Thermally Induced α-Helix to β-Sheet Transition in Regenerated Silk

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Thermally Induced r-Helix to β-Sheet Transition in Regenerated Silk Fibers and Films Lawrence F. Drummy, David M. Phillips, Morley O. Stone, B. L. Farmer, and Rajesh R. Naik* Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Dayton, Ohio 45433 Received May 24, 2005; Revised Manuscript Received August 4, 2005

The structure of thin films cast from regenerated solutions of Bombyx mori cocoon silk in hexafluoroisopropyl alcohol (HFIP) was studied by synchrotron X-ray diffraction during heating. A solid-state conformational transition from an R-helical structure to the well-known β-sheet silk II structure occurred at a temperature of approximately 140 °C. The transition appeared to be homogeneous, as both phases do not coexist within the resolution of the current study. Modulated differential scanning calorimetry (DSC) of the films showed an endothermic melting peak followed by an exothermic crystallization peak, both occurring near 140 °C. Oriented fibers were also produced that displayed this helical molecular conformation. Subsequent heating above the structural transition temperature produced oriented β-sheet fibers very similar in structure to B. mori cocoon fibers. Heat treatment of silk films at temperatures well below their degradation temperature offers a controllable route to materials with well-defined structures and mechanical behavior. Introduction There is considerable interest in spiders and silkworms as model fiber processors, and silk fibers have been the subject of intense study over the past several decades because of their remarkable mechanical properties.1,2 Thin films of silk are of interest for both biomedical3 and optical4 applications. Precise control of the microstructure in natural silk fibers is maintained by the sequence of amino acids in the proteins and the processing conditions during spinning inside the spider5-8 or silkworm.9-11 Similar control is desirable when processing silk into solutions and thin films. Several unique crystal structures have been characterized for Bombyx mori silk, the most well-known being the silk I and silk II structures. Both structures form hydrogen-bonded β-sheets; however, they are formed by very different processing methods and have very different characteristics. The silk I structure is metastable and water-soluble. It is commonly produced by drying the silk gland contents or by casting a film at room temperature from an aqueous solution in the absence of mechanical stress. Silk I was first characterized by powder X-ray diffraction (XRD).12 Later, a structure was proposed on the basis of single-crystal electron diffraction data,13 and more recently, a similar structure was proposed on the basis of NMR data.14 The molecule is thought to adopt an unusual “crankshaft” conformation in silk I crystals.13 The silk II structure is waterinsoluble and is naturally found in silkworm cocoon fibers. Crystal structure models were first proposed by Marsh et al.15 and later refined by Takahashi et al.16 In silk II the molecules exist in a more extended conformation, forming β-pleated sheets. * Corresponding author. E-mail: [email protected]. 10.1021/bm0503524

Research has shown that the crystal structure of silk films can be controlled during the casting process. Magoshi et al. developed a concentration-substrate temperature phase diagram for films cast from aqueous solutions.9,17 It was found that concentrated solutions (>3%) cast onto warm substrates (>50 °C) produce the silk II structure, while those cast onto cooler substrates produce the silk I or random coil forms. Structural changes can also be induced after silk fiber or film formation by heat treatment in various controlled atmospheres, mechanical deformation, or treatment with organic solvents. Methanol has been used to induce a transition from silk I or random coil to silk II by acting as a dehydrating agent. The heat treatment of silk films and fibers has been studied by several researchers.18-22 These studies have shown changes in the color, crystal structure, and mechanical behavior that occur in silk at elevated temperatures. A temperature dependence of the silk glass transition as a function of water content has been reported. It was proposed that excess solvent remaining in cast films acts as a plasticizer, lowering the Tg.22 Tg in dry silk fibers and films has also been reported by many researchers to be ∼180200 °C.20-23 Changes in Tg with water content have been seen in other fibrous proteins, such as elastin.24 In this study, we monitor the structural changes in silk films cast from a nonaqueous solvent, 1,1,1,3,3,3-hexafluoroisopropyl alcohol (HFIP), when heated. The structural reorganization that takes place is monitored using XRD with a temperature resolution of 5 °C between X-ray patterns. Differential scanning calorimetry (DSC) is also used to give insight into the thermodynamic nature of the transitions. The initial molecular conformation in both the films and oriented fibers is consistent with an R-helical structure, and when

