One of nature's wondrous chemical structures is being dissected so that it can be used in human inventions
People have been attempting to obtain and use spider silk for centuries. But success at mass production has been elusive for two reasons: the spiders tend to eat each other when thrown together in silk-making colonies, a characteristic incompatible with good harvests, and their silk is difficult to work with because it hardens on contact with air.
Scientists cannot do much about spider behavior. But their recent nuclear magnetic resonance imaging and genetic engineering studies are revealing the secrets of silk production and may suggest better ways to work with the tricky substance.
"Silking" has never been easy
One of the first references to using spider silk dates to French naturalist René-Antoine Ferchault de Réaumur. In 1709, his government asked him to find uses for spider silk. So he diligently collected the material from egg sacs and attempted to make stockings and gloves. He gave upñit took too many spiders to get enough silk. Another Frenchman, Bon de Saint-Hilaire, later in the eighteenth century tried to raise spiders. He found that, unlike silkworms, spiders could not be reared in close quarters.
Collecting spider silk required a way to immobilize the animals. An inventor named Daniel Rolt received a silver medal from the Society of Arts in Britain in 1830 for a device consisting of a spool attached to an engine to harvest spider silk. This contraption reportedly wound 18,000 feet of silk from two dozen spiders in two hours.
Civil War surgeon Burt G. Wilder tackled the challenge too, publishing a series of papers on his efforts between 1866 and 1873 in the Proceedings of the American Association for the Advancement of Science, the Proceedings of the Boston Society of Natural History, and the Atlantic Monthly. Wilder built a device that resembled the stocks used to hold criminals in colonial New England. The stocks held spiders still while Wilder gently drew silk from their rear ends. He succeeded in extracting 150 yards from one particularly cooperative arachnid, then calculated that it would take 5000 animals to retrieve enough material to make one dress.
Until World War II, spider silk was used for crosshairs in optical devices, including microscopes, telescopes, guns, and bomb guiding systems. Single strands were excellent for this purpose because they are thinñthe average spider silk is only 1/20,000 of an inch across, compared to 1/250 for a human hair. Today, some military facilities still keep a semidomesticated black widow spider around to provide silk to repair the crosshairs in old instruments. Spider silk crosshairs, however, have largely been replaced with etched marks or metal filaments.
Various societies have managed to collect enough spider silk to use. The ancient Greeks stanched bleeding wounds with cobwebs. Australian aborigines use the silk of a giant tropical spider for fishing lines, rubbing parts of the spider's body onto the silk to attract small fish, which become caught in the sticky silk when they bite the spider tidbits. New Guinea natives fashion fish nets, head gear, and bags from spider silk.
Today, spider silk is attracting the attention of a range of researchers, including geneticists, protein biochemists, and materials scientists intent on understanding and replicating spider silk's unusual properties. Spider silk has the strength of steel (although its thinness makes it workable), yet it also has the ability to billow out on a breeze. It can stretch to 40% of its normal length, yet shorten back with great resiliency. Spider silk easily withstands low temperatures. The substance is a soluble fluid in the aqueous environment of a spider's abdomen, but it turns into an insoluble solid after it exits the body.
Spiders use their silk in a variety of ways. Webs entice prey, silken nests enshroud eggs, and offspring tentatively venture from their webs, exploring beyond the nest yet still tethered to it by a lifeline. Reproduction requires silk too. A male weaves a small sperm web and deposits sperm onto it. He then transfers the web to his palps, which he uses to place the sperm web in a female's genital opening.
Some species use silk in even more specialized ways. A species that lives in freshwater ponds throughout Europe weaves a diving bell of sorts. The animal anchors the balloonlike structure it creates to an aquatic plant and then submerges, ferrying air bubbles in the bell. The entrappedàir, which rises to the top of the silken diving bell, sustains the spider. A type of sea spider builds a silken hideaway at the intertidal zone, retreating to the protected shell when the tide comes in.
Humans have in mind different uses for spider silk. They envision medical products such as surgical thread and replacement ligaments, and military applications including flak jackets, parachute cords, and tethers for planes on aircraft carriers.
Raising spiders has a long history of failure because of their cannibalistic tendencies. So researchers today are investigating ways to replicate spider silkñwhich means better understanding the biochemistry that gives rise to its incredible package of characteristics. Two research groups, led by Lynn Jelinski, professor of engineering at Cornell University in Ithaca, New York, and Randy Lewis, professor of molecular biology at the University of Wyoming in Laramie, are among those deciphering spider silk's complex structure.
