker of the environment of the hydroxyl groups and the strength of hydrogen bonds involving these groups.56,57 For the coagulated film and the fiber, the 853/828 cm-1 intensity ratios are 1.9 and 1.4, respectively, indicating that for both samples, the tyrosine side chains are exposed, and consequently are either proton donors or acceptors in weak to moderate hydrogen bonds. The smaller intensity ratio observed for the fiber indicates that hydrogen bonds involving tyrosyl residues are stronger in the fiber compared to the treated film. Similar conclusions were drawn by comparing the Raman spectra of B. mori fibroin and lyophilized silk.23 In the case of the regenerated film, no information can be extracted from the tyrosine doublet since a strong unassigned band overlaps with the 853 cm-1 component. The new component at 853 cm-1 may be due to the same vibrational mode as the β-sheet band at 881 cm-1, but further experiments should be performed to validate this assumption. Orientation Measurements Qualitative Analysis. Figure 4A presents the polarized Raman spectra obtained for the methanol treated film of B. mori that, as discussed above, has a high β-sheet content. As can be seen, the film is isotropic since the spectra for the parallel polarizations (IXX and IZZ) and the crossed polarizations (IXZ and IZX) overlap almost perfectly within the experimental error. This observation also validates the spectral correction procedure applied to the different spectra (see Experimental Section). The polarized spectra of Figure 4B show that, as opposed to the isotropic regenerated film, the B. mori fibroin fiber is a highly oriented sample. Several bands are sensitive to the polarization of the incident laser, particularly the amide I and III bands. As seen in this figure, the amide I band is stronger in the IXX spectrum whereas the amide III band is stronger in the IZZ spectrum. Since the amide I band is mostly associated with the CdO stretching vibration, whereas the amide III mode has a major contribution from the C-N vibration, these results clearly show that the β-sheets are predominantly parallel to the fiber axis in agreement with the proposed models for the structural organization of silk1-4,58 and with previously published Raman results.22 Figure 5 shows the four polarized spectra obtained for the spider dragline of N. edulis (A) and the fibroin of S.c. ricini (B). The relative intensities of the amide bands indicate that


Figure 4. Polarized Raman spectra of (A) the coagulated film and (B) the fibroin fiber of B. mori silk.

Figure 5. Polarized Raman spectra of (A) the N. edulis spider dragline and (B) the S.c. ricini fibroin silk thread.

the β-sheets are highly oriented toward the fiber axis, as for B. mori fibroin. Although N. edulis dragline and S.c. ricini


