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J. Phys. Chem. B 1999, 103, 11382-11392
Methionine Redox Controlled Crystallization of Biosynthetic Silk Spidroin R. Valluzzi,† S. Szela,† P. Avtges,† D. Kirschner,‡ and D. Kaplan*,† Tufts Biotechnology Center, Department of Chemical Engineering, Tufts UniVersity, 4 Colby Street, Medford, Massachusetts 02155, and Department of Biology, Boston College, Chesnut Hill, Massachusetts 02467 ReceiVed: April 26, 1999; In Final Form: October 4, 1999
The formation of intractable β-sheet crystallites is a major cause of insolubility in proteins that can form β-sheets. To study this phenomenon, recombinant DNA techniques were used to prepare a protein modeling the consensus sequence of Nephila claVipes spider dragline silk, incorporating redox “triggering” residues. X-ray diffraction, electron diffraction, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) were used to characterize the ability of the recombinant protein to form β-sheet crystals dependent on the redox trigger oxidation state. Changes in the crystallinity were observed when triggered (oxidized/soluble) and untriggered (reduced/insoluble) protein samples were compared. The β-sheet content was undetectable in the triggered state, while clear evidence of β-sheet crystallinity was observed in the untriggered state. TEM and electron diffraction data of thin films of the untriggered protein indicated that spontaneous local orientation of needlelike crystalline aggregates occurs over small regions, suggestive of morphologies analogous to native dragline silk. There was also evidence of a liquid crystalline or oriented amorphous phase in the untriggered protein, but the d-spacings observed for the liquid crystal did not match any structure reported for the natural spider silk. To elucidate the behavior of the amorphous phase, we synthesized a water-soluble 27-residue peptide model of the dragline silk consensus amorphous sequence. The interchain packing distance observed for crystals of this peptide matched the d-spacing observed for the amorphous phase in the untriggered recombinant dragline silk protein. The results suggest that methioninemodified silks assemble into a silk-like structure in the reduced state (untriggered), structure which is lost upon oxidation or activation of the triggers.
Introduction Among engineering fibers, desirable properties include high modulus, high extension to break, good compression strength, and processability. Many synthetic fibers, such as Kevlar fibers and ultrahigh density polyethylene fibers, achieve a high modulus and tensile strength through very high crystallinity. These fibers tend to be brittle rather than tough and fail in compression. Spider dragline silks, while not achieving the extremely high moduli of some synthetic fibers, have a high elongation to break and are stronger in compression.1,2 The various failure modes are coupled to the detailed microscopic structure of the entire fiber, not simply to the degree of crystallinity. Spider dragline silk has achieved a good combination of properties through an apparent hierarchy of organized structures. The primary structure (sequence) contains repetitive motifs, such as short runs of polyalanine, that are expected to readily form β-sheet crystallites. These highly repetitive motifs are interspersed among less repetitive sequences that are believed to form the amorphous phase.3 The primary structure of native spider silk is thus somewhat blocky, while the repetitive nature of the sequences within each block facilitates the formation of a consistent regular helical conformation. The exact nature of the helical conformation will depend on the environment of the protein and its sequence. Continuing this theme, the primary * To whom correspondence should be addressed. Fax: 617-627-3900. E-mail:
[email protected]. † Tufts University. ‡ Boston College.
structure and environment would then cause portions of these regular helical structures to aggregate into β-sheet crystals. The less repetitive amorphous blocks, which may also adopt helical secondary structures, would form the matrix of the fiber, aggregating or self-assembling into a liquid crystalline mesophase, nonperiodic β-sheet (paracrystalline structure), or other oriented amorphous (or not fully crystalline) structure. There may also be aggregated unoriented amorphous matrix present. The relative proportions of amorphous, oriented amorphous/ paracrystalline, and crystalline material phases will influence the properties of a hierarchically organized material, such as a spun fiber. The relative arrangement of these phases, the nature of the interphase between crystalline and noncrystalline material, and the manner in which the protein chains are dispersed among the various phases will also help determine the overall properties of the fiber. The interaction between the different phases of material in spider silk and the nature and function of the noncrystalline material in spider silks is not fully understood. To understand the coupling between macromolecular architecture and the self-assembly processes and interesting macroscopic functional properties observed in silks, protein models simplifying the problem are needed. One of the hurdles in characterizing the process of macromolecular organization of spider silks such as the dragline from Nephila claVipes is low solubility due to rapid formation of fibrillar structures. The general intractability of the as-spun fiber and the difficulties encountered in control of solubility suggest that alternative approaches are needed to render the intermediate steps in the silk spinning process accessible. To address this need, we designed and synthesized a recombinant protein, incorporating
10.1021/jp991363s CCC: $18.00 © 1999 American Chemical Society Published on Web 12/07/1999
