Secondary Structures and Conformational Changes in Flagelliform

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Secondary Structures and Conformational Changes in Flagelliform, Cylindrical, Major, and Minor Ampullate Silk Proteins. Temperature and Concentration Effects Cedric Dicko,*,†,‡ David Knight,† John M. Kenney,§,| and Fritz Vollrath†,‡ Department of Zoology, Oxford University, Oxford OX1 3PS, United Kingdom, Department of Physics, East Carolina University, North Carolina 27858, and Institute for Storage Ring Facilities and Department of Zoology, University of Aarhus, 8000 Aarhus C., Denmark Received November 23, 2003; Revised Manuscript Received August 28, 2004

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperatureinduced transitions of the various silk structures toward β-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced β-sheet formation. Introduction Spiders have the ability to produce a wide range of different silks for a variety of purposes including ballooning, egg protection, or prey capture. Each silk’s role, functionality, and diversity raises the interesting question whether selection pressures on the final fiber properties are mirrored (at, e.g., the molecular level) in the precursor liquid proteins. Orb web spiders produce up to seven silks each with a specific function.1-3 This compares well with non-orbweavers such as the much more ancestral mygalomorphs with a limited silk armory produced from up to three specific gland types. Craig4,5 speculated that the principal silk proteins in the orb spiders may have evolved precise silk protein structure/function relationships to allow the silk threads to fulfill specific ecologically dependent functions. For the more ancestral spiders, Craig assumed that the principal silks were used for less demanding purposes (e.g., lining burrows or covering eggs) with little selective advantage for high differentiation of the silk proteins. Craig argued as a corollary that the diversity of silks and glands from orb-weaver spiders may suggest that silk proteins have evolved under selection for the expanded uses to which spiders put each silk.5,6 Unlike most other silk-producing animals7 such as insects, spiders possess the ability to not only combine fibers from different glands to make composites8 but also to control, adjust, and rapidly fine-tune the (i) silk protein composi* To whom correspondence should be addressed at Oxford University. Tel.: + 44 1865 271216. Fax: + 44 1865 281253. E-mail: cedric.dicko@ zoology.ox.ac.uk. † Oxford University. ‡ Department of Zoology, University of Aarhus. § East Carolina University. | Institute for Storage Ring Facilities, University of Aarhus.

tion;7,9 (ii) fiber diameter;10 and (iii) drawing rate, all leading to the production of silks with highly “tuned” mechanical properties.10,11 Most of this is achieved by constantly adjusting the physicochemical environment in the spinning duct.10,12 This ability to control spinning and, thus, thread parameters has stimulated a number of studies aimed at unraveling the structure/function relationship of various silks.10,13-31 But very little is known about the secondary and tertiary structure, assembly, and behavior of the native soluble proteins constituting the bulk of all spider silks.10,32 Genetic studies of various spider silk proteins demonstrated that they have modular designs consisting of specific motifs (see Table 1), elastic and nonelastic, with different predicted secondary structures.21,33-36 These predicted structural blocks could potentially be used as predictors of the macroscopic behavior of the silk fiber. A better understanding of silk building blocks has led to the hypothesis that “spider proteins may be selected for their propensity to undergo a structural transition to β-sheet-rich structures”.5,37 To test this hypothesis, we need to evaluate the dynamic properties of the precursor silks in light of their mechanical properties and molecular design to establish the proposed relationships between structure and function. This paper gives such an evaluation by investigating the secondary structures of flagelliform (FLAG), cylindrical (CYL), major ampullate (MA), and minor ampullate (MI) gland proteins at “in vivo” concentrations. We hoped that these data would also lead to insights into the spider’s spinning process. Indeed, we are able to show that (i) silk function is reflected at the molecular level in the precursor proteins; (ii) the temperature-induced conversion to the β-sheet-rich state provides a possible model for in vivo fiber formation; and (iii) concentration is a key factor in the natural processing of silks.

10.1021/bm034486y CCC: $27.50 © 2004 American Chemical Society Published on Web 10/16/2004

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Table 1. Types, Functions, and Predicted Secondary Structures of Four Silks from N. edulis gland MA

MI

repeatsa

function dragline silk

(GA)n/(A)n

web radial threads dispersion thread

GPGGX/GPGQQ GGX

auxiliary radial threads

(GA)n/(A) n

secondary structureb

amino acidsc,d

crystalline domain β sheets elastic β spiral 310 helix

Gly (38%), Ala (29%), small side chains (70%), polar (21%)

GGX

crystalline domain β sheets 310 helix

Gly (40%), Ala (35%), small side chains (80%), polar (19%)

extensione (%) 35

>35

FLAG

sticky spiral capture spiral

GPGGX GGX

elastic β spiral 310 helix

Gly (36%), Pro (14%), small side chains (51%), Polar (27%)

200

CYL (tubuliform)

cocoon silk

?

