Structure of a protein superfiber: spider dragline silk.

Abstract

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...

Silk-based biomaterials

“…Genetic engineering is being actively explored to construct, clone and express native and synthetic genes encoding recombinant spider silk proteins to overcome limits to use of the native organisms. This strategy has provided new opportunities in fundamental studies of spider silk genetics, silk protein structure and function, and materials processing [2,[4][5][6]. In general, interest in spider silk has increased in recent years due to the differences in mechanical properties when compared to silkworm silk and the presence of the multi-gene family encoding this group of silks as a basis for the study of protein structurefunction relationships [4,[6][7][8][9][10][11].…”

“…Cloning and expression of native and synthetic silks has been achieved in a variety of host systems. The sequences of cDNAs and genomic clones encoding spider silks illustrate the highly repetitive structures [4,6,[8][9][10][11], which can be readily exploited to construct genetically engineered spider silk-like proteins using synthetic oligonucleotide versions of the consensus repeats or variants of these repeats. This highly repetitive sequence was also recently fully described for the fibroin heavy chain from the silkworm, B. mori [12].…”

The hidden link between supercontraction and mechanical behavior of spider silks

“…Although very few silk genes have been completely cloned (Xia et al, 2004;Ayoub et al, 2007), due to their large size (Xu and Lewis, 1990), the existence of a small number of simple motifs of sequence extensively repeated (Gatesy et al, 2001) has led to the synthesis of artificial analogs that are believed to capture the essential features of the natural proteins, though so far no process has resulted in silk fibers that perfectly mimic the mechanical properties of natural silks.…”

“…The polypeptides were labeled as rcSpl and rcSp2, referring to the spidroin on which are inspired (Xu and Lewis, 1990). The production of the recombinant proteins, preparation of the spin dopes and spinning of the fibers are described in detail elsewhere (Karatzas et al, 2005).…”

Orientational order of Australian spider silks as determined by solid‐state NMR

“…10,11 From amino acid analysis of cDNA, a repeating unit made up of three segments (consisting of 6, then 13 and 15 amino acids in length) dominated by an Ala-rich sequence of 5-7 residues has been found. 12 Regions in which Gly is abundant are proposed to compose the amorphous domains that impart elasticity to silk, 1 while a pentapeptide GPGXX and a GGX repeat motif have been found in the noncrystalline regions of major ampullate silk. 13 However, debate on the structure of the poly-Gly regions continues.…”