Spider major ampuilate (dragline) silk is an extracellular fibrous protein with unique characteristics of strength and elasticity. The silk fiber has been proposed. to consist of pseudocrystalline regions of antiparallel 13-sheet interspersed with elastic amorphous segments. The repetitive sequence of a fibroin protein from major ampullate silk of the spider Nephila clavipes was determined from a partial cDNA clone. The repeating unit is a maximum of 34 amino acids long and is not rigidly conserved. The repeat unit is composed of three different segments: (i) a 6 amino acid segment that is conserved in sequence but has deletions of 3 or 6 amino acids in many of the repeats; (ii) a 13 amino acid segment dominated by a polyalanine sequence of 5-7 residues; (ii) a 15 amino acid, highly conserved segment. The latter is predominantly a GlyGly-Xaa repeat with Xaa being alanine, tyrosine, leucine, or glutamine. The codon usage for this DNA is highly selective, avoiding the use of cytosine or guanine in the third position. A model for the physical -properties of fiber formation, strength, and elasticity, based on this repetitive protein sequence, is presented.One distinctive feature of spiders is the ability to produce silk threads. The silk is synthesized in specialized silk glands in the abdomen. The orb-web-spinning spiders can produce silk from six different types of glands (1). Each of the six fibers has different mechanical properties. However, they all have several features in common. They are (i) composed completely of protein; (ii) undergo a transition from a soluble to an insoluble form that is virtually irreversible; (iii) composed of amino acids dominated by alanine, serine, and glycine (2) but, unlike silkworm silk, have substantial quantities of other amino acids, such as glutamine, tyrosine, leucine, and valine; and (iv) composed of protein(s) of unknown number and molecular weight.Due to its size and accessibility the major ampullate gland has been the focus of most studies. Synthesis of the silk protein occurs in specialized cells at the tail of the gland, and the silk protein is secreted into the lumen of the gland where it is stored in a soluble form (1). The (Big Pine Key, FL). The first step was to get pure silk from a single type of silk gland. By using an idea from Work (13), an apparatus was designed to draw a single silk fiber from one spinnerette of the spider. A single silk fiber was wrapped on a spool, and a variable-speed electric drill was used to pull 0.5-1 mg of pure silk from the spinnerette.Protein Cleavage, Peptide Purification, and Sequencing. Preliminary results showed a "ragged" amino terminus when the whole silk fiber was sequenced. Thus, the silk protein was cleaved to provide peptides for partial sequencing. Peptide fragments were generated by cleavage in 6 M HCl at 1550C for 3 min. The peptide fragments were purified by HPLC using a C18 reverse-phase column with a pyridine/acetate buffer (pH 4.0) and 1-propanol as the organic modifier (14). The *To whom reprint reques...

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Spiders (Araneae) spin high-performance silks from liquid fibroin proteins. Fibroin sequences from basal spider lineages reveal mosaics of amino acid motifs that differ radically from previously described spider silk sequences. The silk fibers of Araneae are constructed from many protein designs. Yet, the repetitive sequences of fibroins from orb-weaving spiders have been maintained, presumably by stabilizing selection, over 125 million years of evolutionary history. The retention of these conserved motifs since the Mesozoic and their convergent evolution in other structural superproteins imply that these sequences are central to understanding the exceptional mechanical properties of orb weaver silks.

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Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each (13)C-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 3(1)-helical conformation and an ordered beta-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.

Spider flagelliform silk is one of the most elastic natural materials known. Extensive sequencing of spider silk genes has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution. The intron sequences are more homogenized within a species than are the exons. This pattern can be explained by extreme mutation and recombination pressures on the internally repetitive exons. The iterated sequences within exons encode protein structures that are critical to the function of silks. Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences.

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