Crystallization of Biosynthetic Silk Spidroin solubility “triggers”, based on a modification of the repetitive consensus sequence from the dragline of N. claVipes. The model protein incorporates methionine residues flanking the pentaalanine β-sheet forming regions of the protein to create a redox solubility trigger. Using different oxidation and reduction conditions, we were able to control β-sheet crystal formation and thus the ability of this modified protein to reproduce N. claVipes dragline silk’s crystal structure. The designed synthetic protein was also capable of self-organizing into a hierarchically ordered structure. The complex behavior observed for the modified protein may provide insight into the assembly steps related to the formation of spider silk’s macroscopic structure. Experimental Section Recombinant Protein. Gene Design and Protein Expression. A synthetic gene of roughly 100 base pairs (bp) was constructed based on the consensus sequence from the highly repetitive dragline silk gene of N. claVipes, using strategies we have previously reported.4,5 The major difference in this construct was the inclusion of codons for methionines flanking a pentaalanine repeat in the sequence. The 100-bp sequence was used as a building block for the construction of a larger-sized gene encoding ∼25 kDa silk protein. The methionines were included to act as a redox trigger, as has been demonstrated for globular proteins.6 The methionine side chain can be oxidized to the sulfoxide state to disrupt hydrophobic interactions between overlaying sheets, thus maintaining solubility of the protein. The procedure can be reversed by reducing methionine sulfoxide with β-mercaptoethanol to its original state, leading to hydrophobic interactions in the polyalanine regions. Escherichia coli strain BLR(DE3) (F- ompT hsdSB (rB mB-) gal dcm ∆(srl-recA)306::Tn10 (DE3)) (Novagen) was used as a host for cloning and expression. The silk gene was cloned in the BamH1 site of the expression vector pET29a (Novagen) and expressed and purified using standard procedures.4,5 Cells were harvested by centrifugation and lysed by French Press. Complete Mini Protease Inhibitor Cocktail (Boehringer Mannheim) and lysozyme (200 µg/mL) were used in the lysate buffer. The supernatant of the lysate was stored at -70 °C for further processing. An S-Tag Purification System (Novagen) was used to purify recombinant proteins from the lysate according to manufacturers recommendation. The purified protein solution was subsequently dialyzed extensively against water to remove buffer salts and other contaminants. Characterization of Purified Protein. All characterization was carried out on a protein containing eight repeats of the building block: eight penta-alanine domains with 16 methionines within the ∼25 kDa protein. The recombinant protein was characterized by SDS-PAGE, amino acid analysis, and MALDI-TOF to validate size and composition (data not shown). The amino acid analysis and MALDI-TOF were carried out at the Tufts University Medical School core protein facility. Circular Dichroism (CD) spectra were recorded in a 1-mm path length cell at concentrations of 0.2 mg/mL on an Aviv60DS spectrophotometer. All spectra were recorded at 25 °C. Protein solution concentrations were calculated using the standard BCA assay. The instrument was routinely calibrated with d-camphorsulforic acid. Secondary structure analysis was performed using algorithms provided by the manufacturer (Aviv Associates, Lakewood, NJ). Synthetic Peptide. To better understand the behavior of the amorphous phase in the recombinant dragline silk-like protein, a model spidroin peptide incorporating only the consensus sequence of the amorphous blocks of spider dragline silk was
J. Phys. Chem. B, Vol. 103, No. 51, 1999 11383 synthesized at the Tufts University Core Protein Chemistry Laboratory. The sequence of this peptide was (Arg)3 (Gly-GlyAla-Gly-Gln-Gly-Gly-Tyr-Gly-Gly-Leu-Gly-Ser-Gln-Gly-AlaGly-Arg-Gly-Gly-Leu-Gly-Gly-Gln-Gly-Ala-Gly) (Arg)3 (in single letter amino acid codes, R3GGAGQGGYGGLGSQGAGRGGLGGQGAGR3) with the arginine residues added to the ends of the peptide to render it soluble in water.7 The consensus sequence of this amorphous region was based on clones of the native gene.3,8 The peptide was synthesized using standard solid-phase Fmoc chemistry on an Applied Biosystems 431A using HBUTAT activation. Reverse-phase HPLC with a C18 column was used to purify the peptide, and the peptide composition and purity were verified using mass spectrometry. Protein Modification. Oxidation of the recombinant protein was performed by adding a solution of phenacyl bromide in ethanol to an S-Tag purified and dialyzed protein solution sample (approximately 1 mg/mL). The reaction was allowed to proceed at room temperature in the dark for 2.5 h and was stored at -20 °C. Solutions of the recombinant protein were examined (a) in the as-expressed/reduced form, (b) in the reduced form using β-mercaptoethanol, and (c) with phenacyl bromide added to oxidize the methionine triggers. The as-expressed protein is expected to be in the reduced form due to the reducing environment in the host cell. The solutions of oxidized recombinant dragline silk protein were dialyzed against pure water for 3 days to remove the phenacyl bromide oxidant. The peptide synthesized to mimic the spidroin amorphous sequence was also examined. The peptide was soluble at 7 and 40 mg/ mL in 18 MΩMillipore pure filtered water. No solubilityenhancing additives were needed. The following solutions were examined: (1) reduced (as expressed) recombinant protein; (2) stoichiometric (1:1) ratio of oxidant to methionine, recombinant protein; (3) 100:1 ratio of oxidant to methionine, recombinant protein; (4) 500:1 ratio of oxidant to methionine, recombinant protein; (5) 1000:1 ratio of oxidant to methionine, recombinant protein; and (6) synthetic peptide, “amorphous” region of dragline silk. Fourier Transform Infrared Spectroscopy (FTIR). FTIR studies were performed using a Bruker equinox FTIR spectrometer with an IR microscope attachment. Samples were prepared