?

Ser (20%), Ala (27%), small side chains (56%), polar (45%)

25

a Repeats from N. clavipes.21,47 b Predicted secondary structures.21 c From amino acid composition in Table 2. d Small side chains ) glycine + alanine + serine; polar ) aspartic acid + threonine + serine + glutamic acid + tyrosine + lysine + histidine + arginine. e MA and MI,48 FLAG from Araneus spiders,19 CYL from unpublished data.

Materials and Methods Sample Preparation. Mature female “Golden Silk” spiders, Nephila edulis (Tetragnathidae), were reared semifree-range under controlled conditions.38 The proteins from all glands (MA, MI, FLAG, and CYL) were obtained by crushing the cephalothorax of the adult female spider and immediately dissecting the whole gland under spider ringer39 (adjusted to pH 7.4) before transferring it into 10 mM phosphate buffer at pH 7. The gland epithelium was carefully stripped off, and the contents of the lumen were gently blotted and collected in preweighed Eppendorf tubes, with the exception of the FLAG gland, where we used whole glands after thorough rinsing because it proved impractical to remove the epithelium from this gland. After reweighing, the material was then dissolved overnight at room temperature, 20 ( 2 °C in double-distilled water. Concentrations of 1, 5, and 20% blotted (w/w) were prepared for the MI, FLAG, and CYL glands and 0.5, 1, 2, 5, 10, and 20% for the MA gland. Throughout the paper, protein concentrations are given as percent blotted (w/w), because it is not practical to provide more absolute measurements of protein concentration for silk protein solutions. Care was taken to avoid shearing the protein solutions at all times, because this material is very sensitive to strain, particularly when concentrated.40 The tubes were left to stand vertically to allow epithelial debris to fall to the bottom of the tube. To assess the influence of the epithelial cell contents on the circular dichroism (CD) signal, separate extracts of the MA and the FLAG glands were prepared by placing 10 mg wet weight of the epithelial layer in 1 mL of distilled water. We did not observe any structural contribution from the solubilized epithelia. Protein Solution pH. The pH of the solutions was not actively controlled but carefully measured using a pH microelectrode.12 The average pH for each gland lay between 6.5 and 7. Absorption Spectra. The absorption profile of the different silk gland proteins was taken for a 1 wt % solution in double-distilled water in a 1-mm-path-length rectangular cell. The spectra were recorded at 20 °C on a Perkin-Elmer UV spectrometer.

Fourier Transform Infrared Spectroscopy (FTIR). All infrared spectra were recorded on a Nicolet Magna 550 FTIR spectrometer. To eliminate the spectral contribution due to atmospheric water vapor and CO2 the instrument was continuously purged with dry air from a purge gas generator, model PG85L from Peak Scientific. The spectra were recorded using a liquid nitrogen cooled mercury cadmium telluride detector. For each measurement, 400 interferograms were collected and Fourier transformed using a Genzel-Happ apodization function to yield spectra with a nominal resolution of 2 cm-1. FTIR measurements were conducted on low concentration proteins at 2 wt % in D2O to determine the band content in the amide I region of MA, MI, FLAG, and CYL gland proteins. The sample preparation was similar to the one described above except that the gland extracts were left to dissolve in fresh D2O instead of double-distilled water. The samples were loaded in cells made of two rectangular pieces of BaCl2 separated by a 100-µm Teflon spacer. The experiments were repeated three times for each gland. The spectra were analyzed using Peak fit software. A Fourier self-deconvolution (FSD) was performed on backgroundcorrected spectra. The background subtraction was carefully inspected for flatness in the 2000-1800 cm-1 region and fitted with mixed Gaussian and Lorentzian peak shapes. A linear baseline was used for the curve-fitting calculation. The subsequent iteration process was carried out allowing frequencies, intensities, widths, and shapes to vary. The best fit was sorted according to how the residual values were normally distributed. The goodness of fit was assessed by calculating the normalized root mean square deviation (NRMSD): NRMSD )

x

(Yobserved - Ycalculated)2 Yobserved2

where Yobserved and Ycalculated are the observed and the calculated signals, respectively. Secondary structure estimates were made by calculating the secondary structure band percentage of the total amide I (1600-1700 cm-1) using the following frequency ranges in D2O: 1613-1637 cm-1, β sheet; 1637-1646 cm-1, random; 1646-1662 cm-1, R helix;

Silk Protein Structure and Conformational Change

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Figure 1. Absorption spectra of 1 wt % MA, MI, FLAG, and CYL silk proteins in water in a 1-mm-path-length rectangular cell.