This article not subject to U.S. Copyright. Published 2005 by the American Chemical Society Published on Web 09/08/2005

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heated above approximately 140 °C, a transition from R-helix to β-sheet was observed. Experimental Section Silk cocoons were obtained from two sources, Dr. Masuhiro Tsukada at the National Institute of Agrobiological Sciences, Japan, and from silkworms grown in our laboratory on an artificial diet of Silkworm Chow (Mulberry Farms, Fallbrook, CA). Silk from both sources showed very similar responses to the heating experiments described in this paper. To degum the silk, the sericin was removed by boiling the cocoons in 0.05% (w/v) Na2CO3 for 30 min and then rinsing thoroughly. The degummed silk cocoons were dried and bundles of fibers were gently removed from the cocoons and oriented for X-ray analysis. Regenerated silk solutions were obtained by dissolving the dried cocoons in 9.3 M LiBr (99+%, Sigma-Aldrich) to a concentration of 10% (w/w) by heating to 100 °C for 15 min. The dissolved silk was dialyzed against distilled, deionized water to remove the LiBr and subsequently lyophilized. The lyophilized silk cake was directly dissolved in HFIP (99+%, Sigma-Aldrich, St. Louis, MO) to a concentration of 10% (w/w). Silk films approximately 100-500 µm in thickness were cast onto release paper from a 5% (w/w) solution in HFIP. Circular dichroism (CD) spectroscopy was performed on a Jasco J-715 spectropolarimeter. The CD Pro software package25 was used to fit the spectrum and to deconvolute the data. XRD was performed at the Brookhaven National Synchrotron Light Source beamline X-27C with an X-ray wavelength of 0.1371 nm. Films were placed in a furnace with Kapton windows for transmission X-ray measurements, and the samples were held under a nitrogen atmosphere. Heating ramps were from approximately room temperature to 250 °C at 5°/min. X-ray exposure time was 1 min. Diffraction patterns were collected on a Mar-CCD large area detector, and sample to detector distances were calibrated using an aluminum oxide standard. Some XRD patterns were also collected using a Rigaku rotating anode Cu-KR source (λ ) 0.15418 nm), a Statton camera, and 2D image plate detectors. The Fit2D software package was used for analysis of the two-dimensional diffraction patterns. For electron microscopy analysis, a Delong Instruments LVEM5 was used in both transmission electron microscopy (TEM) and scanning electron microscopy (SEM) modes, at a nominal operating voltage of 5 kV. Droplets of silk in HFIP were atomized onto amorphous carbon-coated Cu grids using a nebulizer kit from Ted Pella. Ultrathin sections (25-30 nm) were produced from B. mori cocoon fibers embedded in epoxy using a RMC PowerTome and a 35° diamond knife. No staining procedures were used. Modulated DSC measurements were made on a TA Instruments Q1000. The heating rate was 3 °C/min, the modulation amplitude was (1°, the period was 60 s, and the experiments were carried out in conventional MDSC mode. Results and Discussion To investigate the silk molecular conformation in solution, we used circular dichroism (CD). The CD spectrum of 0.5%

Figure 1. Circular dichroism (CD) spectrum from a 0.5% (w/w) silk solution in HFIP.