by dropping a small amount of 7 mg/mL solution onto ZnSe crystals and examining the dried drop in the FTIR microscope in transmission mode. In this manner, each droplet could be examined for the presence of large precipitates. Separate spectra were obtained for the dried solution and for the precipitates in samples where large precipitates were observed upon drying. X-ray Studies. Solutions of protein were placed in siliconized thin-walled capillaries (Charles Supper Co., Natick, MA). Diffraction experiments were performed using nickel-filtered and double mirror focused Cu KR radiation from an Elliot GX20 rotating anode generator operating at 35 kV and 25 mA. Diffraction patterns were recorded on DEF-5 (Kodak Direct Exposure Film). The specimen-to-film distance, or camera length, was calibrated using an internal known standard. The data are summarized in Table 1. Transmission Electron Microscopy and Electron Diffraction. Air-water interface surface films of the recombinant protein were transferred onto uncoated gold TEM grids and allowed to dry as free-standing films. Precipitates from the protein were
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TABLE 1: Summary of X-ray Diffraction Resultsa protein unmodified/as expressed recombinant model dragline silk
oxidized 1:1 recombinant model dragline silk reduced recombinant model dragline silk
native spider dragline silk
lattice spacings (Å)
descriptionb
∼7.7 ∼4.76 3.95 3.81 3.48 2.81 2.66
vw diffuse ms ms ms mw mw
3.88 3.51 3.04 2.81
doublet ms vs mw
13.3 4.66 3.94 3.78 3.50 2.82
diffuse halo ms ms ms mw mw
7.49 4.62 3.90 3.46 2.79 2.62 2.36
vs, broad sampled ring ms ms mw mw mw
a Data indicate a loss of β-sheet crystallinity in the oxidized sample. Unless noted otherwise, the reflections were sharp. vs ) strong, s ) strong, ms ) medium strong, mw ) medium weak, vw ) very weak.
b
collected onto carbon-substrate TEM grids. The amorphous sequence peptide was prepared by dropping the solution onto carbon-substrate TEM grids. Additional samples of the peptide were prepared by gently rubbing carbon-substrate TEM grids along a dried solution of the peptide on ZnSe crystals. This latter sample provided a control between the IR characterization of the peptide and the TEM analysis. A JEOL 2000 Mark 2 transmission electron microscope, operated at 200 kV, was used to characterize the samples. Brightfield images, electron diffraction patterns, and defocused diffraction images were obtained. In addition, qualitative beam stability studies were used to assess the presence of salt crystals in the films. A cryogenic sample stage was used to improve the quality of diffraction data obtained and extend the working lifetime of the protein. Because some ice formation was observed, the major reflection from ice at 3.7 Å was used as an internal calibration standard for electron diffraction camera lengths. Separate electron diffraction studies on the same peptide and protein used gold as an internal diffraction standard. DEF-5 X-ray film (Kodak) was used to capture the images and diffraction patterns because its high sensitivity (roughly 5× that of EM film) mitigates the problems of protein degradation by the beam during image acquisition. The shorter exposure times available when X-ray film is used also minimizes the beaminduced crystallization of amorphous and cubic ice to the hexagonal form. Results Solution State Conformation (CD). CD spectra obtained from the reduced/unmodified recombinant protein and stoichiometrically oxidized samples in the solution state indicated that the only secondary structural elements present in both cases were the β-sheet and the random coil. Solutions of the reduced recombinant protein precipitated during purification and were not suitable for CD. In the unmodified sample, the proportion
Figure 1. IR spectra for the unmodified and oxidized recombinant proteins. The amide I region is indicated with an arrow.
of β-sheet was 56%, whereas in the oxidized sample the proportion of β-sheet was 43%. Since these measurements were performed on solutions of the recombinant protein, they are not in any way an indicator of β-sheet crystallinity. Also, since the protein measured using CD spectroscopy in both cases was soluble, the influence of β-sheet structure on the protein’s solubility cannot be easily inferred from these data. Nonetheless, the data did indicate that the presence of oxidized methionine residues reduced the amount of β-sheet conformation present. It is likely that the oxidized methionine residues prevented adjacent residues from adopting the β-sheet conformation, even in solution. Fourier Transform Infrared Spectroscopy of Dried Films. It was only possible to obtain comparable infrared spectra from the oxidized and unmodified as-expressed proteins due to severe problems with precipitation of the reduced recombinant protein. The infrared spectra for the oxidized samples all showed a strong amide I band at 1640 cm-1; this position correlates with a silk I-like or a “random coil” (amorphous) structure in silks and silk-like proteins. The spectra obtained for the most highly oxidized samples were characterized by extremely weak bands corresponding to a typical β-sheet absorbance of 1629 cm-1, a strong absorbance at a position indicating “amorphous” protein (1640 cm-1), and the appearance of an absorption band at 1615 cm-1, as can be seen in the lowest curve of Figure 1. The stoichiometrically oxidized sample has stronger absorbance bands in the β-sheet region and has an absorbance at 1660 cm-1 in addition to the “amorphous” band at 1640 cm-1. Amide I bands at 1660 are typical of fibrous proteins in a 3-fold polyglycine II or polyproline II conformation.9,10 The approximately (GGX) repetitive sequence in the amorphous blocks of the recombinant protein is similar to other repetitive
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Figure 2. Comparison of the IR spectra for the short amorphous peptide and the recombinant protein.