The stuff of silk
All 36,000 described species of spiders, which range everywhere except Antarctica, produce silk in glands located in their abdomens. Once made, the silk passes through spinnerets and exits the spider via spigots. It is during this journey, researchers think, that the silk proteins begin to become insoluble, perhaps as they are repeatedly pressed by the animal's abdominal muscles.
The silk-producing parts of spiders are ancient. The earliest fossil evidence of a spider's silk-spinning activity is a fossil from 380 million-year-old rocks near Gilboa, New York, which reveals an animal with a single spinneret with attached spigots.
Many spiders today have six sets of silk glands, each producing a different type of fiber. Secretions from different glands feed into the same spinnerets, like a soft ice cream machine that can make chocolate or vanilla. A spinneret consists of hundreds of microscope tubes coming from the silk glands. The glands are called major ampullate and minor ampullate, and the number of each varies in different species. The terms minor ampullate and major ampullate are also used to describe certain silks and silk proteins. "The major and minor ampullate silks are different, both in mechanical properties and in protein components," says Lewis. "The minor ampullate silk has only about half the tensile strength of major ampullate silk, and it is not as elastic," he adds.
Although researchers are just now working out the precise chemical structures of silk proteins, silks have long been named according to their macroscopic functions. Bridgeline silk is the first strand, on which an entire web is constructed. Trapline silk, extending from the center of a web, vibrates when prey makes contact, telling the spider that dinner has arrived. The focus of much research is dragline silk, known to consist of two types of major ampullate proteins. This particular thread always extends from the spider to the web. Should the animal encounter danger, it scurries back to the safety of the web on the dragline. Dragline silk also makes up the spokes of a web.
The complexity of dragline silk
Jelinski and Lewis work primarily with dragline silk from the golden orb weaver, Nephila clavipes, the subject of most spider silk research. Named for the distinctive golden hue of its silk, the animal lives from southern Brazil to Florida
The dramatically different solubility of silk inside and outside the animal's body makes it tricky for scientists to handle. It is notoriously difficult to solubilize in the laboratory, a step necessary for many analytical methods. "A lot of the properties of silk derive from its processing. The silk is a liquid in the gland, and it journeys through a torturous route through ducts, and it comes out a strong and oriented solid. The orientation process must be very important, and this is what is difficult to mimic in the lab," says Jelinski, referring to a condensation of its structure as its state changes.
To study this intractable yet marvelous stuff of nature, Jelinski uses nuclear magnetic resonance (NMR) to probe silk protein conformation, and Lewis uses genetic engineering techniques. Each has devised clever, low-tech ways to collect the silk, which is easily teased from the spider's ampullate glands.
Lewis winds the silk 50-100 times around a cylinder, like thread on a spool, then cuts the silk at one point, and next tapes it down on a piece of black construction paper. He then teases free strands on the paper and tests them with a device called a universal test bed, which is used in mechanical engineering. Specifically, he measures strain, which is the percentage of its original length that a silk strand can elongate before it breaks. This is a measure of strength.
Jelinski, who uses a similar approach but substitutes Band-Aids for tape, hand-feeds her spiders deuterium (a heavy isotope of hydrogen) so they will secrete labeled silk proteins that can be analyzed using NMR.
Lewis and his coworkers have identified two similar proteins that make up dragline silk, formerly thought to be just one protein. He calls them spidroin 1 and spidroin 2. The two proteins differ predominantly in their levels of the amino acids proline and tyrosine. Because the spider mixes the two proteins in dragline silk, it is difficult to distinguish between them functionally. Lewis used the naturally mixed silk for his experiments but is now evaluating the proteins individually in genetic studies.
Jelinski works with the two spidroins together. Their extensive amino acid sequences are repetitive, suggesting a highly ordered structure. Approximately 42% of the amino acids of the combined proteins are glycine, and 25% are alanine. Much of the alanine occurs in stretches of five to ten alanines in a row. The remaining amino acids are mostly glutamine, serine, leucine, valine, proline, and tyrosine.