(w/w) silk solution in HFIP (Figure 1) shows that the structure is predominantly helical, similar to other published CD spectra from silk in HFIP.26,27 In particular, there is a strong negative peak at 205 nm with a shoulder at 222 nm and a strong positive peak at 190 nm. The deconvolution of the spectrum shows that a significant portion (∼70%) of the molecule is in a helical conformation, while the remainder is random coil. This result is consistent with solid-state XRD from silk films cast from HFIP. CD spectra taken at elevated temperatures, even near the boiling point of the solvent (see Supporting Information, Figure S1), did not show any significant conformational changes in the molecule, demonstrating the strong secondary structure stabilizing capability of HFIP. This result clearly differs from spider silk in aqueous solution, which has been shown to undergo an irreversible conversion to β-sheet upon heating above 40 °C.28 This transition to β-sheets was linked to the formation of amyloid-type fibrils evident in TEM micrographs of the silk-producing glands in the spiders. Films were cast from a more concentrated solution (5% (w/w)), and the resulting thickness of the films was between 100 and 500 µm. The morphology of the cast silk films was initially investigated using optical microscopy. It was found that the films were optically clear and in some cases birefringent. We have found the degree of birefringence to be related to the thickness of the cast films, with thicker films exhibiting more apparent birefringence. This is presumably due to an increase in the drying stresses for thicker films causing a slight degree of orientation. The films were also birefringent when mounted in cross-section, with the molecules lying nominally parallel to the substrate (see Supporting Information, Figure S2). In-situ XRD was used to monitor the silk secondary structure during heating. The wide-angle X-ray scattering (WAXS) pattern from an as-cast silk film during in situ heating is shown in Figure 2. Although the films were birefringent, no significant amount of orientation was seen in the X-ray patterns. The strong diffuse reflections present in films cast from HFIP are located at 0.47 and 0.56 nm. These are not representative of a silk I or silk II structure. In particular, the pattern does not show the relatively sharp 0.45 nm (210) and 0.37 nm (211) reflections of the silk II β-sheet structure.16 Also, the characteristic 0.72 nm (110) reflection of the silk I structure12,13,29 is absent. The observed

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Figure 2. WAXS data from a silk film cast from HFIP during the in situ heating experiment at three different temperatures (45, 140, and 195 °C).

Figure 3. Radial intensity scans of the WAXS data as a function of temperature. A clear transition is seen at ∼140 °C.

diffraction is consistent with an R-helical structure. There is evidence that fluorinated alcohols such as HFIP induce helical structures in other proteins.30 As the temperature is increased at a rate of 5 °C/min to 140 °C, clear changes in the pattern were seen. The R-helix peak at 0.56 nm disappears and relatively sharp peaks at 0.45 and 0.37 nm begin to appear. These peaks correspond to the (210) and (211) reflections of the silk II β-sheet structure, respectively.16 Crystallization of β-sheets continues after heating past 140 °C, and these peaks become more defined up to a temperature of 210 °C. The WAXS curves are plotted in Figure 3 as intensity vs 2θ for temperatures from 40 °C up to 215 °C. Above 210 °C the films began to degrade, and this was seen in small-angle X-ray scattering patterns taken simultaneously with the WAXS data during heating. As the mass loss began to increase significantly above 220 °C, the apparent scattering from voids increased significantly. Low-voltage electron microscopy was used to investigate the morphological changes that occurred in silk thin films after heat treatment at high resolution. This technique generates high-contrast TEM images from low atomic number materials, such as biological materials, without staining.31 A 0.5% (w/w) silk solution in HFIP was atomized into droplets onto amorphous carbon-coated TEM grids. Figure 4, parts A and B, show low-magnification TEM and SEM images, respectively, of the same droplet. At higher magnifications in a thin part of the droplet (Figure 4C), little contrast is visible above the grainy phase contrast generated by the underlying amophous carbon support film. After heat

Figure 4. (A) LVTEM image of an atomized silk droplet from a solution in HFIP lying on a copper grid. (B) SEM image of the same droplet. (C) High-magnification LVTEM image from a thin region of the droplet, as cast from HFIP. Little contrast is visible except for the grainy phase contrast from the carbon support film. (D) LVTEM image of a silk droplet, annealed in a vacuum at 190 °C. More contrast is visible due to crystallization of β-sheets. (E) Low-magnification LVTEM of a longitudinal thin section of a B. mori cocoon fiber. (F) At higher magnification, anisotropic density fluctuations, corresponding to the β-sheet crystallites, are seen with their long axis running nominally parallel to the fiber axis.

