Structural evolution of regenerated silk fibroin under shear: Combined wide- and small-angle x-ray scattering experiments using synchrotron radiation

Structural Evolution ofRegenerated Silk FibroinUnder Shear: CombinedWide- and Small-Angle X-rayScattering Experiments UsingSynchrotron Radiation

Manfred Rossle1

Pierre Panine2

Volker S. Urban3

Christian Riekel21 European Molecular Biology

Laboratory,Hamburg Outstation,

Notkestrasse 85, D-22603Hamburg, Germany

2 European SynchrotronRadiation Facility,

B. P. 220,F-38043 Grenoble Cedex,

France

3 Oak Ridge NationalLaboratory,

P. O. Box 2008 MS6100Oak Ridge,

TN 37831-6100 USA

Received 17 September 2003;accepted 2 March 2004

Published online 30 April 2004 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20083

Abstract: The structural evolution of regenerated Bombyx mori silk fibroin during shearing witha Couette cell has been studied in situ by synchrotron radiation small- and wide-angle x-rayscattering techniques. An elongation of fibroin molecules was observed with increasing shear rate,followed by an aggregation phase. The aggregates were found to be amorphous with �-conforma-tion according to infrared spectroscopy. Scanning x-ray microdiffraction with a 5 �m beam onaggregated material, which had solidified in air, showed silk II reflections and a material withequatorial reflections close to the silk I structure reflections, but with strong differences in reflectionintensities. This silk I type material shows up to two low-angle peaks suggesting the presence ofwater molecules that might be intercalated between hydrogen-bonded sheets. © 2004 WileyPeriodicals, Inc. Biopolymers 74: 316–327, 2004

Keywords: Bombyx mori silk fibroin; small-angle x-ray scattering; wide-angle x-ray scattering; insitu shearing experiment; silk I/II formation; synchrotron radiation

INTRODUCTION

During the spinning process of silk worms, solvatedrandom-coil fibroin protein, which is assumed to bestored in the silk gland as a liquid crystalline phase,1,2

is transformed into a semicrystalline silk threadcomposed of two fibers with a sericin coating.1,3

The Bombyx mori fibroin protein consists of twodifferent polypeptide chains, the light (L-) and theheavy (H-) chain, which are linked together by disul-

Correspondence to: Christian Riekel; email: [email protected], Vol. 74, 316–327 (2004)© 2004 Wiley Periodicals, Inc.

316

fide bridges.4,5 The core sequence of the fibroin chain,which corresponds to a –[G–A–G–A–G–S]8–– motif,6

forms domains of crystalline antiparallel �-sheets ofthe so-called silk II structure.7–9 Drying of the native� regenerated fibroin protein results in what is knownas the silk I structure, which can be transformed intothe silk II structure by weak tensile stress.10–12 Pro-posed silk I structures differ considerably in the chain-conformation ranging from crankshaft,13 extendedchains,14 to repeated �-turns.15 It is frequently as-sumed that silk I is transformed into silk II during thespinning process, although an experimental proof islacking as the onset of fiber formation has not yetbeen studied in vivo. In vivo x-ray microdiffractionstudies using synchrotron radiation during forced silk-ing of Nephila spiders shows that the solidificationstep must take place already inside the spinning duct,which is not accessible to x-ray diffraction studieswithout major destruction of the organism.16–18 It isthought that the solidification step in silk worms andspiders occurs during shearing of the liquid crystallinefibroin solution within the tapered spinning duct.3,19,20

In order to shed more light on this step several shear-ing experiments using cone-and-plate21–23 or Couetteviscosimeters,24,25 have been performed on regener-ated Bombyx mori silk. Most rheological experimentswere, however, not coupled with an in situ analyticaltechnique, which would allow the study of the evolu-tion of microstructural parameters. A notable excep-tion are rheo-optical studies in which shear rates of�200 s�1 did not produce observable solidification.24

The current article will address the evolution of mo-lecular parameters of fibroin during shearing using aCouette viscosimeter in combination with in situ small-and wide-angle x-ray scattering techniques (SAXS/WAXS). SAXS provides information on the molecularparameters in solution while WAXS allows determina-tion of the crystallinity of solidified phases. Sampleswere removed from the Couette cell after shearing andexamined by Fourier-transformed infrared spectroscopy(FTIR) to detect secondary structural elements such as�-helices, �-sheets, or random coil structures. Scanningmicrobeam SAXS/WAXS26,27 was used to study theheterogeneity of the solid material after crystallizationupon weak tensile stress. X-ray scattering experimentsmade use of the ID02 and ID13 beamlines at the Euro-pean Synchrotron Radiation Facility (ESRF).

