Spidroins, the major silk proteins making up the spider's dragline silk, originate in two distinct tissue layers (A and B) in the spider's major ampullate gland. Formation of the complex thread from spidroins occurs in the lumen of the duct connected to the gland. Using pH-sensitive microelectrode probes, we showed that the spidroins traveling through the gland and duct experience a monotonic decrease in pH from 7.2 to 6.3. In addition, circular dichroism spectroscopy of material extracted from the gland showed a structural refolding concomitant with position in the gland and post-extraction changes in pH. We demonstrate that lowering the pH in vitro causes a dramatic conformational change in the protein from the A zone, converting it irreversibly from a coil to a predominantly beta-sheet structure. Furthermore, amino acid analyses have indicated that there are at least two distinct, though similar, proteins secreted in the A and B zones suggesting a potential factor in the progressive acidification as well as a pH sensitivity of the folding of spidroins in the gland. Thus, we provide, for the first time, a quantitative map of the pH value and position correlated with molecular structural folding in the silk gland characterizing the crucial role that pH plays in spider silk formation.

Unlike man-made fibers, the silks of spiders are spun from aqueous solutions and at atmospheric pressure in a process still poorly understood. The molecular mechanism of this process involves the conversion of a highly concentrated, predominantly disordered silk protein (spidroin) into beta-sheet-rich structures. To help store and transport the spidroins in solution, as well as probably control their conversion, a liquid crystalline arrangement is established in the storage region in the ampulla and persists into the duct. Although it has been suggested that changes in the concentration of hydrogen and metal ions play a role in the formation of the solid thread, there is no reported evidence that these ions influence the secondary structure of native spidroin in solution. Here, we demonstrate that pH values between approximately 3.5 and 4.5 induce a slow change of conformation from the disordered to the beta-sheet-rich form. We also report that Al(3+), K(+), and Na(+) ions induce similar changes in structure, while Ca(2+) and Mg(2+) stabilize the predominantly disorder state of the protein. Cs(+) and Li(+) have no apparent effect. Direct volumetric and spectrophotometric titration showed a pI of 4.22 +/- 0.33 and apparent pK values of 6.74 +/- 0.71 and 9.21 +/- 0.27, suggesting a mechanism for the effect of low pH on the protein and a rationale for the observed reduction in pH in the duct. We discuss the importance of these findings for the spinning process and the active role played by the spider to alter the kinetics of the transition.

Aerosols are significant to the Earth’s climate, with nearly all atmospheric aerosols containing organic compounds that often contain both hydrophilic and hydrophobic parts. However, the nature of how these compounds are arranged within an aerosol droplet remains unknown. Here we demonstrate that fatty acids in proxies for atmospheric aerosols self-assemble into highly ordered three-dimensional nanostructures that may have implications for environmentally important processes. Acoustically trapped droplets of oleic acid/sodium oleate mixtures in sodium chloride solution are analysed by simultaneous synchrotron small-angle X-ray scattering and Raman spectroscopy in a controlled gas-phase environment. We demonstrate that the droplets contained crystal-like lyotropic phases including hexagonal and cubic close-packed arrangements of spherical and cylindrical micelles, and stacks of bilayers, whose structures responded to atmospherically relevant humidity changes and chemical reactions. Further experiments show that self-assembly reduces the rate of the reaction of the fatty acid with ozone, and that lyotropic-phase formation also occurs in more complex mixtures more closely resembling compositions of atmospheric aerosols. We suggest that lyotropic-phase formation likely occurs in the atmosphere, with potential implications for radiative forcing, residence times and other aerosol characteristics.

Orb weaver spiders use exceptionally complex spinning processes to transform soluble silk proteins into solid fibers with specific functions and mechanical properties. In this study, to understand the nature of this transformation we investigated the structural changes of the soluble silk proteins from the major ampullate gland (web radial threads and spider safety line); flagelliform gland (web sticky spiral threads); minor ampullate gland (web auxiliary spiral threads); and cylindrical gland (egg sac silk). Using circular dichroism, we elucidated (i) the different structures and folds for the various silk proteins; (ii) irreversible temperature-induced transitions of the various silk structures toward beta-sheet-rich final states; and (iii) the role of protein concentration in silk storage and transport. We discuss the implication of these results in the spinning process and a possible mechanism for temperature-induced beta-sheet formation.

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Silks spun by spiders and insects are independently evolved proteinaceous biomaterials with fascinating physical properties attracting scientists from a wide range of disciplines using a wide range of analytical tools. Here we demonstrate the suitability of neutron small angle scattering (SANS) to investigate the morphology and structure of native silk fibroin at near in vivo conditions. Comparing native and reconstituted silk we observed significant differences in sizes, molecular weights, refolding and interactions. These observations question the validity of a presently widespread approach in silk analysis i.e. studying reconstituted silk with the goal to gain important insights into the mechanisms involved in the formation (storage and spinning) of native silks.

Spider silk is made and spun in a complex process that tightly controls the conversion from soluble protein to insoluble fiber. The mechanical properties of the silk fiber are modulated to suit the needs of the spider by various factors in the animal's spinning process. In the major ampullate (MA) gland, the silk proteins are secreted and stored in the lumen of the ampulla. A particular structural fold and functional activity is determined by the spidroins' amino acid sequences as well as the gland's environment. The transition from this liquid stage to the solid fiber is thought to involve the conversion of a predominantly unordered structure to a structure rich in beta-sheet as well as the extraction of water. Circular dichroism provides a quick and versatile method for examining the secondary structure of silk solutions and studying the effects of various conditions. Here we present the relatively novel technique of synchrotron radiation based circular dichroism as a tool for investigating biomolecular structures. Specifically we analyze, in a series of example studies on structural transitions induced in liquid silk, the type of information accessible from this technique and any artifacts that might arise in studying self-assembling systems.

Spider silks combine basic amino acids into strong and versatile fibers where the quality of the elastomer is attributed to the interaction of highly adapted protein motifs with a complex spinning process. The evaluation, however, of the interaction has remained elusive. Here, we present a novel analysis to study silk formation by examining the secondary structures of silk proteins in solution. Using the seven different silks of Nephila edulis as a benchmark system, we define a structural disorder parameter (the folding index, gamma). We found that gamma is highly correlated with the ratio of glycine present. Testing the correlation between glycine content and the folding index (gamma) against a selected range of silks, we find quantitatively that, in order to achieve specialization with changes in mechanical performance, the spider's silks require higher structural flexibility at the expense of reduced stability and consequently an increased conversion-energy cost. Taken together, our biophysical and evolutionary findings reveal that silk elastomericity evolved in tandem with specializations in the process of silk spinning.