treatment at 190 °C (Figure 4D), much more contrast can be seen on a ∼10-20-nm length scale, which is most likely attributed to crystallization of the films into the β-sheet structure. Also shown for comparison is a LVTEM image of a longitudinal thin section from a single B. mori cocoon fiber (Figure 4E), with the long axis of the fiber running approximately bottom left to top right in the image. In the middle of the low magnification image (Figure 4E), the sericin can be seen as a dark band surrounded by the two silk fibroin structural cores of the fiber. At higher magnification (Figure 4F) anisotropic density fluctuations are visible in the silk fibroin, with the darker, more electron dense regions corresponding to the β-sheet crystallites in the fiber. Similar images have been obtained from stained samples of Nephila edulis spider silk32 as well as bovine elastin filaments.33 The as-cast and heat-treated films from HFIP do not show any features reminiscent of amyloid structures. Amyloidic features have been seen in certain regions of the silk storage glands in N. edulis. Native B. mori fibers do show more elongated amyloid-type fibrils when looking

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Figure 6. (A) WAXS pattern from an oriented fiber from HFIP as spun. (B) The same fiber annealed at 190 °C for 1 h shows a β-sheet structure. (C) β-sheet structure of an oriented bundle of B. mori cocoon fibers.

Figure 5. (A) Optical micrograph of fiber spun from HFIP solution. Scale bar in 100 µm. (B) WAXS pattern from an oriented fiber. The fiber axis is vertical. (C) Equatorial and meridional intensity scan from the pattern shows a high degree of alignment and three peaks visible at 0.95, 0.56, and 0.47 nm.

perpendicular to the long axis of the fibers, and this is related to the elongated nature of the β-sheet crystallites themselves.34 By pulling fibers from a concentrated silk/HFIP solution (>5%), highly oriented fibers were produced that remained in this metastable helical secondary structure. To our knowledge this is the first example of an oriented B. mori silk fiber that is not in a β-sheet conformation. Once oriented the d spacings between chains and along chains can be identified. Figure 5 shows a WAXS pattern from a vertically oriented fiber and intensity scans along the equator and meridian of the pattern. We observe a 0.56-nm d spacing between chains on the equator, and this reflection shows a high degree of orientation, as indicated by the low azimuthal angular width (fwhm ∼75°). The peak on the meridian at 0.47 nm shows a significantly larger azimuthal width (fwhm ∼125°) than the peak on the equator, suggesting that there are two underlying peaks on the first layer line of the fiber pattern. This is the diffraction signature of a helix, which shows splitting of intensity on the (00l) layer lines. The layer line intensity distribution in diffraction patterns from oriented helices can be described by a series of Bessel functions.35 The 0.47-nm d spacing along the chains, measured from the peak on the meridian of the fiber pattern, is consistent with a helix having a pitch of 0.54 nm (similar to an R-helix) and a diameter of 0.95 nm. There is a slight shoulder in the WAXS data near 2θ ) 9° (d ) 0.95 nm) on the equator arising from the lateral