EXPERIMENTAL

Preparation Of Regenerated SilkIn order to remove the sericin layer, silk from openedBombyx mori (Bivoltin) cocoons were cut and degummed

by boiling in a solution containing 10% soap and 1%Na2CO3. After several washing steps with hot and coldwater, the silk was dried for 24 h at room temperature. Someresulting impurities were extracted by boiling the silk withpetrol-ether (boiling range of 40–60°C; Sigma 77397) un-der backflow in a Soxhlet apparatus. For the preparation ofthe fibroin solution 1 g of the degummed silk was dissolvedat 60°C in 5 mL saturated LiBr (Merck 105669) solution.The highly viscose LiBr–silk solution was diluted with 2mL of hot water (60°C) before dialysis in a cellulose acetatedialysis tube (SpectraPor� 2: 12–14 KDa cutoff) against2000 mL of demineralized water. The water was exchangedseveral times until no bromide was traceable in the dialysantusing AgNO3 as test reagent. The fibroin concentration ofthe resulting solution was measured spectroscopically at��276 nm with a coefficient of Alcm

1% (w/v)�11.3. The highlyconcentrated fibroin solutions (�80 mg/mL) were dilutedfor the shearing experiments to 5 mg/mL. This concentra-tion was a compromise to obtain a reasonable precipitationspeed and a sufficiently strong SAXS signal in the shearinggeometry described below.

Rheometry

The fibroin solution with a concentration of 5 mg/mL wassheared with a Couette-type viscosimeter (Haake RT20rotovisco), which consists of a cylinder (stator) in which ahollow cylinder is rotated (rotor). The custom made shear-ing cell was made out of polycarbonate with a gap of 1 mmbetween stator and rotor. This rheometer setup allows onlinemeasurements of the viscosity �,28,29 while the x-ray trans-parent windows permit in situ scattering studies (Figure 1).The shear rate � was increased in steps of 10 s�1 and was

FIGURE 1 Schematic design of Couette cell geometryused for x-ray scattering experiments.

Evolution of Regenerated Silk Fibroin 317

kept constant at every single step for 30 s. Given an expo-sure time of 0.55 and a readout time of �10 s up to threeexposures could be recorded for every shear step.

Attenuated Total Reflectance–FourierTransform Infrared (ATR-FTIR)Spectroscopy

Spectra were obtained with an FTIR setup (Bruker IFS55)equipped with an attenuated total reflectance (Golden GateDiamond ATR unit) accessory, which allows investigatingthe sample at room temperature and normal pressure. Theunsheared fibroin solution and the amorphous insolublematerial produced by shearing this were examined in sep-arate experiments on the ATR window without furtherprocessing. In both cases the water background was sub-tracted.

SAXS/WAXS Setup

Experiments were performed at the ESRF ID02 beam-line.28–31 The x-ray beam size was 100 �m (h) � 100 �m(v) and the wavelength � �0.0995 nm. The configuration ofthe SAXS/WAXS setup used in this study provided a SAXS