TABLE 2: Amide I and II Band Positions Are Correlated to Secondary Structure for Extended Chain Fibrous Proteins and Synthetic Model Molecules amide bands recombinant spider silksreduced recombinant spider silksoxidized spidroin1 peptidea parallel β accepted value antiparallel β accepted value unordered accepted value β turn accepted value Poly(AGG) II synthetic, Rippon and Waltonb Poly(AGG) I synthetic, Rippon and Waltonb Poly(AGG) III synthetic, Rippon and Waltonb silk I protein, solid membrane, Ishidac silk II protein, solid membrane, Ishidac silk “R” fibroin “amorphous” peptide Glu5(GAGAGY)6Glu5a fibroin “crystallizable” peptide (GAGAGS) “crystalline sericin1 peptidea a
A
I
II
3345 3265 3308, 3228h
1613 s,d 1655hd 1681 s, 1649 1663 1640 1629, 1696 1640 1680 1656 1621, 1697 1655 1650 1625 1657, 1538 1651 1627, 1647h 1627, 1655h
1410, 1458h 1531 1543
β random + turn PPII
1550 1515 1535 1535 1528
PGII β “random” coil 12/5;5/2 helix? pleated β “random” coil silk I β β
3295 3290 3293
3340, 3292h 3292 3292
1547 1531 1527
Structure confirmed by electron diffraction on sample dried on identical substrate. b See ref 9. c See ref 30. d h ) shoulder, s ) strong.
sequences that can form 3-fold helices; poly(GGA) is a notable example.9,10 This amide I band position has also been observed for Bombyx mori fibroin in dried thin Langmuir Blodgett films, where a 3-fold helical conformation is observed with an amide I absorbance at 1662 cm-1.11-14 The synthetic model amorphous peptide has a strong IR amide I band at 1660 cm-1 (Figure 2, third curve from the top) and an IR spectrum that closely resembles reported data for Poly(GGA), as shown in Table 2. In contrast, the as-expressed recombinant protein had strong amide I bands at 1624 and 1660 cm-1. This suggests that the reduced form of the recombinant dragline silk protein contained regions in a β-sheet conformation and other regions that are in a 3-fold helical conformation. The behavior of the short peptide
modeling the amorphous blocks of N. claVipes spider dragline silk suggests that it is the amorphous blocks which adopt the 3-fold helical conformation. NMR studies by Meier support a 3-fold conformation for the amorphous regions in natural spider silk.15 The polyalanine domains are expected to adopt a crystalline β-sheet conformation. Deconvolution of the amide I peaks, using a Lorentzian peak profile, was performed to obtain relative changes in the amount of each conformation. The results are summarized in Table 3. The stoichiometrically oxidized biosynthetic spider silk yielded very noisy spectra, possibly due to the difficulties in forming uniform films or fibers of this sample. In the other samples, there was a trend toward decreasing β-sheet content and
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TABLE 3: Proportions of Each Conformation from Deconvolution Using Curve Fitting silk I or random coil
3-fold helix
β turn
native spider dragline silk biosynthetic dragline silk (as-expressed)
31 63
15 n/obsa
34 13
21 24
oxidized biosynthetic dragline silk 1:1 1:100 1:500 1:1000
9 23 9 n/obs
n/obs 3 12 67
68 27 46 15
23 46 32 18
protein and conditions
a
β sheet
Not observed.
