Spider Silk Formation Research Articles

Engineered a novel pH-sensitive short major ampullate spidroin

The pH diversification has been proved as an important factor affecting the self-assembly of spidroin. Herein, we constructed a novel spider silk protein (NT-MaSp1s-CT) with the pH-dependent secondary structures, containing pH-sensitive N-terminal, C-terminal domains and a repeated core region with merely 191 amino acids. Then pH sensitivity of NT-MaSp1s-CT was detected at different pH conditions and NT-MaSp1s-CT displayed pH-dependent conformational transitions consistent with rational designed objective. Besides, the micelles theory was employed to inquiry the assembly mechanism of NT-MaSp1s-CT in high concentration spinning dope. As expected, NT-MaSp1s-CT protein can be spun into continuous and uniform fibers with the pH ranging from 2 to 11, which is the largest pH boundary for artificial spider silk formation, simplifying the assembly conditions and paving a broad path for spinning process. Moreover, the hemolysis and cytotoxicity of NT-MaSp1s-CT fibers were also determined and the novel fibers exhibit excellent biocompatibility, providing wider potential applications in the biomedical and pharmaceutical fields.

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

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.

Probing the Impact of Acidification on Spider Silk Assembly Kinetics.

Spiders utilize fine adjustment of the physicochemical conditions within its silk spinning system to regulate spidroin assembly into solid silk fibers with outstanding mechanical properties. However, the exact mechanism about which this occurs remains elusive and is still hotly debated. In this study, the effect of acidification on spider silk assembly was investigated on native spidroins from the major ampullate (MA) gland fluid excised from Latrodectus hesperus (Black Widow) spiders. Incubating the protein-rich MA silk gland fluid at acidic pH conditions results in the formation of silk fibers that are 10-100 μm in length and ∼2 μm in diameter as judged by optical and electron microscope methods. The in vitro spider silk assembly kinetics were monitored as a function of pH with a (13)C solid-state MAS NMR approach. The results confirm the importance of acidic pH in the spider silk self-assembly process with observation of a sigmoidal nucleation-elongation kinetic profile. The rates of nucleation and elongation as well as the percentage of β-sheet structure in the grown fibers depend on the pH. These results confirm the importance of an acidic pH gradient along the spinning duct for spider silk formation and provide a powerful spectroscopic approach to probe the kinetics of spider silk formation under various biochemical conditions.

Evaluation of Functionalized Spider Silk Matrices: Choice of Cell Types and Controls are Important for Detecting Specific Effects.

Evaluation of Functionalized Spider Silk Matrices: Choice of Cell Types and Controls are Important for Detecting Specific Effects.

How spiders spin silk.

Spider silk is wonderful stuff—light as the breeze and stretchy yet stronger than steel. People can manufacture synthetic fibers, such as Kevlar, that come close but can't begin to match the process spiders use. Their silk proteins, called spidroins, rapidly convert from the soluble form to solid fibers at ambient temperatures and with water as the solvent. Not only is this beyond us, we don't even know how spiders do it. Now, in this issue of PLOS Biology, new research by Anna Rising, Jan Johansson, and colleagues shows that silk formation involves structural shifts at either end of the spidroin and that these shifts are completely different, overturning the hypothesis that these protein terminals play similar roles. Spidroins are big proteins of up to 3,500 amino acids that contain mostly repetitive sequences, and the nonrepetitive N- and C-terminal domains at opposite ends are thought to regulate conversion to silk. These terminal domains are unique to spider silk and are highly conserved among spiders. Spidroins have a helical and unordered structure when stored as soluble proteins in silk glands, but, when converted to silk, they contain β-sheets that lend mechanical stability. We know that there is a pH gradient across the spider silk gland, which narrows from a tail to a sac to a slender duct, and that silk forms at a precise site in the duct. However, further details of spider silk production have been elusive. To discover the basis for the pH gradient in the silk gland, the researchers studied the orb weaver spider Nephila clavipes, which reaches 7.6 centimeters wide and spins webs up to 3 meters across. Poking ion-selective microelectrodes into the silk gland revealed that the pH fell from 7.6 to 5.7 between the base of the tail and halfway down the duct, which was as far as the electrodes fit. This pH gradient is much steeper than previously thought. The microelectrodes also showed that bicarbonate ions rise between the base of the tail and the far end of the sac. Taken together, these ion patterns suggest that the pH gradient is due to carbonic anhydrase, an enzyme that converts carbon dioxide and water to bicarbonate and hydrogen ions. In support of this idea, a carbonic anhydrase inhibitor called methazolamide collapsed the pH gradient. Next, the researchers investigated how the pH range across the silk gland affects silk proteins, using N- and C-terminal domains isolated from the spidroins of another orb weaver, Araneus ventricosus (Figure 1). They found that both domains undergo structural changes at the pH found in the duct. Importantly, this is also where carbonic anhydrase activity is concentrated. Figure 1 An Araneus spider in its orb web. The researchers also found pH had opposite effects on the two domains' stability to temperature and urea, which was a surprise given that the domains had been suggested to have a similar impact on silk formation. The N-terminal dimerized at pH 6—which is found in the beginning of the duct—and became increasingly stable as the pH dropped along the duct. In contrast, the C-terminal domain destabilized as the pH dropped, gradually unfolding until it formed the β-sheets characteristic of silk at pH 5.5. What makes the C-terminal domain unfold? This end of the spidroin has a salt bridge joining two helices, and mutations that interfere with this bridge also destabilize the C-terminal domain. Underscoring its importance, the salt bridge is conserved in spidroins from several spider species. The researchers believe that protonating amino acids in the salt bridge would also destabilize the C-terminal domain, facilitating conversion of spidroins from helices to β-sheet fibrils. These findings led the researchers to propose a new “lock and trigger” model for spider silk formation. Gradual dimerization of the N-terminal domains could lock spidroins into multimers, while the β-sheet fibrils at the C-terminals could serve as nuclei that trigger rapid polymerization of spidroins into fibers. Interestingly, the C-terminal β-sheets are similar to those in the amyloid fibrils characteristic of diseases such as Alzheimer disease. This work brings us closer to unraveling the mystery of spider silk, explaining how it can form so quickly—faster than a meter per second—as well as how its formation can be confined to the spinning duct. Moreover, because the N- and C-terminal domains of spidroins are found nowhere else, this lock and trigger formation is likely unique to spider silk. Besides being essential to producing biomimetic spidroin fibers, knowing how spiders spin silk could give insights into natural ways of hindering the amyloid fibrils associated with disease. Andersson M, Chen G, Otikovs M, Landreh M, Nordling K, et al. (2014) Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains. doi:10.1371/journal.pbio.1001921