range of 0.05�Q (nm�1) � 0.6 nm�1 and a WAXS rangeof 7 �Q (nm�1) �50 nm�1. The SAXS data and theWAXS data were recorded simultaneously by means of twoCCD cameras. The SAXS data were recorded with a FrelonCCD camera coupled to an image intensifier system. Thisdetector was operated at a sample-to-detector distance of10 m. The distance of the WAXS detector to the sample(20.6 cm) was calibrated by a standard (4-bromobenzoicacid). The x-ray beam was oriented normal to the cylinderaxis of the rheometer. The scattering data were recorded attwo positions of the x-ray beam with respect to the rheom-eter cell. (Figure 1) The tangential position (gap position) isshown in Figure 1. The second position was along thecylinder radius (center position). In the center position thex-ray beam had to pass the two additional walls of the rotor,which produced a significantly higher scattering back-ground as compared to the gap position. Data recorded atthe center position showed in general the same behavior asthose obtained at the gap position, but with poorer statistics(data not shown below). The SAXS data were correctedonline for spatial distortion and further reduced using theESRF program package SAXS.32 The further data analysisof the 1-dim scattering data was done using the softwareIGOR� (WaveMetrics, Inc.) on a standard PC. The radius of

FIGURE 2 Change of the viscosity (�) on increase of the shear rate (�). Characteristic SAXSpatterns recorded in the 4 zones (see text) are shown above. Selected azimuthally averaged SAXScurves obtained in zones I/II are shown on the left side (Q�4�sin�/�). An azimuthally averagedWAXS pattern of the aggregated material is shown on the right together with a WAXS pattern ofthe fibroin solution precipitated in a methanol bath.

318 Rossle et al.

gyration Rg was derived using the Guinier approximation.33

This approach was also used to determine the changes in themolecular weight of the scattering particles. Three corre-sponding data points were averaged for every single shearrate step. The p(r) analysis as well as the calculation of thesurface models from the scattering data were performedwith the program packages GNOM and DAMMIN.34,35

Spinning of Liquid Fibroin into Methanol

For the spinning of fibroin into a methanol bath a thin glassnozzle was produced by a standard pipette puller procedure(Sutter Instruments). The nozzle diameter was �40 �m andthe inner diameter of the initial tube 0.58 mm. The tube wasglued into a syringe needle and the initial highly viscousfibroin solution was pressed with a 2 mL syringe throughthe nozzle into the methanol bath. The formation of fibroinfibrillar precipitates was visible immediately after the noz-zle. A coil of the brittle fiber precipitate was measured withthe SAXS/WAXS setup of the ID02 beamline (see above).In contrast to the shearing process (see below), aggregationwas not observed upon mixing fibroin and methanol.

Micro-SAXS/WAXS Setup

Experiments were performed at the ESRF ID13 beamlineusing a monochromatic beam with a wavelength of��0.0975 nm.26 A 5 �m beam with a beam divergence atthe sample position of about 0.2 � 0.2 mrad2 was obtainedby a condensing mirror and a 5 �m diameter definingaperture. A 20 �m guard aperture enabled the recording ofcombined SAXS/WAXS patterns using a high-resolution

CCD detector.27 The solid obtained after drying the shearedmaterial was mesh-scanned for an area of 100 �m �100�m with a step resolution of 10 �m(h) � 10 �m(v). Afterevery step a 20 s pattern was obtained with a MAR CCDdetector. The sample-to-detector distance (138.3 mm) wascalibrated by an Ag-behenate sample.36 Further data anal-ysis was performed with the FIT2D program package.37

RESULTS AND DISCUSSION

Rheometry

A typical viscosity scan of the fibroin solution isshown in Figure 2. This dataset is representative ofsimilar datasets obtained at other fibroin concentra-tions in the range described above. In all cases amultistep behavior of the viscosity � upon changingthe shear rate � was observed. The change in viscositycan roughly separated into four zones. The preforma-tion zone (I) starting from � �100 to �250 s�1 witha nearly Newtonian flow where the viscosity � ispractically independent of the shear rate �. The sec-ond zone (II) from ��250 to �450 s�1 shows anon-Newtonian behavior with a fast increase of � bya factor of about 4. In the subsequent zone (III) up to��600 s�1 the appearance of wiggles can be related

FIGURE 3 Shear rate dependent increase of the radius ofgyration Rg (■ radius of gyration; ● viscosity).

FIGURE 4 Variation of average molecular weight Mw

with increasing shear rate. The first data points were used asa reference.