1662-1682 cm-1, turns. These assignments for curve fitting estimates of secondary structures were made according to those used by other investigators.41-45 Amino Acid Composition. Samples (at least five glands from different spiders) from MA, MI, FLAG, and CYL glands were isolated after dissection (see above) into preweighed Eppendorf tubes and left to dry at room temperature in a desiccator. After several weeks and no further measurable changes in the sample mass, amino acid analyses were conducted using an ABI 420A derivatizer/ analyzer (PE Biosystems, Warrington, U.K.) with a narrowbore HPLC system (Applied Biosystems 130A) after hydrolysis for 24 h at 110 °C in 5.7 N hydrochloric acid. The ABI 420A utilizes precolumn derivatization46 with phenylisothiocyanate to form phenylthiocarbamyl amino acids. Data handling was performed using Dionex Chromeleon software (version 6.40 from Dionex UK, Ltd., Macclesfield, U.K.). CD. Solution samples for CD were transferred to a Suprasil quartz sandwich cell (Hellma 124-QS). Spectra were collected at 20 °C on the synchrotron radiation based CD facility at ISA, Aarhus, Denmark. For the 0.5% concentration, the far-UV CD was collected in a 0.2-mm sandwich cell, in a 0.1-mm sandwich cell for 1 and 2% concentrations, and in a 10-µm sandwich cell for 5, 10, and 20%. Great care was taken to check and avoid bubbles and shear (the loaded cells were checked with a polarizing microscope) when using the short path length cell. The control voltage that determines the high-tension (HT) dynode voltage of the photomultiplier tube was recorded with each CD spectrum to indicate the reliability of each CD spectrum. In all our measurements, the control voltage was -5 V (corresponding to a 600-V HT) for a completely transmitting sample and +5 V (1600-V HT) for a totally absorbing sample. All samples were scanned three times with a 3-s accumulation time, and the results were averaged from at least three repeated experiments. Temperature-Induced Transition. Samples were loaded into sandwich cells (see above) and sealed with quick-drying

nail varnish to prevent evaporation. The samples were scanned from 20 to 80 °C, with a 10 °C step size. At each step, the sample was allowed to equilibrate for 20 min. The CD scans were recorded once with a 6-s accumulation time, and the experiments were repeated at least three times with different spiders’ samples. At the end of each temperature melt, the system was slowly cooled back to 20 °C. In all cases we did not observe a recovery of the starting spectra; thus, the heat-induced changes were nonreversible. Fractional changes in conformation were plotted against temperature using the following equation: fu )

θ0 - θmin θmax - θmin

where θ0 is the ellipticity at a temperature T and θmax and θmin represent the ellipticities of the final and initial states, respectively. Because the temperature effects are not reversible, no quantitative thermodynamics of the change could be obtained. The transition midpoint Tm, independent of the model, was determined by fitting the fractions fu with a simple Boltzmann sigmoid. Results UV Absorption and Amino Acid Composition. In the initial stage of the study, we collected the UV absorption spectra of MA, MI, FLAG, and CYL gland proteins at a concentration of 1 wt % in distilled water (Figure 1). Specific features were identified: in the MA proteins, three distinct bands at 274, 365, and 440 nm and a shoulder at 280 nm; in the MI proteins, a band at 275 nm with a shoulder at 280 nm; in the FLAG, a single peak at 270 nm; and in CYL, four bands at 275, 315, 358, and 440 nm. Only the MA and CYL were yellow in color, a feature that was supported by the presence of absorption bands in the region of 300-500 nm in both glands; these bands were absent in MI and FLAG samples. The amino acid composition of all four glands (see

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Figure 2. FTIR spectra of MA (a), MI (b), FLAG (c), and CYL (d) at 2 wt % in D2O and deconvolved bands (see text for attribution). Table 2. Amino Acid Compositions (%) of MA, MI, FLAG, and CYL Glandsa amino acid