display similar Raman spectra (Figure 2), the polarized spectra exhibit major differences (Figure 5). In particular, the intensity of the amide I band at 1669 cm-1 in the IXX spectrum is stronger for S.c. ricini fibroin compared to N. edulis dragline, suggesting that the β-sheet content is higher for S.c. ricini silk. This is further supported by the fact that the width of the amide I band is 26 cm-1 for the N. edulis compared to 18 cm-1 for S.c. ricini, indicating that the conformation of S.c. ricini fibroin is more homogeneous with a higher β-sheet content. Figure 5 also shows that the band at 904 cm-1, associated with the poly(alanine) segments, exhibits the same polarization dependence as the amide I band. Since the scattering is more intense in the IXX polarization, the Raman tensor for this vibration may be preferentially aligned perpendicular to the fiber axis. Several spectral components can be distinguished in the amide III region of the polarized spectra of N. edulis dragline and S.c. ricini fibroin, presented in Figure 5. A major component is observed at 1226 cm-1 in the IXX polarization for both samples. A similar band appears at around 1230 cm-1 in B. mori samples and has been assigned to β-sheet structures (Table 2). In contrast, the component at 1242 cm-1 is quite strong in the crossed-polarized spectra (IXZ and IZX) of N. edulis and S.c. ricini silks (Figure 5) and only appears as a shoulder in the IXX and IZZ spectra. Normal mode calculations performed by Krimm and co-workers47,59 on polypeptides forming the antiparallel β-sheet structure, which belongs to the D2 symmetry group, have shown that bands observed at 1230 and 1242 cm-1 are due to the amide III modes with A and B2 symmetry, respectively. Since the A mode at around 1230 cm-1 is stronger in the IZZ spectrum than in the IXX spectrum, the Raman tensor associated with this vibration appears to be predominantly oriented along the fiber axis. The band due to the symmetric stretching vibration of the carboxylate group of the aspartic (Asp) and glutamic acid (Glu) residues in the ionized state60 appears at 1401 cm-1 in the spectrum of B. mori fibroin (Figure 4B) and around 1397 cm-1 in the spectra of both N. edulis (Figure 5A) and S.c. ricini (Figure 5B). The observed shift to lower frequency might indicate that those side chains contribute to hydrogen bonding within the fibers. These bands are stronger in the IZZ spectra showing that the carboxylate side chains are preferentially oriented along the fiber axis. According to the model generally accepted for the protein organization in silk,8,61 the Asp and Glu bulky side chains are more abundant in the amorphous segments of the silk proteins. In the case of B. mori fibroin, Mita61 and Zhou et al.62 have shown that these residues are only present in the short amorphous segments found between crystallizable poly(alanylglycine) sequences and are most likely involved in β-bend structures. Our results are in good agreement with this model which implies that the carboxylate side chains are mostly parallel to the β-sheet backbone. Quantitative Analysis of the β-Sheet Orientation. As described in the Theoretical Section, it is possible for systems showing uniaxial symmetry to determine quantitatively the order parameters P2 and P4 from the intensity ratios R1 and R2 that can be obtained by polarized Raman microspectroscopy for a Raman tensor showing cylindrical symmetry.

2254


Rousseau et al.

Table 2. Calculated Intensity Ratios R1 and R2, Order Parameters P2 and P4, and Lagrange Multipliers λ2 and λ4 for the Different Silk Samples species