Evolution of Regenerated Silk Fibroin 319

to rheological instabilities due to the appearance of asolid phase in coexistence with the liquid phase. Theformation of solid particles was also optically visible.In zone IV the viscosity drops by a factor of 2 withina small increase of the shear rate. This behavior canbe explained by the formation of large fibroin aggre-gates, which swam up and aligned at the top of therheometer cylinder (Weissenberg effect).

SAXS/WAXS During Shearing

SAXS data showed different behavior for the differentzones of the viscosity change. For the zones I/II thescattering patterns are isotropic and characteristic forindependent, randomly oriented particles in solution(Figure 2). The formation of aggregates at highershear rates leads to refraction effects at the particle–water interface. The resulting intensive streaks in thescattering patterns impede further analysis of theSAXS data for the subsequent following regions IIIand IV. It is interesting to note in this context that anucleation-dependent aggregation process has beenproposed for silk spinning based on CD spectroscopyexperiments on regenerated silk solutions.38 The ag-

gregation does not, however, produce a crystallinematerial, as WAXS peaks are absent in region IV(Figure 2). This is also true for shear rates �3000 s�1

(not shown). In agreement with other reports,39,40 theformation of silk II could, however, be provoked bypressing a regenerated fibroin solution through a smallnozzle into a methanol bath (Figure 2). Silk II forma-tion by only mixing of fibroin solution with methanolwas, however, not observed.

Evolution of Fibroin Shape Prior toAggregation

The SAXS scattering curves for zones I/II (Figure 2)were analyzed assuming the presence of independentparticles in solution. The radius of gyration (Rg) wasextrapolated from the low-angle tail of the scatteringcurves.33 Figure 3 shows the evolution of Rg withincreasing shear rate � and the corresponding changeof the viscosity. Rg remains constant within the errormargins up to ��350 s�1. The increase in viscosity iscorrelated with an increase of Rg values from �6.8 to�8 nm, which levels off at ��400 s�1. The corre-sponding change for the average molecular weight(Mw) is shown in Figure 4. These values suggest thatfibroin molecules do not associate or aggregate up to

FIGURE 5 Kratky plot of the initial fibroin state (●) andafter applying shear stress (�}); the � symbols indicate thestate prior to aggregation and } an intermediate shear point(Q�4�sin�/�).

FIGURE 6 Pair distance distribution functions of theinitial fibroin state (�) and after applying shear forces (■).Beside the change in the maximal distance the maximum ofthe distribution is changed from �11 nm (initial state) to�8 nm in the final state.

320 Rossle et al.

��400 s�1. At higher shear rates, where the forma-tion of aggregates results in streaks (Figure 2) Mw

changed by a factor of 2 for a small � change.Information on the shape evolution of the fibroin

molecules can be obtained in the medium Q rangefrom a Kratky plot.41 The weighting of the scatteringfunction I(Q) with Q2 enhances the changes fromrandom coil to more prolate and higher ordered pro-tein structures (Q�4�sin�/�). Figure 5 shows Kratkyplots of the initial state, an intermediate state for��300 s�1 and one of the last accessible states priorto aggregation at about ��400 s�1. The developmentof a maximum at Q�0.25 nm�1 suggests a transitionfrom a random-coil fibroin3 to a more compactfolded fibroin, which implies the emergence of inter-chain hydrogen-bond interactions. The emergence ofa more compact structure is also reflected in thepair-distance-distribution function p(r). This functionindicates the probability of finding a distance r withinthe particle and provides therefore direct structuralinformation. In Figure 6 a comparison of p(r) func-tions for the initial and the sheared states are shown.One of the main differences in the p(r) functions is thechange in the maximal dimension of the molecule.The increase in the maximal dimension from 25 nmfor the initial state to 30 nm indicates an extension ofthe structure under shear. On the other hand, changeof the distribution function maximum from 11 to 8 nmsuggests the development of shorter distances relatedto a more compact state. This finding is in line with

the data deduced above from the Kratky plots. Itfurther corroborates the assumption that the proteindoes not form larger assemblies in this state. Weattempted, therefore, to obtain more structural infor-mation by low-resolution solution scattering analysis.For this the scattering functions were fitted using theprogram packages GNOM and DAMMIN.42,43 Re-

FIGURE 7 Low-resolution fibroin structures derived from SAXS solution scattering. A: initialstate of fibroin molecule before the shear; B: fibroin molecule after shearing in the rheometer. Thetwo structures are on scale.