MA

MI

Asxb Glxc Ser Gly His Arg Thr Ala Pro Tyr Val Met Cys Ile Leu Phe Lys

1.44 ( 0.38 9.3 ( 0.49 2.91 ( 0.13 38.17 ( 1.44

1.87 ( 0.08 3 ( 0.03 4.38 ( 0.03 39.86 ( 0.65

a

FLAG

4.25 ( 0.51 5.18 ( 0.45 5.57 ( 0.92 35.48 ( 2.03 0.17 ( 0.35 2.09 ( 0.32 1.92 ( 0.09 1.99 ( 0.11 0.68 ( 0.16 1.52 ( 0.04 3.32 ( 0.58 28.87 ( 3.01 34.68 ( 0.21 10.13 ( 0.15 6.24 ( 4.46 13.82 ( 0.48 3.83 ( 0.29 5.14 ( 0.09 2.6 ( 0.14 0.89 ( 0.21 1.82 ( 0.09 2.87 ( 0.19 0.23 ( 0.02 0.50 ( 018 0.99 ( 0.26 0.05 ( 0.02 0.04 ( 0.01 0.18 ( 0.03 0.56 ( 0.14 0.95 ( 0.03 2.94 ( 0.16 2.74 ( 0.47 1.9 ( 0.06 3.26 ( 0.18 0.49 ( 0.14 0.78 ( 0.03 1.46 ( 0.15 0.90 ( 0.44 1.1 ( 0.17 4 ( 0.41

Table 3. Amount of Secondary Structure from FSD of FTIR Spectra of MA, MI, FLAG, and CYL at 2 wt % in D2O

CYL 3.87 ( 0.13 8.59 ( 0.27 20.18 ( 0.02 8.61 ( 0.55 0.32 ( 0.04 2.18 ( 0.06 5.89 ( 0.08 27.31 ( 0.47 2.05 ( 0.11 2.75 ( 0.05 1.44 ( 0.23 0.09 ( 0.01 2.79 ( 0.05 7.41 ( 0.17 3.85 ( 0.18 1.64 ( 0.13

Average of five glands. b Asx: Asp and Asn. c Glx: Glu and Gln.

Table 2) showed the presence of tyrosine residues and phenylalanine that have a characteristic absorption band in the 270-285-nm region. The UV spectra gave no indication of the presence of tryptophan residues in any of the silk solutions. Table 1 summarizes the amino acids abundances in the lumen content of all four glands (see Table 2 for details). MA and MI were dominated by amino acids with small side chains, mainly alanine and glycine, and with an overall low polarity. FLAG had a balance of small side chains as well as polar groups, with a predominance of glycine and proline residues. CYL had a smaller amount of small side chains and a larger proportion of polar groups, with predominance of alanine and serine residues. Infrared Data. Solution-state conformational information was obtained in D2O using FTIR spectroscopy; the deconvolved spectra of MA, MI, FLAG, and CYL are presented in Figure 2, with a secondary structure assignment in Table 3 (see Materials and Methods for band assignments). The FSD yielded at least four major bands in all four glands. In

MA MI FLAG CYL

helices (%)

sheets (%)

turns (%)

disordered (%)

NRMSDa

38.2 59.5 47 57

11 6 5 5

24.8 9.5 25 11

26 25 23 27

0.006 0.005 0.004 0.007

a NRMSD: normalized root mean square deviation (see Materials and Methods).

MA the bands were 1608, 1620, 1632, 1651, 1670, and 1690 cm-1; in MI the bands were 1615, 1636, 1657, and 1683 cm-1; in FLAG the bands were 1614, 1632, 1650, and 1671 cm-1; and in CYL the bands were 1613, 1633, 1652, and 1675 cm-1. The spectra in Figure 2 were approximately centered at 1650 cm-1, suggesting that helical or disordered structures dominate. In general in H2O the FTIR spectra will not discriminate between helices and disordered, but in D2O a split occurs with the disordered structures at 1644 cm-1 and the helical structures at about 1648-1657 cm-1. This is due to the extreme sensitivity of disordered structure to deuteration,49-51 as a result of a favorable H-D exchange of its amide N-H groups that is not involved in stabilizing H bonds in helical structures. However, in all four spectra we did not observe any split, suggesting that the proton from the amide groups was predominantly undissociated. Although we could not assess the extent of the H-D exchange, the absence of a split suggested that silk proteins have at least some short-range ordered structures. Table 3 shows that all four glands had approximately the same composition of disordered structures, and that MI and CYL have the highest helical content compared to MA and FLAG. On the other hand, MA and FLAG had the highest turn content when compared to MI and CYL. The content of β-sheet structures although not predominant was similar in MI, FLAG, and CYL (about 5%) but higher in MA (11%).