increasing random coil and turn contents as the synthetic silk was oxidized with increasing amounts of oxidant. At very high oxidation levels, the 3-fold helical conformation is also less evident. It is also interesting to note that at moderate oxidation levels, the turn and 3-fold helix contents are higher than in the unmodified (reduced) and highly oxidized silks, suggesting that an intermediate state may have been captured. The same trend has been observed in methanol-induced transitions of silk-like model peptides.11 X-ray Diffraction Studies. A series of X-ray diffraction experiments were performed on the protein to assess the effectiveness of the trigger in inhibiting β-sheet formation. X-ray diffraction data were recorded from the unmodified recombinant protein, the protein dried in an oxidizing environment, and the protein dried in a reducing environment. If the methionine triggers were functioning as designed, β-sheet crystallinity would be expected to decrease as a function of increasing oxidation of the recombinant protein. In the oxidizing environment, the steric interference from the oxidized methionine triggers was expected to block β-sheet crystal formation. There may be a small proportion of oxidized methionine residues present in the unmodified recombinant protein sample, but β-sheet crystals are nevertheless expected. The reduced sample would be expected to have the smallest proportion of oxidized methionine residues and thus to be the most crystalline. While β-sheet crystalline content is not a direct measure of solubility, insolubility of fibrous proteins and proteins with a high glycine content can often be traced to the formation of β-sheet crystallites. In B. mori silk fibroin and in native spidroins, crystallization in the β-sheet conformation is associated with a concomitant loss of solubility. As a baseline, native N. claVipes dragline silk X-ray diffraction patterns were obtained for comparison. The X-ray diffraction patterns obtained for the proteins are compared in Figure 3. The native spider silk was used “as spun”. X-ray diffraction revealed a number of polycrystalline rings (Table 1). Strong or sharp reflections without evidence of salt contamination were observed at 7.49, 4.72, 3.90, 3.75, 3.46, 2.79, 2.62, and 2.36 Å. Weak but well-defined arcs were also observed, corresponding to d-spacings of roughly 14-15 Å. The X-ray diffraction pattern for the native spider dragline silk is shown in Figure 3a. The reduced recombinant dragline silk protein yielded X-ray diffraction polycrystalline rings with d-spacings indicative of β-sheet crystalline spider silk (Figure 3b). Sharp well-defined rings were observed corresponding to d-spacings of 4.66, 3.94, 3.78, and 2.64 Å, which are very similar to the reflections observed for the native spider silk. There was no X-ray diffraction evidence to support a significant oriented amorphous phase in this sample. However, X-ray diffraction is a bulk “average” measurement of structure, and observation of an oriented amorphous phase would be difficult if the amorphous material existed in multiple domains that are not easily reoriented along one common axis.
Figure 3. X-ray diffraction reveals changes with increasing oxidation. There are salt reflections overlapping several of the protein reflections: (a) native spider dragline silk; (b) reduced recombinant spider dragline silk (there are a number of reflections attributable to the β-sheet, including a sharp reflection at ∼4.7 Å, the 200); (c) unmodified as-expressed recombinant spider dragline silk (the β-sheet reflections, especially the 200, are less clear than in (a); and (d) oxidized recombinant spider dragline silk (1:1 ratio oxidant to methionine) (the β-sheet reflections are absent).
X-ray diffraction from the unmodified recombinant protein revealed two polycrystalline rings, corresponding to d-spacings of 4.7 and 3.8 Å (Figure 3c). There was an additional reflection observed at 3.95 Å in the X-ray diffraction pattern that was not observed by electron diffraction, but scattering from ice may have obscured weak reflections in this region in the cryogenic electron diffraction experiment. Similarly, a 7.7-Å spacing observed in X-ray diffraction, but not corroborated by electron diffraction, would be obscured by the center spot under the conditions used to obtain the latter pattern. There were reflections characteristic of a β-sheet structure observed in the unmodified recombinant protein in both types of diffraction analysis, but a larger number of well-resolved reflections would be expected for a highly crystalline sample. The unmodified recombinant protein sample contained some β-sheet crystallites, but the crystallites were less evident than in the reduced recombinant protein. The oxidized recombinant protein yielded very few reflections. Two polycrystalline diffraction rings were evident, corresponding to d-spacings of 3.88 and 3.51 Å (Figure 3d) which are characteristic of the salts observed in this series of samples. The typical interchain spacing for a β-sheet, 4.6-4.7 Å, was not observed, nor were there any other crystalline reflections attributable to protein. Thus, there was no observable β-sheet crystallite formation in the oxidized sample, demonstrat-
Crystallization of Biosynthetic Silk Spidroin
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Figure 4. X-ray diffraction pattern from the reduced recombinant protein (a), schematic diagram showing lattice spacings of the observed reflections (b) and Miller indices assigned to the reflections (c).