packing of helices. If the helices are packed locally on a hexagonal lattice with a lattice constant of 1.1 nm, this would give three peaks on the equator of 0.95 nm corresponding to the (100) plane spacing, 0.55 nm corresponding to the (110) plane spacing, and 0.47 nm corresponding to the (200) plane spacing. This model is in good agreement with the observed data. Poly(L-alanine) R-helices are known to pack hexagonally with a lattice constant of 0.86 nm.36 The silk helical structure identified here has an effective helical radius 0.1 nm larger than a poly(L-alanine) R-helix. This is likely due to larger R-groups present in the silk helix (serine and tyrosine are common residues ending the silk repeat sequence GAGAGX, where X ) S or Y).11 Figure 6 shows WAXS patterns taken from oriented fibers pulled from HFIP solution at room temperature (Figure 6A) and after annealing at 190 °C under vacuum for 1 h (Figure 6B). When these fibers are annealed above the 140 °C transition temperature, highly oriented β-sheet structures can be formed that are similar to those present in B. mori cocoon fibers (Figure 6C). The most notable difference between the two patterns is the sharpness of the (002) reflection in the B. mori cocoon pattern and the broad nature of this reflection in the fiber from HFIP. This difference in peak width arises from the larger extent of the crystals along their c-axis (parallel to the fiber axis) in the cocoon fibers. Silk cocoon fiber β-sheet crystals are developed with significant elongational forces along the fiber axis, while the β-sheets in the fiber from HFIP crystallize through a heat treatment alone. Figure 7A shows an azimuthal integration of all peaks inside of 2θ ) 35° for both samples, and relatively good agreement is seen. In particular, the (002) and (211) are both clearly resolved in the 2D pattern (Figure 5B), but when the intensity is azimuthally integrated, the two overlap and are no longer distinctly visible (Figure 7A). Figure 7B shows the orientation comparison of the annealed fiber from HFIP and the natural cocoon fiber. This azimuthal intensity scan was taken at a reciprocal space distance of 2.2 nm-1. This corresponds to the region in the diffraction pattern of the strongest reflections on the equator, the (200) and (210) at approximately 0.45 nm. The full width at half-maximum (fwhm) of the peaks taken from the azimuthal intensity scan is 20° for the cocoon fibers and 30° for the regenerated fiber. Also, the intensity of the (211) reflections can be seen in both curves as slight shoulders to the main peak at azimuthal angles of 40°, 150°, 220°, and 320°.

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Figure 8. DSC and TGA data from a silk film cast from HFIP. The left axis shows the DSC data separated into total, reversing, and nonreversing portions of the heat flow. The right axis shows the total weight loss of the sample (TGA) during heating. DSC results show an endothermic melting of the R-helical structure at 137 °C and an exothermic recrystallization into the β-sheet structure at 146 °C. TGA shows a weight loss of slightly less than 10% up to a temperature of 200 °C.

Figure 7. (A) Azimuthally integrated radial intensity scans of the twodimensional XRD patterns in Figure 5B,C of both the annealed fiber spun from HFIP (Figure 5B) and the B. mori fiber bundle (Figure 5C). (B) Comparison of the degree of orientation in both patterns.

Modulated DSC was used to investigate the thermodynamic nature of the structural transition. Figure 8 shows the DSC results taken from silk films cast from HFIP. In the total heat flow curve, a broad endothermic peak centered at approximately 90 °C can be attributed to loss of HFIP from the film. The boiling point of HFIP is near 60 °C in air under ambient conditions. A relatively sharp endotherm can be seen centered at 137 °C, followed by an exotherm at 146 °C. The endotherm can be correlated with the disappearance of the 0.56-nm R-helix X-ray peak. The exotherm at 146 °C corresponds to the crystallization into the β-sheet structure. Separation of the total heat flow data into the reversing and nonreversing portions appears to show a second-order transition in the films located at 137 °C. It is not clear if this is a glass transition in the amorphous phase, however, as it is so closely associated with the melting of the R-helicies. TGA shows less than a 10% weight loss up to a temperature of 200 °C. Loss of solvent through this temperature range may have an effect on the precise location of the R-helix to β-sheet transition temperature in the films. Some chemical changes, such as oxidation, of the silk protein