type of sample

R1 ) IZX/IZZ

R2 ) IXZ/IXX

P2

P4

λ2

λ4

B. mori B. mori N. edulis S.c. ricini

coagulated film cocoon dragline cocoon

0.21 ( 0.01 0.28 ( 0.02 0.31 ( 0.02 0.29 ( 0.02

0.22 ( 0.01 0.09 ( 0.01 0.11 ( 0.01 0.09 ( 0.01

0.03 ( 0.02 -0.36 ( 0.03 -0.32 ( 0.02 -0.36 ( 0.03

-0.01 ( 0.01 0.19 ( 0.02 0.13 ( 0.02 0.19 ( 0.03

0.15 -2.34 -2.16 -2.34

-0.09 1.71 0.77 1.71

Since the amide I vibration that is mainly due to the CdO stretching vibration of the peptide group should meet these criteria, we have used the intensity of the amide I band at around 1667 cm-1 to determine the orientation of the carbonyl groups adopting the β-sheet conformation in silk fibers. The parameter a of the Raman tensor (eq 11) was first determined by measuring the depolarization ratio, Riso, for the regenerated film treated with methanol since this film is isotropic as shown in Figure 4A and displays a high β-sheet content, like in silk fibers. An average value of the depolarization ratio of 0.21 ( 0.01 was obtained for such films over a series of 9 measurements (3 replicates of 3 different films). By solving eq 19, two values of a were obtained but only 0.16 gave an acceptable result with physical meaning. This value shows that R3 is about 5 times larger than R1 ) R2. Similar values of Riso were obtained for isotropic regenerated films of S.c. ricini in either the R-helical or β-sheet conformation (unpublished results) showing that the shape of the Raman tensor for the amide I vibration is rather independent of the protein conformation. Since the β-sheet component clearly dominates the amide I band in the spectra of silk fibers, we have calculated the intensity ratios from the height of the amide I band. The ratios R1 and R2 for the three types of fibers studied and the corresponding values of the order parameters P2 and P4 calculated using eqs 7 and 8 are listed in Table 2. For the B. mori fiber, the values of P2 and P4 obtained are equal to -0.36 ( 0.03 and 0.19 ( 0.02, respectively. Knowing that the limiting values of P2 are 1 and -0.5 for perfect orientation at 0° and 90° from the fiber axis, respectively, the P2 value obtained for B. mori fibroin indicates that the carbonyl groups are highly oriented perpendicular to the fiber axis and that the β-sheets are oriented parallel to the fiber axis. The values obtained for the N. edulis dragline and for the S.c. ricini fibroin are P2 ) -0.32 ( 0.02 and P4 ) 0.13 ( 0.02 and P2 ) -0.36 ( 0.03 and P4 ) 0.19 ( 0.03, respectively. As can be seen, the reproducibility over 9 measurements obtained from 3 different points on 3 different fibers for each species is quite good. It is interesting to note that the degree of orientation of the β-sheets is identical for the cocoon silk produced by the silkworms S.c. ricini and B. mori (Table 2) even though their primary amino acids sequence is quite different. The above results show that the degree of orientation of the β-sheets is slightly lower for the dragline of N. edulis than that of the fibroin produced by the silkworms. In principle, a higher level of orientation of the β-sheets should lead to a higher tenacity of the fibers. Nevertheless, previous studies have shown that spider dragline has better mechanical properties in terms of tenacity and extensibility than B. mori fibroin.25 Thus, our results indicate that fibroin and spider dragline silk have different structural organization and justify further investigations

concerning the level of orientation of other segments in silk to account for the interesting combination of mechanical properties found in silk. The combined values of P2 and P4 provide valuable information on the shape of the orientation distribution function. For a given P2, only certain P4 values are possible. The domain of variation of P4 is given by the Schwarz’s inequalities63-65 〈cos2θ〉2 e 〈cos4θ〉 e 〈cos2θ〉

(25)

which gives P4min )

1 (35P22 - 10P2 - 7) 18

P4max )

(26)

1 (5P + 7) 12 2

The shape of the orientation distribution function corresponding to different values of P4 has already been discussed.30,31,40,64,65 If P4 ) P4min, the orientation distribution function is unimodal and is given by the delta function centered at θ0 ) arccos

(

)

2P2 + 1 3

1/2

(27)

This angle also corresponds to the mean value of the orientation distribution that may be calculated when only P2 is known. If P4 ) P4max, the orientation distribution function N(θ) is bimodal and corresponds to a double delta function centered at θ ) 0° and θ ) 90°. Another particular value of P4 results from the information entropy model presented in the Theory Section by setting λ4 ) 0 in eqs 23 and 24. In this case, the orientation distribution function is unimodal and Gaussian.30,31 Therefore, the P4 value allows the discrimination between different possible shapes of the orientation distribution function. Figure 6 shows the maximum and minimum limiting values of P4 as a function of P2. The region located between both curves represents the domain of allowed P4 values. The line corresponding to (P2, P4) couples for which λ4 ) 0 is also drawn. By plotting the experimental values of P2 and P4 in this graph, one can qualitatively assess the type of corresponding orientation distribution function. As seen in Figure 6, the couples values of (P2, P4) are located very near the λ4 ) 0 line for the three types of silk investigated (B. mori and S.c. ricini fibers have identical P2 and P4 values, thus they cannot be discriminated in the graph). This indicates that the orientation of the carbonyl groups is Gaussian around the fiber axis for all three fibers. The most probable orientation distribution function, Nmp(θ), has been determined from the information entropy


Figure 6. Maximum and minimum limiting values of P4 as a function of P2. The line corresponding to (P2, P4) couples for which λ4 ) 0 is also drawn as well as the values of P2 and P4 obtained experimentally for the fibroins (B. mori and S.c. ricini) and the spider dragline (N. edulis).