ing the ability of the oxidized methionine triggers to block β-sheet formation. The X-ray reflections obtained from the unmodified and reduced recombinant proteins were indexed on an orthorhombic unit cell where a ) 9.3 Å, b ) 7.0 Å, and c ) 11.0 Å. This represents an asymmetric antiparallel β-sheet unit cell, where a is the interchain distance (two chains), b is the distance along the chain (two residues), and c is the intersheet distance (two sheets). The unmodified recombinant spider dragline silk X-ray diffraction pattern is shown in Figure 4a. Figure 4b shows the d-spacings calculated for each observed reflection, and Figure 4c shows the Miller indices assigned to the diffraction pattern. Because there was residual salt present, yielding X-ray reflections overlapping the reflections from the protein, small differences were expected in the observed and predicted d-spacings. The relative intensities observed are less reliable than the d-spacings because of the possible contributions from strongly diffracting salt. The X-ray data and the choice of unit cell were corroborated by electron diffraction studies (where salt was more easily distinguished from protein, discussed in the next section). Transmission Electron Microscopy and Electron Diffraction. TEM morphology images of precipitates obtained from aqueous solution of the triggered and untriggered recombinant proteins revealed a loss of fine structure with increasing levels of oxidation. The featureless precipitate shown in Figure 5a was from protein triggered with a 1000:1 molar ratio of phenacyl bromide to oxidize the methionine residues. When a 1:1 ratio of phenacyl bromide was used, the precipitates have a reticulated morphology (Figure 5b). In Figure 5b, features from the individual crystallites are discernible, but a clear needlelike habit and aspect ratio are not developed. The unmodified recombinant protein, which is expected to be reduced due to the reducing environment in the cell, generated precipitates with a fibrous or needlelike appearance (Figure 5c). These precipitated clusters of needlelike crystallites consistently yield polycrystalline unsampled electron diffraction rings indicative of a β-sheet structure, with the same strong reflections (at 4.6 and 3.8 Å-1)
Figure 5. Precipitates from recombinant proteins with different degrees of triggering: (a) untriggered, 1000:1 ratio oxidant to methionine residues, (b) untriggered 1:1 ratio oxidant to methionine, and (c) triggered.
as were observed in the X-ray diffraction experiments on unmodified and reduced samples. The reduced recombinant protein generated large precipitates, which were not amenable to TEM imaging. In addition to the precipitates observed, thin films obtained from the surface of the solutions, the air-water-interface, were studied for those cases where such films could be obtained. The electron diffraction data enabled us to distinguish between different crystalline textures in the samples: (1) a polycrystalline protein, yielding smooth diffraction rings; (2) larger orientated crystallites, probably salt, yielding sharp spots arranged in arcs; and (3) a poorly crystalline or “oriented amorphous” phase yielding a diffuse row of spots, which are aligned with the bands in the morphology. Unfortunately, the poor solubility of the reduced recombinant protein made it impossible to study as a film. In films prepared from the oxidized solutions of the recombinant protein, there were no discernible features, except for the occasional salt crystal. The only sharp electron diffraction rings observed were assigned to the salt crystals. A very diffuse symmetric electron diffraction ring, centered on 4.5-4.8 Å, was observed, which was indicative of protein present. At 200 kV, an electron has a deBroglie wavelength of 0.025 Å, which was extremely short (in a typical X-ray experiment the Cu KR radiation has a wavelength of 1.545 Å). The observed breadth of a diffraction reflection due to finite crystallite size is also strongly inversely correlated to the wavelength of the diffracting radiation. Thus, in a high voltage electron diffraction experiment, the absence of distinct diffraction rings indicates no crystallinity and no organization in the amorphous phase. Films of the unmodified recombinant protein (Figure 6) had a “banded” appearance in the transmission electron microscope, with dark and light alternating bands/areas visible. The width
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Figure 6. A sinusoidally textured film containing 200-nm-long needlelike crystallites is observed for unmodified as-expressed recombinant protein at the air-water interface.
of these bands/areas was ∼200 nm. Whether the appearance of the bands was due to changes in film thickness or changes in crystallinity (mass/thickness contrast vs phase/ diffraction contrast) is not yet certain. However, in the darker bands there were numerous small needlelike crystallites that were 200400 nm long and 74.42 88.60 94.31 ?? 112.30
5.5 3.67 4.5 4.52 4.56 5.35 5.8 5.9-7.0 6.7
anti, asym
a
Data show a strong correlation between average residue volume and average intersheet distance for fibrous proteins and their corresponding synthetic model polypeptides. b sym ) symmetric, asym ) asymmetric, anti ) antiparallel. c glu(Oet) ) ethyl glutamate.
crystallizable portions of a number of molecules coalesce into an ordered domainswith poor orientation and coherence at distances larger than a few nearest neighborssseems more likely. The needlelike objects were too large to be chain-folded β-sheet crystallites of a pentapeptide alanine run, and there was no diffraction evidence for a small population of highly oriented single β-sheet crystallites in the sample region. These objects must be aggregates of smaller, unoriented or poorly oriented crystallites. The electron diffraction data showed a pattern of rings at 4.6, 3.75, 2.83, and 2.31 Å. Since protein β-sheet structures have very consistent interchain and rise per residue (intrachain) distances, the only lattice parameter expected to vary greatly between β-sheet structures was the intersheet distance. The sheets in the β-sheet conformation interact largely through relatively weak van der Waals forces, and this conformation places the amino acid side chains in the intersheet region.23 Thus, a larger intersheet lattice parameter is expected for a protein with larger side chains. The 4.6-4.7 Å diffraction ring was consistent with the interchain distance expected for a β-sheet, while the expected residue rise was 3.5 Å. The observed reflections can be indexed on a β-sheet unit cell with an intersheet distance (the only truly variable parameter) of 5.5 Å. There are a number of fibrous proteins and recombinant proteins which crystallize as β-sheets and whose β-sheet crystallizable sequences are known, and a number of these reported crystal structure results are summarized in Table 4. For the B. mori silk II structure, the average intersheet spacing is 4.7 Å per sheet (for the two sheets in the unit cell, which represents an asymmetric β-sheet structure).24-27 The crystallizable sequence of B. mori silk, believed to comprise the β-sheet crystals, is approximately GAGAGS.24,28,29 Alternation of glycine and alanine/serine in this sequence results in β-sheets that have one surface (intersheet region) decorated with alanine methyl group side chains and serine methoxy side chains, while the opposite side of the sheet contains the glycyl hydrogen. The uneven sterics result in an asymmetric β-sheet structure, with different intersheet distances for the glycine and alanine/serine intersheet regions. This situation does not exist in the proposed crystallizable sequence, all alanine residues, for N. claVipes dragline silk and for the recombinant protein. The two sides of the β-sheet should be identical in this case, leading to a symmetric structure. It was interesting to compare the intersheet distances for some symmetric and asymmetric β-sheets of silks and silk-like proteins, using the average intersheet spacing for the asymmetric cases. For polyglycine I, which contains only glycine residues, the distance between sheets was 3.7 Å. For B. mori silk II, which was approximately half glycine and half alanine and serine, this distance was 4.7 Å. The recombinant protein spider dragline silk model, which has a pentapeptide of
Figure 9. Graph showing the linear relationship between average residue volume and β-sheet intersheet distance.
