Degree of Biomimicry of Artificial Spider Silk Spinning Assessed by NMR Spectroscopy.

Biomimetic spinning of artificial spider silk requires that the terminal domains of designed minispidroins undergo specific structural changes in concert with the ß-sheet conversion of the repetitive region. Herein, we combine solution and solid-state NMR methods to probe domain-specific structural changes in the NT2RepCT minispidroin, which allows us to assess the degree of biomimicry of artificial silk spinning. In addition, we show that the structural effects of post-spinning procedures can be examined. By studying the impact of NT2RepCT fiber drying, we observed a reversible beta-to-alpha conversion. We think that this approach will be useful for guiding the optimization of artificial spider silk fibers.

Mass spectrometry captures structural intermediates in protein fiber self-assembly.

Self-assembling proteins, the basis for a broad range of biological scaffolds, are challenging to study using most structural biology approaches. Here we show that mass spectrometry (MS) in combination with MD simulations captures structural features of short-lived oligomeric intermediates in spider silk formation, providing direct insights into its complex assembly process.

Biomimetic spinning of artificial spider silk from a chimeric minispidroin.

Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

Silk Spinning in Silkworms and Spiders.

Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.

Carbonic anhydrase generates CO2 and H+ that drive spider silk formation via opposite effects on the terminal domains.

Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive ß-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO2) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.

Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation.

The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.

Morphology and composition of the spider major ampullate gland and dragline silk.

Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout dragline silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.

Color vision and occupational chemical exposures: I. An overview of tests and effects.

The paper presents a summary of the literature published until December 2000 on effects from some industrial chemical exposures on color perception, as well as short descriptions of the tests applied. Several different tests have been used to study acquired alterations of color vision. These changes are frequently found in the blue-yellow axis. Many of the tests were originally designed to detect congenital alterations in the red-green axis, and thus have relatively low sensitivity when studying chemically induced deficits in color perception. At present, the Lanthony D15-desaturated panel seems most suitable for application in industrial settings, since it is clearly the most sensitive and easily administered test. Color vision seems to be a physiological function very sensitive to several chemicals. The potency of industrial chemicals to induce color vision deficiencies has often been investigated during the last two decades. The chemicals most frequently studied are different solvents and mercury. Pronounced effects on color perception have been reported following chronic exposure to organic solvents such as styrene, carbon disulphide, perchloroethylene, n-hexane and solvent mixtures, and to organic as well as inorganic mercury. The effect of occupational toluene exposure seems not as well established, since only slight effects and several negative studies have been reported. For some of these compounds the effect on color vision has been further established through the finding of clear dose-effect relationships. In a few cases, even acute exposure situations, e.g. exposure to toluene for a few hours or acute alcohol intake, seem to affect color perception. Follow-up studies are needed to investigate the possible reversibility of effects in relation to discontinued or reduced exposures.

Color vision and occupational chemical exposures. II. Visual functions in non-exposed subjects.

This paper presents data on visual functions (visual acuity, contrast sensitivity, and several tests of color vision), in a group of 199 non-exposed healthy subjects with an even distribution over the age range 18-65 years, and sex. Although subjects with obvious congenital color vision deficiencies were removed from the analyses (four males), females were superior to males on several of the color vision tests applied. Age influenced visual acuity and contrast sensitivity, while color discrimination was less affected. Correlations between functions of the right and the left eye in the individual subjects were rather low, ranging from 0.40 to 0.73. Correlations between visual acuity and contrast sensitivity on the one hand and color discrimination ability on the other hand were still lower (r < 0.20). These low correlations between functions in the two eyes support the need for testing each eye separately.