Silk Protein Structure and Conformational Change Table 4. Concentration Effects on Band Shifts MAa

a

0.5%

1%

2%

5%

10%

20%

217.6 ( 0.6 198.9 ( 0.7

218.6 ( 0.7 199.4 ( 1.0

219.6 ( 1.0 200.5 ( 0.9

220.0 ( 1.0 200.3 ( 1.8

219.5 ( 0.9 201.3 ( 0.8

219.8 ( 1.1 203.6 ( 1.3

MI

217.8 ( 0.6 206.2 ( 1.6

218.4 ( 0.4 203.7 ( 0.2

218.9 ( 0.7 201.9 ( 1.8

218.1 ( 0.6 199.1 ( 2.3

FLAG

221.5 ( 0.3 206.9 ( 0.4

221.2 ( 0.5 207.5 ( 1.1

219.1 ( 0.5 205.8 ( 1.0

220.8 ( 1.0 205.2 ( 0.9

CYL

221.1 ( 0.6 209.1 ( 0.6

220.1 ( 0.5 208.7 ( 0.4

220.6 ( 0.4 208.7 ( 0.1

See Figure 4 for plots.

Figure 3. Comparison of the CD and the HT spectra of MA, MI, FLAG, and CYL at 5 wt % in distilled water in a 10-µm sandwich cell at 20 °C (see text).

CD. CD Signature of MA, MI, FLAG, and CYL Silk Gland Proteins. Figure 3 shows the CD and HT spectra of all four glands at 5 wt % in distilled water. All four spectra were markedly different from each other and presented specific spectral features. In MI, there were two negative bands at approximately 218 and 204 nm and a positive band at 190 nm. In MA, as previously reported,12,38,52,53 we observed a negative plateau at 217 nm and a strong negative band at 199 nm (and a putative band at 185 nm). In FLAG, we observed an inflection point at 220 nm and a negative band at 206 nm. In CYL, we found two negative bands at 220 nm and 208 nm and a strong positive band at 192 nm. In general the bands at 220 and 208 nm are associated with helical structure and are always found with a positive band at 192 nm. The plateau at approximately 217 nm indicates the presence of β-sheet and/or turn structures. The strong negative band at 199 nm is commonly associated with the presence of disordered structures. Thus, CYL and MI were helix-dominated while FLAG gave predominantly “helix/turns/sheet” spectra thought to arise from β-spiral folds.34,54-57 MA spectra showed predominantly disordered and β-sheet structures. However, polyproline II type structure is also a valid interpretation of the MA CD spectrum. The difficulty in properly attributing conformations to MA protein is illustrated by the apparent contradiction between the FTIR and the CD data (see above). Concentration Effect. Table 4 shows the effect of increase in protein concentration on the CD band positions. We interpreted the shifts in wavelength with concentration as

Figure 4. Concentration effects on the bands shifts and folds of MA protein CD spectra: (a) band 1 (nm), (b) band 2 (nm), and (c) ratio of band 1 to band 2. This ratio, also referred as the r value,58 is independent of the protein concentration and is a good indicator of changes in secondary structures.58 Here we observe that an r value of about 0.3 remained unchanged as concentrations increased to 10 wt % but increases to 0.6 at a concentration of about 20%. This change corresponded to an increase in helicity in the CD spectra of MA protein in water.

followed: a shift to longer wavelength (red shift) implied more interactions, a shift to shorter wavelengths (blue shift) implied less interactions, and no changes with concentration suggested that intramolecular interactions were dominant compared to intermolecular interactions. We observed that within the experimental error CYL and FLAG did not shift their band position with concentration. However, in MI we observed a blue shift in a single band from 206 nm at 1 wt % to 199 nm at 20 wt %. The overall shape of the MI CD spectrum was slightly changed, suggesting that at a low concentration the protein environment favors folded structures whereas at a high concentration looser structures would be favored. In MA, we observed a shift from 198 to 204 nm and a shift from 217 to 220 nm with increasing concentration to a more helical-type structure. Figure 4 shows the change in band position in MA glands. At both 217 and 200 nm the variations were nonlinear and could result from a large change at lower concentration followed by a slower change at higher concentrations. In Figure 4, panel c, the r value changed slowly at low concentrations but more rapidly as concentrations exceeded

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Figure 5. CD spectra of temperature-induced transition in MA (a), MI (b), FLAG (c), and CYL (d) at 20 wt % in water at different temperatures. The arrows indicate the direction of change with increasing temperatures. The temperature was varied from 20 to 80 °C in steps of 10 °C. The MA and MI gave β-sheet-rich structures, whereas FLAG and CYL unfolded to a β-turn-like structure.

