Spider silk proteins have mainly been investigated with regard to their contribution to mechanical properties of the silk thread. However, little is known about the molecular mechanisms of silk assembly. As a first step toward characterizing this process, we aimed to identify primary structure elements of the garden spider's (Araneus diadematus) major dragline silk proteins ADF-3 and ADF-4 that determine protein solubility. In addition, we investigated the influence of conditions involved in mediating natural thread assembly on protein aggregation. Genes encoding spider silk-like proteins were generated using a cloning strategy, which is based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences. Comparing secondary structure, solubility, and aggregation properties of the synthesized proteins revealed that single primary structure elements have diverse influences on protein characteristics. Repetitive regions representing the largest part of dragline silk proteins determined the solubility of the synthetic proteins, which differed greatly between constructs derived from ADF-3 and ADF-4. Factors, such as acidification and increases in phosphate concentration, which promote silk assembly in vivo generally decreased silk protein solubility in vitro. Strikingly, this effect was pronounced in engineered proteins comprising the carboxyl-terminal nonrepetitive regions of ADF-3 or ADF-4, indicating that these regions might play an important role in initiating assembly of spider silk proteins.

Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other. The absence of prolines in one component was previously speculated to be essential for the thread structure. Here, we analyzed dragline proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, which are both proline rich, by employing the baculovirus expression system. Whereas ADF-3 represented an intrinsically soluble protein, ADF-4 was insoluble in vitro and self-assembled into filaments in the cytosol of the host insect cells. These ADF-4 filaments displayed the exceptional chemical stability of authentic silk threads. We provide evidence that the observed properties of ADF-3 and ADF-4 strongly depend on intrinsic characteristics such as hydropathicity, which differs dramatically between the two proteins, as in most other pairs of dragline silk proteins from other Araneoidea species, but not on their proline content. Our findings shed new light on the structural components of spider dragline silk, allowing further elucidation of their assembly properties, which may open the door for commercial applications.

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In applications such as flavor encapsulation, drug delivery, and biomedical devices, microencapsulation is developing into an increasingly effective method for the protection and delivery of active ingredients. Herein, we show that spider-silk proteins are well-suited for producing responsive microcapsules with high mechanical stability. Emulsion interfaces are harnessed to induce the controlled self-assembly of these proteins into predominantly b-sheet configurations, resulting in a mechanically stable thin polymer shell. Capsules transferred into a continuous phase can readily encapsulate large molecules, while allowing small molecules to permeate freely. The capsules demonstrate good chemical stability, which is attributed to the b-sheet-rich structure of the self-assembled spider-silk proteins. These microcapsules represent a new class of biomimetic materials, exhibiting functionalities that can be further modified and controlled on the molecular level.