alanine as its core crystallizable sequence, had an intersheet spacing of 5.5 Å, which was consistent with the progression toward larger intersheet distances as the proportion of larger residues increases. However, as can be seen from Table 4, the crystal structure formed by β-sheets of polyalanine has a smaller average intersheet distance. In fact, when the intersheet distances (average for 1 sheet) obtained for the β-sheet-forming fibrous proteins and protein models are plotted against the average volume of the residues known to participate in the β-sheet crystal, a straight line results, as seen in Figure 9. The crystallographic data obtained for polyalanine falls on this line, as does that for natural silkworm silk and the proteins used to model silkworm silk’s repetitive crystallizable sequence. If the crystal structure observed for the recombinant dragline silk protein incorporates only the alanine pentapeptide portion of the sequence, the observed average intersheet spacing would be anomalously large. If, however, the residues flanking the alanine pentapeptide run (AAGM) are also incorporated into the β-sheet crystallite, the ratio of average residue volume to average intersheet distance becomes consistent with data reported for the well-characterized β-sheet structures and falls on the same straight line (Figure 9). Considering the small size of the individual alanine domains (five residues), it is unlikely that an individual alanine domain could organize itself into a chain-folded crystal, even if the residues flanking the alanine pentapeptide sequence are incorporated. It is more likely that the crystalline regions are formed through participation of multiple alanine domains, with irregular folds and loops at the edges of the crystalline region resulting in a large crystalline/amorphous interphase, as illustrated schematically in Figure 10. Such a model for the crystalline domains could also easily accommodate the methionine residues, in the disordered regions at the edges of the crystallites, and would account for the observed crystal morphology and diffraction features.
Crystallization of Biosynthetic Silk Spidroin
J. Phys. Chem. B, Vol. 103, No. 51, 1999 11391 The amorphous matrix is itself believed to be organized or oriented, but the details regarding the type and degree of organization are still the subject of some contention. The recombinant model mimics the pertinent aspects of the sequence and secondary structure and approximates the higher order selfassembled organization of native spider silk. However, the design of a recombinant protein with solubilizing triggers has allowed the use of experimental approaches for which native silk would be intractable. The simplified (as compared to native silk) sequence of the recombinant silk has rendered the data more accessible to interpretation as well and has allowed approaches, such as the use of a peptide that precisely mimics an interesting portion or block of the protein sequence, to delineate sequence-specific structural self-assembly behavior.
Figure 10. Schematic diagram showing the irregular reentry of macromolecules in the β-sheet regions may result in crystals with a large interphase formed by chain folds and constrained amorphous material.
Conclusions The solution state CD data and the infrared results on dried films clearly show that the oxidation state of the methionine triggers influences the tendency of the recombinant spider dragline silk model protein to adopt a β-sheet conformation. The X-ray and electron diffraction results further demonstrate that the ability of the oxidized methionine triggers to inhibit formation of the β-sheet conformation is even more pronounced when β-sheet crystallinity is considered. The oxidized triggers were observed to completely block the formation of β-sheet crystallites at high levels of oxidation. The oxidized solutions also exhibited fewer problems with precipitation, which was an insurmountable obstacle for the reduced recombinant protein in several of the sample preparation techniques used. These data strongly suggest that there is a direct path from the oxidation state of the methionine triggers, to the tendency to adopt the β-sheet conformation and the subsequent ability to form β-sheet crystallites and the solubility or intractability of the protein. The structural data obtained for the reduced and unmodified recombinant protein indicate that when the methionine triggers are reduced or not highly oxidized, the protein is capable of forming the β-sheet conformation and crystallizing, forming β-sheet crystallites. The data also indicate that the degree of crystallinity can be controlled, or tuned, by varying the degree of oxidation of the triggers. This implies that not only is control of solubility possible, but control of the materials properties, such as viscoelastic moduli, should also be possible through use of the oxidation trigger methodology. In addition to forming β-sheet structures, the unmodified protein was observed to form an oriented amorphous phase incorporating a roughly 3-fold helical conformation. Comparison with the model amorphous spider dragline peptide suggests that the nonpolyalanine portions of the protein sequence are responsible for generating this structure. Furthermore, it is very likely that the 2-3 residues adjacent to the alanine pentapeptide block in the sequence participate in the β-sheet crystal structure. These results are all in accordance with other studies on native spider silk, which suggest crystallites with a variable β-sheet crystal structure oriented and interspersed within an amorphous matrix.