10%. We interpreted this increase in the r value as an increase in helicity in the MA protein. In other words, the storage form of MA proteins in the ampulla involved ordered structures and intermolecular interactions. Temperature-Induced Conformational Changes. Figure 5 shows the CD spectra at different temperatures of all four glands. With increasing temperature, MA and MI changed irreversibly to β-rich structures. However, the final structures of this transition differed; in MI the characteristic bands from β-sheet structures lay strongly at 196 and 217 nm, whereas in MA the positive band at 196 nm was only just detectable while the 217 nm band was pronounced. In FLAG and CYL, on the other hand, increased temperature led to an irreversible loss of structure. The thermal denaturation of both these glands yielded a structure with a negative band at 217 nm and, for CYL only, an additional positive band at 185 nm. The final structure in CYL suggested β-turn structures. Table 5 shows the midpoint as well as the isodichroic point of the transition. The isodichroic points of MA and MI were similar at approximately 210 nm, while in FLAG and CYL they lay at approximately 202 nm. The midpoint of the transition was the highest in MA with 54.91 ( 6.4 °C, followed by FLAG at 43.96 ( 5.35 °C, MI at 41.02 ( 1.88 °C, and CYL at 37.77 ( 2.38 °C.

Table 5. Transition Temperatures and Isodichroic Points MA MI FLAG CYL

B. morib A. pernyib

isodichroic point (nm)

Tm ( stdeva

210 209 201 203

54.9 ( 6.4 41.0 ( 1.9 43.9 ( 5.3 37.8 ( 2.4 60.5 70

a Standard deviation. b Peak position from DSC study of native silkworm fibroin at 25 wt % in water from ref 59.

The midpoint transitions were determined from the fraction of change in all four glands as presented in Figure 6. Differential scanning calorimetry (DSC) determination59 of temperature transitions in two types of silkworm fibroin is given for comparison. Antheraea pernyi silk fibroin, which is comparable to MA spidroin in its gene sequence,76 shows a higher Tm, whereas Bombyx mori silk fibroin shows a similar value compared to that of MA and higher compared to those of proteins from the other glands. We observed that the fractions of structure change (Figure 6) were comparable at 200 and 217 nm in MI and at 191 and 220 nm in CYL, suggesting a two-state transition in both glands. For MA and FLAG, on the other hand, we observed that the fractions at different wavelengths changed independently, suggesting a multiple-state transition. In MA the



Figure 6. Melting curves of MA (a), MI (b), FLAG (c), and CYL (d) at 20 wt % in water as a function of temperature (see text). In all panels the fractions at 196, 200, 204, 217-218, and 222 nm were attributed respectively to β-sheet structures, disordered structures, partially helical structures, β-sheet structures, and R-helical structures. CYL (d) shows a comparable decrease of its fraction at 222 and 191 nm suggesting that all the protein domains unfold simultaneously. MI (b) shows a direct conversion of the protein to the β-sheet form with increasing temperature. The shift to higher temperatures of some fractions of change in both MA (a) and FLAG (c) may indicate a multiple-step conversion and unfolding, respectively.

Discussion

Figure 7. Effects of MA protein concentration on the transition temperature (Tm) determined by CD spectroscopy. The decrease of the Tm with concentration suggests that the transition to the β-sheetrich structure is promoted by protein-protein interactions. Note that heating did not produce a secondary structure transition in the least concentrated (0.5%) MA solution.

fraction at 217 nm was delayed, suggesting that the formation of the β-sheet structures either required more energy than in MI or occurred only after the conversion of other structures. In FLAG the band at 222 nm was delayed, suggesting that the helical structures were affected only at higher temperatures. The FLAG CD shows two separate behaviors, first a collapse of structure with the absence of an isodichroic point and then, second, a transition with an isodichroic point at 201 nm (Figure 5). This suggests that the β-spiral structure of FLAG unwinds or collapses before undergoing a further transition. Concentration Effects on the Melting Temperature of MA Protein. We investigated the effect of concentration on the Tm in MA (Figure 7). We observed an increase in order in MA protein secondary structures and a decrease of the Tm with concentration. This suggested that the temperatureinduced transition was a cooperative process, involving in this case intermolecular β-sheet structures. Also, most importantly MA proteins were stored in the ampulla as ordered structures.