Figure 7. Most probable orientation distribution function determined for the fibroins (B. mori and S.c. ricini) and the spider dragline (N. edulis).

theory. The values of the Lagrange multipliers calculated from the values of P2 and P4 of the three types of silk are given in Table 2 and the corresponding functions Nmp(θ) are plotted in Figure 7. In each case, Nmp(θ) is Gaussian, unimodal, and centered at θ ) 90°. Since the CdO groups in a β-sheet are perpendicular to the strand and assuming that the Raman tensor of the amide I vibration is oriented along the direction of the CdO bond, our results show that the sheets are oriented along the fiber axis with a Gaussian distribution. As expected from the above data, B. mori and S.c. ricini fibers have identical orientations (i.e., identical values of λ2 and λ4). The spider dragline silk has a broader distribution, its full width at half-height being approximately 40°, whereas that of silkworm silks is approximately 32°. These results are in good agreement with the fact that B. mori fibroin is known to be more crystalline than spider dragline.66 Quantitative orientation measurements have already been done on spider silk using X-ray diffraction16,67-69 and NMR spectroscopy.4 X-ray results have shown that the average crystallite orientation along the fiber axis is approximately 28° for both the silks from Araneus marmoreus and Nephila cruentata,67 whereas for Nephila claVipes silk monofilaments, the orientation distribution of the crystalline fraction was found to be about 23°.16 From 2H NMR measurements, an average angle of 29° was obtained for the alanine regions


of Nephila claVipes dragline.4 No quantitative orientation data is available for silkmoth fibroins. For comparison purposes, we have also calculated an orientation angle from the order parameter P2. One should however keep in mind that such an angle corresponds to an orientation average of the molecules and that a more precise view of the orientation distribution is provided by considering both P2 and P4. Since the carbonyl groups are perpendicular to the β-sheet axis, the average β-sheet orientation angle calculated using the Legendre addition theorem70 is 26° for B. mori and S.c. ricini and 29° for N. edulis. The latter result is in remarkable agreement with that obtained by NMR spectroscopy for Nephila claVipes silk.4 It is important to emphasize that, as opposed to X-ray measurements which allows the determination of the orientation of the crystalline phase only, Raman and NMR spectroscopies give information on the average orientation distribution of the β-sheets without differentiating amorphous from crystalline domains. The β-sheets are considered to form predominantly microcrystals in silk, but it has recently been suggested that β-sheets are also present in the amorphous phase.1 Our results are consistent with this finding since the average angle obtained from the Raman results for dragline silk is larger that the angle obtained from X-ray measurements.16,67 Now that it has been possible to quantify the level of orientation of the β-sheets, mostly associated to the crystalline fraction of silk, it would be interesting to determine the level of orientation of other specific groups found outside the crystalline regions. As a matter of fact, molecular models suggest the presence of an interphase of a thickness of approximately 5 nm around the small crystallites in spider silk in which the amorphous chains are constrained and probably have a preferential orientation to account for the high tenacity of the fibers.2,5 Conclusion The results presented in this paper show that valuable information on both the conformation and orientation of fibroin in silk monofilaments of B. mori, S.c. ricini, and N. edulis can be obtained using polarized Raman microspectroscopy. The comparison between the silk fibers from different species and the regenerated B. mori films has allowed the assignment of some important bands associated to the protein backbone and side chains. As expected, S.c. ricini and N. edulis silk samples, which contain analogous poly(alanine) sequences, give very similar Raman spectra. However, differences in polarization-dependence of the spectra suggest that the orientation level is different. Our results show that it is possible to obtain quantitative information about the distribution of orientation of the β-sheets in silk from the analysis of the polarized Raman spectra. In particular, the order parameters P2 and P4 of the orientation distribution of the β-sheets around the fiber axis has been determined from the amide I band. These calculations show that the β-sheets are well aligned parallel to the fiber axis for the three silk fibers studied. B. mori and S.c. ricini fibroins have similar level of orientation although their primary amino acid sequences are different. The P2 and P4

