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 -sheet crystallites is a major cause of insolubility in proteins that can form -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 -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 -sheet content was undetectable in the triggered state, while clear evidence of -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 methionine- modified 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 -sheet crystallites. These highly repetitive motifs are inter- spersed 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 structure and environment would then cause portions of these regular helical structures to aggregate into -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 me- sophase, nonperiodic -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 archi- tecture and the self-assembly processes and interesting macro- scopic 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 * To whom correspondence should be addressed. Fax: 617-627-3900. E-mail: dkaplan1@emerald.tufts.edu. † Tufts University. ‡ Boston College. 11382 J. Phys. Chem. B 1999, 103, 11382-11392 10.1021/jp991363s CCC: $18.00 © 1999 American Chemical Society Published on Web 12/07/1999