MA and MI Proteins: MA and MI proteins’ CD spectra differed greatly despite the fact that small side chain (alanine and glycine) residues dominated the amino acid compositions of both proteins. A NMR study by Liivak et al.60 showed that approximately half the alanine residues contribute to the crystalline β-sheet regions in MA as compared to only a small fraction in MI fibers. This suggests that the high tensile strength of MI silk is only partially due to β-sheet structures and that MI proteins have different cross-linking mechanisms and matrix properties compared with those of MA. These different cross-linking mechanisms would depend on the protein composition, secondary structures, and molecular interactions. For example, proline residues were virtually absent from MI but accounted for about 6.2% of the amino acid residues in MA. This would allow the MA protein in solution to explore more conformations but would also present a large steric hindrance (similar to collagen), hence, preventing the formation and stabilization of R helices. These differences between MA and MI were supported by the FTIR data in Table 3, where the MI structure was helix-dominated. When compared to Nephila claVipes (Tetragnathidae) and Araneus diadematus (Araneidae), the amino acid composition of MA showed that the ratio of alanine to glycine was higher in Nephila (3:4 and 3:5 respectively for edulis and claVipes) than in the A. diadematus (2:5).20,61-64 N. edulis showed a higher content of small side chains, about 70%, compared to A. diadematus64,65 and N. claVipes63 both at about 62%. The polar chain contents showed similarities between A. diadematus and N. claVipes, respectively 26 and 29% compared to N. edulis at about 21%. We noted as well that in B. mori63 small side chain residues represent about 86% and polar chains about 25%, suggesting that the proportion of alanine to glycine is important to determine the relative ratio of crystalline to amorphous regions in silk.

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Another striking difference was the proline content in MA silks, about 15% in A. diadematus, 1% in N. claVipes, and about 6% in N. edulis, suggesting a higher concentration of turns in A. diadematus silk secondary structures. Surprisingly, the amino acid composition of MI glands was similar in Nephila and Araneus spiders. The noted absence of histidine in MA and MI amino acid compositions, as well as the absence of proline in MI, and the composition of charged and polar residues (Glu/Gln, Asp/Asn, and Ser) showed that although closely related, the three spiders showed speciesspecific differences in their amino acid compositions. FLAG Gland Proteins. FLAG showed an amino acid composition, as well as a different CD spectrum, very different from that of all the other silk gland proteins studied here. In FLAG, the predominance of the small side chain proteins was reduced to 50%, and it consisted mainly of glycine and proline. Proline residues are thought to affect the organization of the noncrystalline regions of the protein and, hence, the silk elasticity.19,34 Craig and Riekel showed that FLAG fibers are structurally different from the other silks and do not show any crystalline fraction.33 This was later confirmed by the absence of crystalline domains in the FLAG gene sequence.34 Our FTIR data supported this view, showing that FLAG was mainly composed of helices, turns, and disordered structures. The CD spectrum of FLAG was similar to that predicted from the β-spiral model of Zhou et al.66 The high polar chain content suggested that the β-spiral structure was favorably stabilized in water. Molecular dynamics simulations on the polypentapeptide of elastin, also forming a β spiral, supported this view.67 But the forces involved in maintaining the FLAG fold in native silk are poorly understood. However, the absence of change of structure with increasing protein concentration suggested that intramolecular interactions predominated. Our FTIR data revealed a low content of β-sheet structures and a relatively high content of helices and turns both stabilized by hydrogen bonds. CYL Gland Proteins. CYL proteins such as FLAG proteins showed a small percentage composition of amino acids with small side chains (mainly alanine and serine) but the largest proportion of polar residues of all four glands. At the moment, there are no gene sequences available for CYL proteins for Nephila spiders; however, in A. diadematus Guerette et al.20,68 showed that the CYL protein gene sequence was partially AdF2 type (A. diadematus Fibroin type 2) and partially novel. The prediction of AdF2 secondary structures21 suggests a mix of β-sheet and 310-helical structures. Such a prediction, however, would be inconsistent with our spectroscopic observations (CD and FTIR) that helical structure predominates in CYL and that the amino acid composition of CYL protein is markedly different from that of AdF2. Direct comparison of CYL full gland contents to those of A. diadematus showed more fundamental differences: no proline and a much lower content of polar residues (Glu/Gln, Asp/Asn, and Ser) in N. edulis. Temperature-Induced Transition and Concentration Effects. The relationship between silk protein sequences and the mechanism of transformation from the storage form in the gland to a β-sheet-rich structure typical of the fiber

Dicko et al.