2256


values indicate the shape of the orientation distribution of the peptide CdO groups in all silk fibers is Gaussian and centered at 90° from the fiber axis. The silkworm fibroins seem to have a narrower orientation distribution, which is in good agreement with the fact that they are more crystalline than spider dragline silk. These results show that Raman microspectroscopy is a promising technique to quantitatively determine in situ the molecular orientation of various types of silk samples and to establish correlations between the silk protein orientation and the mechanical properties of the samples. Acknowledgment. We are grateful to the Insect Production Unit of the Canadian Forest Service (Sault-Ste.-Marie, Canada) for providing us with raw silk from B. mori, and to Prof. Frances Separovic (Melbourne University, Australia) and Prof. Marie E. Herberstein (Macquarie University, Australia) for supplying the spider silk samples. Funding for the Raman microspectrometer was made possible through a grant from the Canadian Fundation for Innovation. This work was also supported by grants from NSERC and the Fonds de recherche sur la nature et les technologies. M.-E.R. greatly acknowledges NSERC, FQRNT, and Fondation de l′Universite´ Laval for the award of graduate scholarships. The authors thank Mr. Serge Groleau for his technical support. We also acknowledge Dr. Claude Sourisseau and Dr. Thierry Buffeteau from Universite´ de Bordeaux I (France) for constructive discussions about orientation calculations. The authors address special thanks to Prof. Michel Lafleur and his group from Universite´ de Montre´al for their assistance in the use of their Raman spectrometer for preliminary measurements. References and Notes (1) van Beek, J. D.; Hess, S.; Vollrath, F.; Meier, B. H. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10266-10271. (2) Termonia, Y. In Structural Biological Materials, Pergamon Material Series; Pergamon: New York, 2000; Vol. 4, pp 337-349. (3) Termonia, Y. Macromolecules 1994, 27, 7378-7381. (4) Simmons, A. H.; Michal, C. A.; Jelinski, L. W. Science 1996, 271, 84-87. (5) O’Brien, J. P.; Fahnestock, S. R.; Termonia, Y.; Gardner, K. H. AdV. Mater. 1998, 10, 1185-1195. (6) In Conformation in Fibrous Proteins, Conformation in Fibrous Proteins and Related Synthetic Polypeptides; Fraser, R. D. B., MacRae, T. P., Eds.; Academic Press: New York, 1973; Chapter 5, pp 94-125. (7) Asakura, T.; Watanabe, Y.; Uchida, A.; Minagawa, H. Macromolecules 1984, 17, 1075-1081. (8) Xu, M.; Lewis, R. V. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 71207124. (9) Simmons, A.; Ray, E.; W., J. L. Macromolecules 1994, 27, 52355237. (10) Yukuhiro, K., personal communication. (11) Asakura, T.; Ito, T.; Okudaira, M.; Kameda, T. Macromolecules 1999, 32, 4940-4946. (12) Ishida, M.; Asakura, T.; Yokoi, M.; Saitoˆ, H. Macromolecules 1990, 23, 88-94. (13) Yang, M.; Yao, J.; Sonoyama, M.; Asakura, T. Macromolecules 2004, 37, 3497-3504. (14) van Beek, J. D.; Beaulieu, L.; Schafer, H.; Demura, M.; Asakura, T.; Meier, B. H. Nature 2000, 405, 1077-1079. (15) Riekel, C.; Madsen, B.; Knight, D.; Vollrath, F. Biomacromolecules 2000, 1, 622-626. (16) Riekel, C.; Mu¨ller, M. Macromolecules 1999, 32, 4464-4466. (17) Warwicker, J. O. J. Mol. Biol. 1960, 2, 350-362. (18) Bower, D. I. J. Polym. Sci. Polym. Phys. 1972, 10, 2135-2153.