Acknowledgment. Funding support from the National Science Foundation (DMR-9708062, BES-972-7401) and facilities support from the NSF MRSEC program at the University of Massachusetts, Amherst and the Keck Foundation Polymer Morphology Laboratory at the University of Massachusetts, Amherst and Biomimetic Materials Laboratory at Tufts University are gratefully acknowledged. The Tufts University Medical School Protein Core Facility provided peptide syntheses. We would also like to acknowledge discussions with Sam Gellman. References and Notes (1) Cunniff, P. M.; Fossey, S. A.; Auerbach, M. A.; Song, J. W. Mechanical Properties of Major Ampullate Gland Silk Fibers Extracted from Nephilia clavipes Spiders. In Silk Polymers; Kaplan, D., Adams, W. W., Farmer, B., Viney, C., Eds.; American Chemical Society: Washington DC, 1994; Vol. 544; p 234. (2) Cunniff, P. M.; Fossey, S. A.; Auerbach, M. A.; Song, J. W.; Kaplan, D. L.; Adams, W. W.; Eby, R. K.; Mahoney, D.; Vezie, D. L. Polym. AdV. Technol. 1994, 5, 401-410. (3) Hinman, M. B.; Stauffer, S. L.; Lewis, R. V. Mechanical and Chemical Properties of Certain spider Silks. In Silk Polymers; Kaplan, D., Adams, W. W., Farmer, B., Viney, C., Eds.; American Chemical Society: Washington DC, 1994; Vol. 544; p 222. (4) Prince, J. T.; McGrath, K. P.; DiGirolamo, C. M.; Kaplan, D. L. Biochemistry 1995, 34, 10879-10885. (5) Winkler, S.; Szela, S.; Avtges, P.; Valluzzi, R.; Kirschner, D.; Kaplan, D. L. Int. J. Biol. Macromol. 1999, 24, 265-270. (6) Dado, G. P.; Gellman, S. H. J. Am. Chem. Soc. 1993, 115, 1260912610. (7) Rothwarf, D. M.; Davenport, V. G.; Shi, P.-T.; Peng, J.-L.; Sheraga, H. A. Biopolymers 1996, 39, 531-536. (8) Arcidiacono, S. A.; Mello, C.; Kaplan, D.; Cheley, S.; Bayley, H. Appl. Microbiol. Biotechnol. 1998, 49, 31-38. (9) Rippon, W. B.; Walton, A. G. J. Am. Chem. Soc. 1972, 94, 43194324. (10) Rippon, W. B.; Walton, A. G. Biopolymers 1971, 10, 1207. (11) Wilson, D.; Valluzzi, R.; Kaplan, D. Biophys. J., in press. (12) Valluzzi, R.; Kaplan, D., unpublished data. (13) Valluzzi, R.; Gido, S.; Zhang, W.; Muller, W.; Kaplan, D. Macromolecules 1996, 29, 8606-8614. (14) Valluzzi, R.; Gido, S. P. Biopolymers 1997, 42, 705-717. (15) Van Beek, J. D.; Kummerlen, J.; Vollrath, F.; Meier, B. H. Int. J. Biol. Macromol. 1999, 24, 173-178. (16) Gaill, F.; Herbage, D.; Lepescheux, L. Matrix 1991, 11, 197-205. (17) Gaill, F. Colloq. Phys. 1990, 51, c7-169-c7-181. (18) Willcox, P. J.; Gido, S. P.; Muller, W.; Kaplan, D. L. Macromolecules 1996, 29, 5106-5110. (19) Giraud-Guille, M. M. Mol. Cryst. Liq. Cryst. Sci. Technol. 1987, 153, 15-30. (20) Valluzzi, R.; He, S. J.; Gido, S. P.; Kaplan, D. Int. J. Biol. Macromol. 1999, 24, 227-236. (21) Revol, J. F.; Marchessault, R. H. Int. J. Biol. Macromol. 1993, 15, 329-35. (22) Valluzzi, R.; Gido, S. P.; Muller, W.; Kaplan, D. L. Int. J. Biol. Macromol. 1999, 24, 237-242. (23) Pauling, L.; Corey, R. B.; Branson, H. R. Proc. Natl. Acad. Sci. U.S.A. 1951, 37, 205-211. (24) Lotz, B.; Cesari, C. Biochimie 1979, 61, 205. (25) Warwicker, J. O. J. Mol. Biol. 1960, 2, 350.
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