FIGURE 8 ATR-FTIR spectra of different silk conforma-tions. A: degummed Bombyx mori silk; B: regeneratedfibroin after shearing in a rheometer; C: regenerated fibroinin solution.

Evolution of Regenerated Silk Fibroin 321

peated runs of DAMMIN produced in general twodifferent structural intermediates. The structure de-rived for the initial (unsheared) state showed a glob-ular, knobby structure, whereas at higher shear ratesthe fibroin is transformed to a more cylindrical andelongated structure as shown in Figure 7. The Rg

values for these two structures increase by �1 nm forthe initial state to the sheared structure as derived inthe Guinier analysis, but with slightly different abso-lute values (7.8 nm for the initial state and 9 nm forthe sheared state). The SAXS data suggest therefore aconformational transition of the fibroin moleculeswith increasing shear rate as the molecule becomeselongated and eventually folds into a higher confor-mational state.

Fibroin Aggregates Contain �Conformation

ATR-FTIR data provide direct evidence for theexistence of �-conformation in the fibroin aggre-gate (Figure 2; zone IV). Thus Figure 8A–C shows

ATR-FTIR spectra of degummed silk (A), the ag-gregate after shearing (B), and the unsheared re-generated fibroin solution (C). The ATR-FTIRspectra for the degummed silk and the shearedfibroin show the same features. The sharp amide Iband at 1619 cm�1 as well as the amide II band at1518 cm�1 indicate a �-conformation.44,45 Thestrong absorption peak of amide II can assigned toa poly(alanylglycine) structure in the �-sheetform.44 The corresponding amide III band is lo-cated at �1230 cm�1 accompanied with a shoulderat 1260 cm�1. For �-sheet containing structures,the amide III band is found at �1235 cm�1 sup-porting the assignment of peaks for the amide I andII bands. The smaller fraction at 1260 cm�1 can beassigned to some unordered or less ordered proteinsubstructures. Note, however, that it would be im-possible to distinguish �-sheets from multiple�-turns by ATR-FTIR. In contrast to these findingsfor the solid silk, the ATR-FTIR spectra of theunsheared fibroin solution indicate a completely

FIGURE 9 A: optical image of sample obtained after shearing with zone scanned by x-raymicrobeam. B: composite SAXS/WAXS image based on a mesh-resolution of 10�m(h)�10�m(v).C1–C4: selected SAXS/WAXS patterns including a zoom of the SAXS range.

322 Rossle et al.

different structural state of the fibroin. The corre-sponding absorption for the amide I, II, and IIIbands are at 1652, 1545, and 1294 cm�1.

Fibroin Aggregate Crystallization

To verify that the sheared, aggregated material wascapable of crystallizing, a freshly aggregated samplefrom the Couette cell was exposed to air for a fewminutes and the solidified material lightly rubbed by a

spatula to enhance orientation. This was then scannedwith a 5 �m x-ray microbeam (see above). An opticalimage of the sample is shown in Figure 9 A. Theresult of the mesh scan is shown in Figure 9B as acomposite image where the individual pixels corre-spond to diffraction patterns. Figure 9 C1–C4 showsselected patterns (pixels), which vary in phase com-position, crystallinity, and small-angle scattering.Strong variations in local texture were obviously in-duced by the rubbing with the spatula. A particularly

FIGURE 10 Selected azimuthally averaged patterns. A: single degummed Bombyx mori fiber. C3

and C4 correspond to the patterns shown in Figure 9. The intensity profiles have been fitted byone-dimensional Gaussian functions for the Bragg peaks (blue lines), a broad Gaussian function forshort-range ordered material, and a first-order polynomial for the background. The upper intensitylimit has been cut in C4 in order to better visualize the weaker reflections.