structure may occur at temperatures significantly above 200 °C, as the XRD experiment was not performed in a 100% nitrogen atmosphere. Zhang et al.19 have examined the color changes that occur in silkworm cocoons heat-treated in air due to oxidation. The films are relatively stable up to a temperature of approximately 220 °C, where the weight loss increases drastically with increasing temperature. Some general comments can be made about silk structure development during heating. Helical structures, which are thermodynamically stable when in a solution of HFIP (Figure 1), persist as a metastable state when the silk is cast into a film and dried. Even as solvent is evaporated, by heating the films at temperatures below 135 °C, the silk remains in this metastable helical conformation. As more thermal energy is put into the system, and the temperature is taken above the transition temperature, the intramolecular hydrogen bonds in the R-helix are broken. The molecules are then free to move into the stable β-sheet conformation. Up to a temperature of approximately 135 °C, no changes in the silk structure are evident in the data. Annealing below this temperature, even for extended periods of time, does not induce the transition. Also, the 0.56-nm R-helix peak completely disappears from the data before the sharp β-sheet peaks appear. These observations indicate that the transition is quasihomogeneous, with β-sheets unable to nucleate until the helical structures have denatured or melted. The mechanism for helix formation in B. mori silk appears to be intimately related to the solvent system, as B. mori fibroin is not helical in its native state. Evidence for a 3-fold helical chain conformation in trigonal crystals has been found in B. mori silk formed at the air-water interface using the Langmuir-Blodgett technique.37 Very few out of the hundreds of other types of natural silks have been shown to be R-helical. Two examples are Apis mellifera (silk from honeybee larvae) and mantid oothecal proteins (silk from

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the egg case of the praying mantis).38 More recently, Fudge and Gosline have investigated the structure of hagfish silk, which is also R-helical and has many similar properties to keratin.39,40 In these silks the glycine content is typically low, and the content of charged residues is high. Researchers have investigated the ability of fluorine-substituted alchohols to induce R-helical structures in proteins.30,41 The low polarity of the solvent (HFIP) decreases local hydrophobic interactions that tend to favor formation of β-sheets. There is also an increase in intramolecular hydrogen-bond interactions that typically stabilize the R-helix. While the there is a great deal of data on the mechanical properties of silk and other protein fibers, relatively little of this knowledge has been directly related to a complete microstructural model of the fibers.42-46 Even less is known about the microstructure and resulting properties of silk and other proteins in the thin film form. Control over the secondary structure and the microstructure through the processing steps used and heat treatment of these films may allow for more systematic property evaluations to be made. Conclusions We have identified a unique R-helical crystal structure for regenerated silk fibers and films produced from solution in HFIP. By heating solution cast films above 140 °C, this structure can be converted to the silk II β-sheet structure commonly found in B. mori cocoon fibers. Oriented silk fibers were produced that remained in the R-helical structure, and upon heating above the structural transition temperature, highly oriented β-sheet fibers were obtained. The transitions identified using DSC showed good agreement with the in situ XRD data. It will be of interest to characterize the mechanical behavior of these R-helical fibers and films and to add to the knowledge of structure-property relationships that exists for silk and other fibrous proteins. Acknowledgment. The authors thank Igors Sics at BNL beamline X27C, for assistance with the synchrotron experiments. We thank Chuck Lawrence and Melanie Tomczak, for collecting portions of the CD data, and Marlene Houtz, for the DSC and TGA. Many thanks also go to Hilmar Koerner and Richard Vaia, for helpful discussions as well as assistance with the synchrotron experiments. We thank the National Research Council, for fellowships (L.F.D and D.M.P), and the Air Force Office of Scientific Research, for funding. Supporting Information Available. Circular dichroism spectra of silk solutions (Figure S1) and polarized optical micrographs (Figure S2) of silk film castings. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Shao, Z.; Vollrath, F. Nature 2002, 418, 741. (2) Cunniff, P. M.; Fossey, S. A.; Auerback, M. A.; Song, J. W. In Silk Polymers: Materials Science and Biotechnology; Kaplan, D. L., Adams, W. W., Farmer, B. L., Viney, C., Eds.; American Chemical Society: Washington, DC, 1994; pp 234-251. (3) Altman, G. H.; Diaz, F.; Jakuba, C.; Calabro, T.; Horan, R.; Chen, J.; Lu, H.; Richmond, T.; Kaplan, D. Biomaterials 2003, 24, 401416. (4) Putthanarat, S.; Eby, R. K.; Naik, R. R.; Juhl, S. B.; Walker, M. A.; Peterman, E.; Ristich, S.; Magoshi, J.; Tanaka, T.; Stone, M. O.; Farmer, B. L.; Brewer, C.; Ott, D. Polymer 2004, 45, 8451-8457.

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