Soup to Silk- How do Spiders do it?

The molecular magic of spider silk production.

One might think that spiders would be an important tool in agricultural pest control. But that doesn't seem to be the case. Like cats, they can't be herded around, and probably are not so interested in the usual aphids, borers, hoppers, and other banes of typical crops. And they get killed off by pesticides even more than the target insects ... the usual horrorshow.

But they are amazing creatures, with stunning design sense, incredible patience and persistence, acrobatic eight-leg coordination, parental instincts, and WWE-worthy mating rituals. Their silk is as strong as high-grade steel, but one-sixth the weight, and comes in numerous varieties, from soft egg protecting, to sticky prey capture, to strong dragline support. Each type comes out of a separate gland and spinneret from a protein brew that is clearly quite special.

Anatomy and specialization of spider silk glands.

There is no spinning involved ... silks are secreted in a high-tech/nanotech process that magically transforms a gooey protein solution into finished silk, ready for the spider to dangle from in mid-air. A recent paper extended a good bit of prior work that shows that a key transformation of the extrusion process is performed by pH- the gradual acidification of the protein solution as it is drawn down the quite lengthy gland, causing crucial changes in the protein components.

Dissected spider silk gland, with secretion and storage gland in yellow and the duct to the outside spinneret in tan. Measured pH values are noted, though measuring in such tiny spaces is difficult and impossible in thinnest parts. So pH values are very approximate, and thought to be a bit lower farther down the duct. Bar is 1 mm. This is the major ampul late gland from Nephila clavipes.

Those proteins are called spidroins, and they are all similar, with variations due to evolutionary divergence. The front and back ends (N- and C-termini of the proteins, respectively) are complex conserved protein domains, while the middle is a highly repetitive alternation of alanine-rich and glycine/proline-rich segments. The alanine-rich segments fold into very strong, almost crystalline, structures called beta-sheets (yellow, below), while the glycine/proline segments form a jumble of turns and tight 3/10 helices. The detailed structure of spider silk is thus a sort of spaghetti interspersed with bundles of uncooked ramen- however, ramen that is intensely crosslinked within its block.

Calculated structure of silk protein after production, with beta sheet structures in yellow.

Calculated structure of silk protein after production, and after stretching just short of breaking.

When pulled, the tangles smoothly stretch to maybe twice their resting length, and ultimately all the beta sheet segments align with the direction of pulling. The key to all this is the hydrogen bond, which is only a tenth to a twentieth as strong as covalent chemical bonds, but when added up over a clever structure full of them, in hierarchical fashion, you get very high strength. In constrast, high-strength fibers like Kevlar or cellulose are fully covalent molecular strings. It is a bit like velcro, where if you have a big enough structure made out of it, it can hold a great deal of weight, even though its individual bonds are weak.

Cartoon of spidroin proteins during extrusion. The dimerized C-terminal domains are in red, while the blue N-terminal domains dimerize only while the proteins are underway through the "spinning" duct, principally under the influence of acidic pH. In this paper, the authors propose that the C-terminal domains do not remain dimerized, but change structure as well, binding to the bulk of the protein's beta sheets while underway through the duct.

"Along the length of the duct, Na+ drops from 3.1 mg g−1 dry weight in the ampulle (where the spidroins are stored) to 0.3 mg g−1 in the fiber, and K+ increases from 0.75 to 2.9 mg g−1 (6). Phosphate concentration also increases at least 5-fold, whereas flow velocity and shear force increase (especially near the end) because of the tapered geometry of the duct (5, 7). Importantly, pH drops from 7.2 in the storage region to 6.3 in the first 0.5 mm of the duct and reaches an unknown value by the end of the ~20-mm duct (8). Collectively, these chemical and physical forces induce the spidroin molecules to align with the direction of flow, form β-sheets, and partition out of the aqueous phase to form a solid fiber"

The spidroins have to join together in a controlled & rapid way, so they don't solidify before coming out of the spinerett. The C-terminal ends of the protein join together into dimers immediately after being synthesized. This paper provides evidence that the C-terminal domain switches dramatically from its alpha-helical dimeric structure in the gland, to something more amyloid-like with beta sheet structures that interweave with and help organize the bulk of the internal repeats as the solution passes down the acidifying duct.

The C-terminal domain re-organized dramatically when pH is lowered. The Y- axis here is the fluorescence of a chemical that binds to beta sheets specifically, indicating that this protein segment, which is originally largely alpha-helical, switches to a beta-sheet conformation, more rapidly as the pH is lowered.

The N-terminal ends undergo a constrasting transformation. They do not dimerize in the storage gland, ensuring that the solution, though highly concentrated, does not turn solid. However as the solution gets pulled down the duct, it gets progressively acidified, which causes the N-termini to dimerize. The solution also gets exposed to a few other ionic changes and squeezing/pulling force that cause the nano-elements to line up and helps them bind to each other. The new paper focuses on this acidification process, finding that the enzyme carbonic anhydrase, which generates carbonic acid from water and CO2, is concentrated all along the duct. With the duct's length and narrowness, it appears that the pH of the nascent fiber plunges down from about 7-8 in the gland to some unknown value far below 5.5 by the time it comes out, sterilizing the silk as well as fashioning it structurally into a fiber.

Cross-sections of some sample silk ducts, stained for cells (blue) and for the acidifying enzyme carbonic anhydrase (black dots).

One might ask, if purely ionic and pH effects generate silk from soluble proteins, why doesn't the silk melt again in the morning dew or any time pH rises again? One answer is that external pH of dew and open air is slightly acidic, due to the ambient CO2, so conditions are typically favorable. But mostly, it is a matter of hysteresis / tangling, where a process that easily goes in one direction has great difficulty going in reverse. Silks need to be boiled in alkaline solution to melt, and even then they are a rather tangled mess, difficult to use for other purposes.

"In summary, the spidroin N- and C-terminal domains show synchronous and opposite structural changes in response to the physiological conditions of the spinning duct. CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins, whereas gradually locked NT dimers alleviate the need for rapid diffusion, firmly interconnect the spidroins, and allow for propagation of pulling forces along the peptide chains. These events are driven by CO2 and proton gradients that ensure temporal and spatial confinement of the divergent structural changes of CT and NT. This novel lock and trigger mechanism elegantly explains how silk formation can occur at a very high speed, more than 1 m/s, and at the same time be confined to the very distal part of the spinning duct."

A good deal remains mysterious, but it is fascinating to learn bit by bit how amazing things such as silk production happen. What seems like magic turns out, as usual, to have very material, explicable, and interesting tricks behind it.

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