requires further elucidation.10,69,70 It has been previously shown (for MA) that increasing temperature drives the initial state to form a β-sheet-rich structure.53 A similar observation has been reported for silkworm fibroin59 and further suggested that the transition to a β-sheet structure is the basis for the silk protein phase separation, the nucleation mechanism, and fiber formation later on in the production pathway.71 Generally the mechanism of temperature-induced β-sheet structure transition is unclear for any protein. The first record of the transition in a model peptide [poly(Llysine)] by Davidson and Fasman72 suggested that upon increase of temperature the transition from helix to β structure is promoted by the random coil intermediates and dominated by intermolecular interactions. Mattice, in a review article,73 suggested that the temperature-induced transition to β structure is promoted by favorable depletion of the random coil by interstrand loops (consecutive strands connected by β bends), leading to the formation of larger β-sheet structures. The disordered interstrand loops would have a lower entropy because the ends are constrained. A recent study on dragline silk proteins by Oroudjev et al.74 proposed similar packing, also postulated by Knight and Vollrath.75 In our study we observed for MA and MI silk proteins that the intermediate forms during the temperature-induced transition went through a “helical state” before reaching their final sheet-rich structure. In this respect, the initial concentration was a crucial parameter to promote transition. It is to be noted that MI had a higher helical content and, hence, a lower Tm while MA at a low concentration was predominantly random coil with a high Tm and at high concentration was dominated by helical structure with a low Tm. This behavior suggested a cooperative mechanism at high concentration. In fact, large random chain entropy would be more easily reduced by local intramolecular interactions at high concentration while at a low concentration the Mattice model would apply. However, at this point we were not able to show that MI proteins followed the same mechanism, particularly because of their apparently contrary dependence of wavelength shift in CD spectra on concentration. In FLAG and CYL we observed very different transition behaviors. FLAG and CYL temperature denaturation leads to structures similar to a β turn that as of yet are not identified further. Both silks showed a classical decrease in CD band intensity, suggesting a loss of structural organization. In FLAG, the loss of structure would appear to contradict the behavior of the elastin β-spiral model peptide that showed an inverse transition temperature forming more ordered structures at a higher temperature.77,78 Two major differences may explain this discrepancy: (i) the higher polarity of FLAG proteins and (ii) the more complex content of the FLAG solution studied compared to the β-spiral model peptide. These differences could help reduce the impact of hydrophobic collapse at high temperature, hence, preventing a hydrophobic formation of further structures. In summary, the absence of change in the CD spectra of CYL and FLAG with increasing protein concentration supported the idea that intramolecular interactions dominated the stability of CYL and FLAG proteins. This showed that


CYL and FLAG compared to MA and MI had different inherent chain connectivities, meaning that a greater molecular cooperation was necessary for internal readjustments in MA and MI. Storage structures and a crystallization mechanism in the respective glands would be dependent on other factors, such as the physicochemical environment and the geometry of the spinning pathway. For example, one can intuitively see that the overall shapes of the proteins, dictated by their interactions, will have a dramatic impact on their rheologies. To this respect, it is worth noting that the four glands examined in this study have processing ducts of variable size: long for the MA and MI but short for CYL and FLAG. Conclusion In this paper we present evidence that different silks, with different in vivo functions, exhibit different folds in solution. However, the extrapolation from the liquid feedstock to the properties of the solid silk fiber is not easy because the transition involves dramatic physical changes including rapid extensional flow and phase separation. But we could clearly establish that silk protein polymerization is based on surprisingly different amino acid compositions and initial secondary structures. Our observations suggest that variation in percentage composition of polar chains and small side chain residues helps to account for specific secondary structure signatures. This in turn suggests that, despite the relatively low information content of silk protein sequences,53 there is a strong bias toward specific interactions and secondary structures such as turns, short-range order, and helices. We present evidence for the existence of a cooperative inverse temperature transition in all four silks, leading to an increase in β-sheet structures. Low Tm and helical-like storage structures provided evidence that the natural fiber formation mechanism is an energy-efficient process allowing easy protein transport and polymerization. Further detailed work using our comparative approach should provide more data to study the relationship between amino acid composition, primary sequence, secondary folding, and fold conversion. This should eventually lead to the production of designer block copolymers with defined mechanical properties. Acknowledgment. We thank the British Biological and Engineering Research Councils (BBSRC-S12778, EPSRCGR/NO1538/01), the European Commission (F5-G5RD-CT2002-00738), AFSOR of the United States of America (F49620-03-1-0111), and the Danish Natural Sciences Research Council (SNF-21-00-0485) for financial support. We further thank the Institute for Synchrotron Radiation, ISA, Aarhus, Denmark, for use of their CD facility through an EC-Human Potential Program Transnational Access to Major Research Infrastructures (EU Contract No. HPRI-CT-200100122). We are grateful to Dr. Ann Terry, Dr. David Porter, and Dr. Alex Sponner for reading the manuscript and for helpful discussion and Else Rasmussen for technical support. We thank three anonymous reviewers for their useful and pertinent comments.


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Secondary Structures and Conformational Changes in Flagelliform

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