Rousseau et al. (19) Schlotter, N. E. In Raman Spectroscopy [as Applied to Polymers]. Encycl. Polym. Sci. Eng.; Kroschwitz, J. I., Ed.; Wiley: New York, 1988; Vol. 14, pp 8-45. (20) Asakura, T.; Iwadate, M.; Demura, M.; Williamson, M. P. Int. J. Biol. Macromol. 1999, 24, 167-171. (21) Shao, Z.; Vollrath, F.; Sirichaisit, J.; Young, R. J. Polymer 1999, 40, 2493-2500. (22) Gillepsie, D. B.; Viney, C.; Yager, P. Silk Polymers. ACS Symp. Ser. 1994, 544, 155-167. (23) Monti, P.; Freedi, G.; Bertoluzza, A.; Kasai, N.; Tsukada, M. J. Raman Spectrosc. 1998, 29, 297-304. (24) Monti, P.; Taddei, P.; Freddi, G.; Asakura, T.; Tsukada, M. J. Raman Spectrosc. 2001, 32, 103-107. (25) Sirichaisit, J.; Brookes, V. L.; Young, R. J.; Vollrath, F. Biomacromolecules 2003, 4, 387-394. (26) Sirichaisit, J.; Young, R. J.; Vollrath, F. Polymer 2000, 41, 12231227. (27) Edwards, H. G. M.; Farwell, D. W. J. Raman Spectrosc. 1995, 26, 901-909. (28) Zheng, S.; Li, G.; Yao, W.; Yu, T. Appl. Spectrosc. 1989, 43, 12691272. (29) Lagugne´ Labarthet, F.; Buffeteau, T.; Sourisseau, C. Macromol. Symp. 1999, 137, 75-82. (30) Lagugne´ Labarthet, F.; Buffeteau, T.; Sourisseau, C. Appl. Spectrosc. 2000, 54, 699-705. (31) Pottel, H.; Herreman, W.; van der Meer, B. W.; Ameloot, M. Chem. Phys. 1986, 102, 37-44. (32) Berne, B. J.; Pechukas, P.; Harp, G. D. J. Chem. Phys. 1968, 49, 3125-3129. (33) Roe, R.-J.; Krigbaum, W. R. J. Chem. Phys. 1964, 40, 2608-2615. (34) Jen, S.; Clark, N. A.; Pershan, P. S.; Priestley, E. B. J. Chem. Phys. 1977, 66, 4635-4661. (35) Pigeon, M.; Prud′homme, R. E.; Pe´zolet, M. Macromolecules 1991, 24, 5687-5694. (36) Citra, M. J.; Chase, D. B.; Ikeda, R. M.; Gardner, K. H. Macromolecules 1995, 28, 4007-4012. (37) Turrell, G. J. Raman Spectrosc. 1984, 15, 103-108. (38) Turrell, G. In Raman Sampling, Practical Raman Spectroscopy; Gardiner, D. J., Graves, P. R., Eds.; Springer-Verlag: Berlin, 1989; Chapter 2, pp 13-54. (39) Buffeteau, T.; Lagugne´ Labarthet, F.; Sourisseau, C.; Kostromine, S.; Bieringer, T. Macromolecules 2004, 37, 2880-2889. (40) Bower, D. I. J. Polym. Sci. Pol. Phys. 1981, 19, 93-107. (41) Chen, X.; Shao, Z.; Marinkovic, N. S.; Miller, L. M.; Zhou, P.; Chance, M. R. Biophys. Chem. 2001, 89, 25-34. (42) Yamada, H.; Nakao, H.; Takasu, Y.; Tsubouchi, K. Mater. Sci. Eng. C 2001, 14, 41-46. (43) Asakura, T.; Kashiba, H.; Yoshimizu, H. Macromolecules 1988, 21, 644-648. (44) Kishore, A. I.; Herberstein, M. E.; Craig, C. L.; Separovic, F. Biopolymers 2002, 61, 287-297. (45) Tsukada, M.; Nagura, M.; Ishikawa, H.; Shiozaki, H. J. Appl. Polym. Sci. 1991, 43, 643-649. (46) Moore, W. H.; Krimm, S. Biopolymers 1976, 15, 2465-2483. (47) Dwivedi, A. M.; Krimm, S. Macromolecules 1982, 15, 186-193. (48) Krimm, S. Biopolymers 1983, 22, 217-225. (49) Krimm, S.; Bandekar, J. AdV. Protein Chem. 1986, 38, 181-364. (50) Yoshimizu, H.; Asakura, T. J. Appl. Polym. Sci. 1990, 40, 17451756. (51) Magoshi, J.; Magoshi, Y.; Nakamura, S. Polym. Commun. 1985, 26, 309-311. (52) Monti, P.; Taddei, P.; Freddi, G.; Ohgo, K.; Asakura, T. Biopolymers 2003, 72, 329-338. (53) Asakura, T.; Ashida, J.; Yamane, T.; Kameda, T.; Nakazawa, Y.; Ohgo, K.; Komatsu, K. J. Mol. Biol. 2001, 306, 291-305. (54) Freddi, G.; Monti, P.; Nagura, M.; Gotoh, Y.; Tsukada, M. J. Polym. Sci. Polym. Phys. 1997, 35, 841-847. (55) Tsukada, M.; Freddi, G.; Monti, P.; Bertoluzza, A.; Kasai, N. J. Polym. Sci. Polym. Phys. 1995, 33, 1995-2001. (56) Yu, N.-T.; Jo, B. H. J. Am. Chem. Soc. 1973, 95, 5033-5037. (57) Siamwiza, M. N.; Lord, R. C.; Chen, M. C.; Takamatsu, T.; Harada, I.; Matsuura, H.; Shimanoxhi, T. Biochemistry 1975, 14, 4870-4876. (58) Shen, Y.; Johnson, M. A.; Martin, D. C. Macromolecules 1998, 31, 1, 8857-8864. (59) Moore, W. H.; Krimm, S. Biopolymers 1976, 15, 2439-2464. (60) Frushour, B. G.; Koenig, J. L. Biopolymers 1974, 13, 1809-1819. (61) Mita, K.; Ichimura, S.; Zama, M.; James, T. C. J. Mol. Biol. 1988, 203, 917-925.