It is not coincidence that alanine and glycine, which make up much of spider silk, are the two smallest amino acids. The absence of bulky amino acid side chains enables the protein to pack tightly, forming a crystal in some parts. Consequently, Jelinski decided to target alanine in her NMR studies.
Jelinski, Alexandra Simmons (now a staff scientist at DuPont Canada), and physics graduate student Carl Michal used NMR to view the orientation and environment of the deuterium-labeled alanines in approximately 90,000 dragline silk fibers. "It's like painting all of the alanines red. We then query the ëred' parts using nuclear magnetic resonance," says Jelinski.
Michal built special hardware to collect the data, which revealed a surprisingly complex three-part organization for silk proteinsña rigid crystal portion, a less rigid crystal part, and an amorphous background material. "The exciting part was that we found two types of crystalline alanines," Michal says, a substance unlike any other in nature, Jelinski adds.
Studies using x-ray diffraction reported that spider silk consists of crystalline and amorphous portions in 1960, but the Cornell work is the first to reveal two types of crystalline regions. "NMR has told us which amino acids form the beta sheets, [which are the] crystalline regions. A beta sheet is mostly alanine, which you can't learn directly from x-ray diffraction. And we now know how very well the alanines are oriented with respect to the fiber axis. It's pretty amazing," says Jelinski.
Approximately 40% of the alanines are highly oriented, and the other 60% are less so, yet still crystalline. "The way we think [the structure] works is that the poorly oriented crystalline segments are like fingers, reaching out to make a good coupling between the highly oriented and the amorphous domains," Michal says. This organization would impart tremendous overall strength to the protein. Repeating the NMR measurements when the silk was stretched to varying degrees did not alter the results, and NMR using labeled carbon in some of the amino acids supported the deuterium NMR results.
There are generally three ways to mass-produce natural proteinsñextract them from a living source, synthesize them chemically, or use recombinant DNA technology to coax bacteria to produce them. The first approach, which would involve raising spiders for their silk, à la silk-worms, is not practical because the animals need to be raised in large, separated areas.
Chemists have not known enough about the structures of silk's constituents to synthesize them, but David A. Tirrel, a professor of polymer science and engineering at the University of Massachusetts at Amherst, has synthesized the glycine- and alanine-rich sections of silk proteins that form the crystal portion.
The genetic engineering route for mass-producing spider silk seems most promising, despite an initial glitch. When Lewis and coworkers inserted DNA sequences encoding key repetitive portions of spidroins 1 and 2 into Escherichia coli, they obtained poor expression, the yield rapidly dropping off.
The problem is that certain amino acids are encoded by more than one codon. For example, the DNA codons CGA, CGG, CGT, and CGC each encode alanine, and the DNA codons CCA, CCG, CCT, and CCC encode glycine.
Lewis discovered that E. coli's protein translation machinery does not efficiently recognize the particular alanine and glycine codons in the spidroin genes. So Lewis altered his gene pieces to more closely resemble the bacterial codon preferences, while maintaining specification of the same amino acid sequences. The changes worked. E. coli produced the truncated spidroins, and the researchers can render the spider proteins into fibers by extruding the material through needles into methanol, an approach that keeps the substance pliable.
Lewis keeps the spidroins supple enough to work with either by dissolving them in formic acid, or by adding genetic instructions that improve the protein's solubility. Tagging a few histidine and arginines onto the ends of the spidroins solubilizes them because these amino acids are hydrophilic (soluble in aqueous solutions). Researchers at the US Army Natick Research Center in Massachusetts, at DuPont Inc. in Wilmington, Delaware, and at several smaller companies and academic institutions also use genetic manipulations to produce spider silk proteins.
Humans have long marveled at, and attempted to recreate, spider silk. A 1928 encyclopedia article concludes, "How they [spiders] originally succeeded in converting food into a natural glue which on entering the air instantly becomes silk, we shall never be able to find out." By using the tools of NMR and genetic engineering, researchers are coming much closer to that elusive goal.
By Ricki Lewis, Ph.D.
Ricki Lewis is a freelance science writer based in upstate New York.
feedback form |
locate your campus rep |
request a review copy
Copyright ©2001 The McGraw-Hill Companies.
digital solutions | publish with us | customer service | mhhe home
McGraw-Hill Higher Education is one of the many fine businesses of the The McGraw-Hill Companies.
Copyright ©2001 The McGraw-Hill Companies.