Evolution of Regenerated Silk Fibroin 323

well-oriented pattern from the edge (C1) shows thatthe strongest equatorial WAXS peak is orthogonal tothe (assumed meridional) SAXS peak (for an assign-ment of the strongest WAXS peak to an equatorialreflection see, for example, Ref. 14). For a moredetailed analysis, the patterns were azimuthally aver-aged and fitted with one-dimensional (1D) Gaussianfunctions for the Bragg peaks and the short-rangeorder.46 Figure 10A shows a comparison of two pat-terns from the scanned area with an azimuthally av-eraged pattern of a single degummed Bombyx morifiber. The C3 pattern from the center of the materialshows a mixture of broad and narrow peaks and a verybroad halo due to short-range order. By comparisonwith the Bombyx mori fiber pattern, we assign thebroad peaks to the silk II structure. The narrow peakshave similar peak positions as reported for silk Istructures but show significant differences for theintegrated intensities.14,47–50 In addition, a strong re-flection in the range at about 0.72 nm observed forsilk I is not observed for the present material (TableI). Assuming a meridional low-angle peak, one canassign the first three reflections in Table I to (hk0)planes as they have the same azimuthal intensitydistribution, which is rotated by 90° from the low-angle peak in pattern C4 (Figure 10). A detailed struc-tural analysis of the sheared material is beyond thescope of the present work. We note, however, that areduction of the b-axis of the silk I unit cell proposedin Ref. 14 from 0.57 to 0.495 nm would allow index-ing the first 3 reflections as 210/310/410. The stron-gest reflection would then have the same index as inRef. 14. This and the high lattice perfection, which

has already been noted previously for dried silk Imaterials,12,14 suggests that the material is of silk Itype and that changes in unit cell parameters areprobably related to the presence of water. The lack ofthe �0.72 nm reflection, which is found in dry silk I,could, for example, be due to water molecules inter-calated between hydrogen-bonded sheets.48

The high structural perfection suggests also thatthe conversion of the amorphous aggregate fibroin tothe silk I type structure takes place without majorstructural rearrangements, which implies that the fi-broin molecule conformation is similar in both mate-rials. Pattern C4 shows that one can get locally ho-mogeneous silk I material at the edge of the samplewhile the material from the center showed an ad-vanced degree of conversion to silk II (e.g., C3). Itshould be noted that the silk I type material retainsshort-range order although to a lesser degree as silk II.The silk I/II transformation, which results in smallcrystalline �-sheet domains and an important amor-phous fraction, is obviously related to major structuralrearrangements. According to a recent model, this isdue to the transformation of chains with multiple�-turns into extended chains forming �-sheets.15 Forthis model the –[G–A–G–A–G–S]8– core sequenceseems to be able to form crystalline domains while theremaining fibroin chain forms short-range order ma-terial.

Figure 11 shows the same mesh scan as in Figure9B but limited to the SAXS range. Selected patternshave been azimuthally averaged. Depending on theposition, the patterns from the outer zones with astrong silk I content show up to two meridional peaksand a strong SAXS signal at lower angles. The 7.1 nmmeridional peak in pattern C4 is similar to the merid-ional peak found for Nephila major ampullatesilk.18,51 We do not, however, observe the character-istic two-point pattern, which is characteristic for ananofibrillar system with a lamellar morphology. Theazimuthal width of the low-angle meridional peak(e.g., C1 in Figure 9) is rather similar to that of theequatorial reflections, which suggests that the peak isdue to a c-axis lattice reflection. We note that a unitcell parameter of 7.3 nm has been proposed for silkI.52 In addition, a 5.1 nm peak is present in pattern C1

and two peaks with 6.9 and 4.7 nm are present inpattern C2. Meridional SAXS peaks have in contrastnot been observed for Bombyx mori silk II and arealso not present in those zones of the mesh-scannedsample where predominantly silk II is found (e.g.,pattern C3 in Figure 9). This supports the assumptionthat the silk I type material related meridional peaksare due to at least one hydrated structure but probablymore as shown by the two peaks in pattern C2 (Figure