Conformation and Orientation in Silk Fibers (62) Zhou, C. Z.; Confalonieri, F.; Medina, N.; Zivanovic, Y.; Esnault, C.; Yang, T.; Jacquet, M.; Janin, J.; Duguet, M.; Perasso, R.; Li, Z. G. Nucleic Acids Res. 2000, 28, 2413-2419. (63) Kooyman, R. P. H.; Levine, Y. K.; van der Meer, B. W. Chem. Phys. 1981, 60, 317-326. (64) Nomura, S.; Kawai, H.; Kimura, I.; Kagiyama, M. J. Polym. Sci. A2 1970, 8, 383-400. (65) Nomura, S.; Nakamura, N.; Kawai, H. J. Polym. Sci. A2 1971, 9, 407-420. (66) Gosline, J. M.; Demont, M. E.; Denny, M. W. EndeaVour 1986, 10, 37-43. (67) Work, R. W.; Morosoff, N. Text. Res. J. 1982, 52, 349-356. (68) Grubb, D. T.; Ji, G. Int. J. Biol. Macromol. 1999, 24, 203-210.

Biomacromolecules, Vol. 5, No. 6, 2004 2257 (69) Grubb, D. T.; Jelinski, L. W. Macromolecules 1997, 30, 2860-2867. (70) Buffeteau, T.; Pe´zolet, M. In Linear Dichroism in Infrared Spectroscopy, Handbook of Vibrational Spectroscopy; Chalmers, J. M., Griffith, P. R., Eds.; John Wiley and Sons: New York, 2002; Vol. 1, pp 693-710. (71) Frushour, B. G.; Painter, P. C.; Koenig, J. L. J. Macromol. Sci.-ReV. M. 1976, C15, 29-115. (72) Tu, A. T. In Proteins, Raman Spectroscopy in Biology: Principles and Applications; John Wiley & Sons: Chichester, U.K., 1982; Chapter 3, pp 65-116.

BM049717V

Study of Protein Conformation and Orientation in Silkworm and Spider

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