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

Author SummaryThe spinning process of spider silk is crucial for making webs or other complex constructions to catch spider's prey. The main components of the silk are spidroins, which are large and repetitive proteins that have conserved nonrepetitive terminal domains (NT and CT). Spiders manage both to store the highly aggregation-prone spidroins in solution at extreme concentrations in the silk glands and then to rapidly convert these spidroins into a solid fiber within fractions of a second as they spin fibres. This process has been extensively studied and is thought to involve a pH gradient, but how this pH gradient is generated and maintained was not resolved. Here, we measured the pH at locations along the ampullate gland and determined that the pH decreases to 5.7 in the middle of the spinning duct. We also observed that the carbon dioxide pressure is simultaneously increased and that its accumulation may affect the stability of CT. We find that active carbonic anhydrase (CA) is crucial to maintain the pH gradient along the gland. Detailed molecular studies of NT and CT under the disparate conditions present along the gland revealed a lock and trigger mechanism whereby in more neutral pH conditions, precocious spidroin aggregation is prevented, and when in more acidic pH conditions, NT dimers firmly interconnect the spidroins and the CT unfolds into β-sheet nuclei that can trigger rapid polymerization of the spidroins. We conclude that this mechanism enables temporal and spatial control of silk formation and may be harnessed in attempts to produce artificial silk replicas.

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.

Spider silk protein refolding is controlled by changing pH.

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.

Amyloid fibrillogenesis of silkmoth chorion protein peptide-analogues via a liquid-crystalline intermediate phase

Chorion, the major component of silkmoth eggshell, consists of the A and B classes of low-molecular weight structural proteins. Chorion protects the oocyte and the developing embryo from environmental hazards and this is due to the extraordinary physical and chemical properties of its constituent proteins. We have shown previously [FEBS Lett. 479 (2000) 141; 499 (2001) 268] that peptide-analogues of the A and B classes of chorion proteins form amyloid fibrils under a variety of conditions, which led us to propose that silkmoth chorion is a natural, protective amyloid. In this work, we present data showing conclusively that, the first main step of amyloid-like fibrillogenesis of chorion peptides is the formation of nuclei of liquid crystalline nature, which is reminiscent of spider-silk formation. We show that these liquid-crystalline nuclei (spherulites) ‘collapse’/deteriorate to form amyloid fibrils in a spectacular manner, important, it seems, for chorion morphogenesis and amyloid fibrillogenesis in general. The molecular ‘switch’ causing this spectacular transformation is, most probably, a conformational transition to the structure of chorion peptides, from a left-handed parallel β-helix to an antiparallel β-pleated sheet. Apparently, these peptides were suitably designed to play this role, after millions of years of molecular evolution.

Deformation induced hydrophobicity: implications in spider silk formation

A theoretical framework, which considers the effect of strong inter-polymer associations on phase separation of a deforming polyacrylamide solution is presented. It is shown that deformation induces effective hydrophobicity in the stretched polymer chains resulting in the formation of strong cooperative hydrogen bonding between the polymer chains. This finding has implications in providing insights into the mechanisms of the way spiders spin silk by rapidly deforming a freshly secreted protein solution. It has been argued that in a manner analogous to the model system showed here, it is likely that a rapid deformation induces phase separation of the solution into a polymer rich phase, which eventually forms the fiber. This work also naturally provides strategic hints for making silk like strong fibers by synthetic means at mild conditions.