Table I Reflection Positions and Relative Integratedintensities (Irel) of Silk I Type Structure Shown inFigure 10C4

a

Sheared Silk I Type Material Dry Silk I14

d (nm) Irel Assignment hkl d (nm) Irel

0.458 7 210 310 0.45 1000.414 100 310 312 0.42 140.373 38 410 410 0.40 260.335 7 — 600 0.37 340.298 2 — 610 0.32 100.261 0.7 — — 0.27 50.249 3 — — 0.24 3

a The relative intensities have been scaled to 100. For compar-ison, silk I values from Ref. 14 are shown (intensities from samplebefore sonication) with an indexation for an orthogonal unit cellwith a � 2.266 nm, b � 0.57 nm, and c � 2.082 nm (fiber axis).The assignment of the sheared silk I type hk0 reflections is based onan orthogonal lattice with a � 2.266 nm and b � 0.495 nm.

324 Rossle et al.

10). Molecular dynamics simulations support the as-sumption that the presence of water molecules in silkI is important for the conversion into silk II.53 It willtherefore be interesting to study the silk I/silk IIconversion process in situ and at different hydrationlevels in order to shed more light on this point.

CONCLUSIONS

Shearing of silk fibroin solution in a Couette cellresults in a molecular elongation, which is associatedwith a compaction of the fibroin molecules normal tothe major axis. A compaction of the fibroin moleculehas also deduced from rheological data.21 The re-ported crystallization at higher shearing rates21 is,however, not observed in the present study but rather

an aggregation process. There is no evidence from theSAXS data for the formation of 100–200 nm fibroinmicelles,54 which would result in short-range correla-tions due to the particle form factor.55 The aggrega-tion process agrees well with other studies suggestinga nucleation/aggregation process during natural silkformation.38 The aggregated material has �-confor-mation, although one cannot distinguish between�-turns or �-sheets. The compaction of the individualfibroin molecules prior to aggregation suggests al-ready the development of stronger intramolecularbonding, which might be of �-turn type, which hasbeen long thought to be an important step in proteinfolding.56 The sheared, aggregated material can beconverted into a silk I type material and silk II duringsolidification in air. Scanning microdiffraction showsthat homogeneous silk I type material can be pro-

FIGURE 11 Same mesh scan as shown in Figure 9B but limited to the SAXS range. Selected,azimuthally averaged SAXS patterns are shown. The numbering of the azimuthally averagedpatterns corresponds to Figure 9. The peaks have been fitted by Gaussian profiles and a second-orderpolynomial for the background.

Evolution of Regenerated Silk Fibroin 325

duced in this way locally. The high lattice perfectionof this material suggests that it is formed withoutmajor structural rearrangements. Differences in re-flection intensities as compared to silk I reflectionsfrom dry materials are probably due to the incorpo-ration of water molecules in the crystal lattice. In viewof the fact that silk spinning occurs from aqueoussolution, it appears that the silk I type pattern ob-served in the present study is closer to the structuresformed during fiber formation than the silk I struc-tures derived from dry materials. The tentativescheme proposed for the structural rearrangementsduring shearing and crystallization is summarized inFigure 12 with hypothetical �-turn formation startingduring molecular compaction and into the silk I phase.

We wish to thank G. Chavancy (INRA, Lyon, France) for agift of Bombyx mori silk cocoons and G. Freddi (StazioneSperimentale per la Seta, Milano, Italy) for introducing thetechnique of preparing regenerated silk fibroin solutions toone of us (MR). We also acknowledge helpful discussionswith both of them. The ATR-FTIR spectra were recorded byH. Muller from the ESRF. D. Svergun provided the softwarepackages GNOM and DAMMIN.

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FIGURE 12 Tentative scheme of structural steps occurring during shearing and solidification offibroin molecules. The assumption of the formation of �-turns in the fibroin molecules duringshearing, which is maintained in the aggregated material and in the silk I type structure, is ahypothesis which is currently only experimentally suggested for silk I.15 Note that there are probablyseveral intermediate hydrated silk I type structures prior to the formation of silk II.

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